Novel ionizable lipids and lipid nanoparticles and methods of use thereof

Abstract

Novel ionizable lipids and lipid nanoparticles are disclosed that can be used to deliver therapeutic cargo.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 402,929, filed on August 31, 2022, and U.S. Provisional Application No. 63 / 502,806, filed on May 17, 2023, which are hereby incorporated by reference in their entirety. Background of the Invention

[0003] Lipid nanoparticles (“LNPs”) formed from ionizable amine - containing lipids can act as therapeutic cargo vehicles to deliver bioactive agents, such as coding RNAs (i.e., messenger RNAs (mRNAs), guide RNAs) and non - coding RNAs (i.e., antisense, siRNAs) into cells. LNPs can facilitate the delivery of oligonucleotide agents across cell membranes and can be used to introduce components and compositions into living cells.

[0004] Bioactive agents that are particularly difficult to deliver into cells include proteins, nucleic - acid - based drugs and their derivatives, especially drugs that include relatively large oligonucleotides (such as mRNAs or guide RNAs). Compositions for delivering promising mRNA therapies or editing techniques into cells (such as for delivering components of the CRISPR / Cas9 system) have received particular attention.

[0005] With the emergence of recent pandemics, messenger RNA therapies have become an increasingly important option for treating various diseases, including viral infectious diseases and diseases associated with a deficiency of one or more proteins. Compositions having useful properties for stabilizing and / or delivering RNA components for in vitro and in vivo delivery have also received particular attention.

[0006] Accordingly, there is still a need in the art for novel lipid compounds to develop lipid nanoparticles or other lipid delivery mechanisms for therapeutic agent delivery. The present invention addresses this need. Summary of the Invention

[0007] Novel ionizable lipids are disclosed herein that can be used in combination with at least one other lipid component (such as neutral lipids, cholesterol, and polymer - conjugated lipids) to form lipid nanoparticle compositions. The lipid nanoparticle compositions can be used to facilitate intracellular delivery of therapeutic nucleic acids in vitro and / or in vivo.

[0008] Ionizable amine - containing lipids useful for forming lipid nanoparticle compositions are disclosed. Such LNP compositions can have properties that are favorable for delivering nucleic acid cargo (such as delivering coding and non - coding RNAs) into cells. Methods of using the disclosed lipid nanoparticles to treat various diseases or conditions (such as diseases or conditions caused by infectious entities and / or protein deficiencies) are also provided.

[0009] Lipids are disclosed below, particularly ionizable lipids having specific tail groups (e.g., geminal functional groups, i.e., geminal difunctional groups bonded to the same carbon next to a biodegradable group E).

[0010] Tail group

[0011] Certain aspects of the invention relate to a lipid comprising at least one head group and at least one tail group of formula (T)

[0012] its pharmaceutically acceptable salts or stereoisomers of any of the foregoing

[0013] wherein:

[0014] E is a biodegradable group;

[0015] R a each independently at each occurrence is a C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl or C2-C5 branched or unbranched alkynyl, said alkyl, alkenyl or alkynyl optionally being interspersed with heteroatoms or being substituted with OH, SH, halogen or NR 7 where each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl; or cycloalkyl or substituted cycloalkyl;

[0016] R b each independently at each occurrence is H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, said alkyl or alkenyl optionally being interspersed with heteroatoms or being substituted with OH, SH or halogen or cycloalkyl or substituted cycloalkyl; C1-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl;

[0017] u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7;

[0018] R t each independently is H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, said alkyl or alkenyl optionally being interspersed with heteroatoms or being substituted with OH, SH or halogen or cycloalkyl or substituted cycloalkyl;

[0019] represents the bond connecting the tail group to the head group; and

[0020] wherein the pKa of the lipid is from about 4 to about 8.

[0021] As used herein, the term "biodegradable" refers to a group that includes one or more bonds that can undergo bond cleavage reactions in a biological environment (e.g., in an organism, organ, tissue, cell, or organelle). For example, a biodegradable group can be metabolized (e.g., by hydrolysis) by the body of a mammal such as a human. Some groups containing biodegradable bonds include, for example, but are not limited to esters, dithiols, and oximes. Non-limiting examples of biodegradable groups are —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(R5)═N—, —N═C(R5)—, —C(R5)═N—O—, —O—N═C(R5)—, —C(O)(NR5)—, —N(R5)C(O)—, —C(S)(NR5)—, —N(R5)C(S)—, —N(R5)C(O)N(R5)—, —OC(O)O—, —OSi(R5)2O—, —C(O)(CR3R4)C(O)O— or —OC(O)(CR3R4)C(O)—. Each R5 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; each R3 and R4 is independently branched or unbranched C1-C 15 branched or unbranched alkyl, C1-C 15 branched or unbranched alkenyl or cycloalkyl or substituted cycloalkyl.

[0022] In some embodiments, R a are each independently C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl, or C2-C5 branched or unbranched alkynyl.

[0023] In some embodiments, R b are each independently H, C1-C 16 branched or unbranched alkyl, or C1-C 16 branched or unbranched alkenyl.

[0024] In some embodiments, R a are each independently C1-C3 branched or unbranched alkyl. In one embodiment, each R a is methyl.

[0025] In some embodiments, R b are each independently H or C1-C3 branched or unbranched alkyl.

[0026] In some embodiments, E is -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(O-R 13 )-O-, -C(O)O(CH2)r -, -C(O)N(R 7 )(CH2) r -, -S-S- or -C(O-R 13 )-O-(CH2) r -, where each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl; R 13 is a branched or unbranched C3-C 10 alkyl, and r is 1, 2, 3, 4 or 5.

[0027] In some embodiments, E is -OC(O)-, -C(O)O-, -N(R 7 )C(O)- or -C(O)N(R 7 )-, where R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl.

[0028] In one embodiment, E is -C(O)O-. In one embodiment, E is -OC(O)-. In one embodiment, E is N(R 7 )C(O)-. In one embodiment, E is or -C(O)N(R 7 )-.

[0029] In some embodiments, there is provided a lipid comprising at least one head group and at least one tail group having formula (TI) or (TI'):

[0030] its pharmaceutically acceptable salts or stereoisomers of any of the foregoing,

[0031] wherein:

[0032] E is each independently a biodegradable group;

[0033] R a each independently at each occurrence is a C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl or C2-C5 branched or unbranched alkynyl, said alkyl, alkenyl or alkynyl optionally being interspersed with heteroatoms or being substituted with OH, SH, halogen or NR 7 , where each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl; or cycloalkyl or substituted cycloalkyl;

[0034] u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7;

[0035] R t is independently H, C1-C 16Branched or unbranched alkyl or C1-C 16 Branched or unbranched alkenyl, said alkyl or alkenyl optionally interrupted by heteroatoms or substituted by OH, SH or halogen or cycloalkyl or substituted cycloalkyl;

[0036] Denotes a bond connecting said tail group to said head group; and

[0037] Wherein the pKa of said lipid is from about 4 to about 8.

[0038] In some embodiments, R a Is each independently at each occurrence a C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl or C2-C5 branched or unbranched alkynyl. In some embodiments, R a Is each independently at each occurrence a C1-C3 branched or unbranched alkyl. In one embodiment, each R a Is methyl.

[0039] In some embodiments, E is each independently -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(O-R 13 )-O-, -C(O)O(CH2) r -, -C(O)N(R 7 )(CH2) r -, -S-S- or -C(O-R 13 )-O-(CH2) r -, wherein each R 7 Is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl; R 13 Is a branched or unbranched C3-C 10 Alkyl, and r is 1, 2, 3, 4 or 5.

[0040] In some embodiments, E is each independently -OC(O)-, -C(O)O-, -N(R 7 )C(O)- or -C(O)N(R 7 )-, wherein R 7 Is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl.

[0041] In some embodiments, E is each independently -C(O)O-. In some embodiments, E is each independently -OC(O)-. In some embodiments, E is each independently -N(R 7 )C(O)-, wherein R 7Independently is H or methyl. In some embodiments, each E is independently -C(O)N(R 7 )-, where R 7 is independently H or methyl.

[0042] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TII):

[0043] where u3 and u4 are each independently 1 - 7 (e.g., 0, 1, 2, 3, or 4). The definitions of the other variables in (TII) are the same as those defined above in (TI).

[0044] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TIII):

[0045] (e.g., where u3 is 0, 1, 2, 3, 4, 5, 6, or 7; and R b is independently H or C1 - C4 alkyl in each case. The definitions of the other variables in (TIII) are the same as those defined above in (TI).

[0046] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TIV):

[0047] where u3 and u4 are each independently 1 - 7 (e.g., 0, 1, 2, 3, or 4). The definitions of the other variables in (TIV) are the same as those defined above in (TI).

[0048] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TV), (e.g., where u3 is 0, 1, 2, 3, 4, 5, 6, or 7; R 7 is independently H or methyl; and R b is independently H or C1 - C4 alkyl in each case. The definitions of the other variables in (TV) are the same as those defined above in (TI).

[0049] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TII'), (e.g., where u3 is 0, 1, 2, 3, 4, 5, 6, or 7; and R bIndependently in each case is H or a C1-C4 alkyl group. The definitions of the other variables in (TII') are the same as those defined above in (TI').

[0050] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TII'), (e.g., wherein u3 is 0, 1, 2, 3, 4, 5, 6 or 7; R 7 each independently is H or methyl; and R b independently in each case is H or a C1-C4 alkyl group. The definitions of the other variables in (TIII') are the same as those defined above in (TI').

[0051] In some embodiments, the lipid comprises at least one tail group of the following formula:

[0052]

[0053] wherein

[0054] R 7 each independently is H or methyl;

[0055] R b independently in each case is H or a C1-C4 alkyl group;

[0056] u3 and u4 each independently are 0, 1, 2, 3, 4, 5, 6 or 7; and

[0057] wherein the pKa of the lipid is from about 4 to about 8.

[0058] In some embodiments, the lipid comprises two or more tail groups having formula (T), (TI), (TII), (TIII), (TIV), (TV), (TII') and / or (TIII'), and each tail group can be the same or different.

[0059] In some embodiments, the lipid comprises three or more tail groups having formula (T), (TI), (TII), (TIII), (TIV), (TV), (TI')(TII') and / or (TIII'), and each tail group can be the same or different.

[0060] In some embodiments, the lipid comprises four or more tail groups having formula (T), (TI), (TII), (TIII), (TIV), (TV), (TI')(TII') and / or (TIII'), and each tail group can be the same or different.

[0061] In some embodiments, in any one of the above formulas (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII') and (TIII'), R a is methyl.

[0062] In some embodiments, in any one of the above formulas (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII') and / or (TIII'), u1 is 3, 4 or 5.

[0063] In some embodiments, in any one of the above formulas (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII') and / or (TIII'), u2 is 0, 1, 2 or 3.

[0064] In some embodiments, in any one of the above formulas (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII') and / or (TIII'), u3 and u4 are each independently 1-7, for example, u3 and u4 are each independently 1, 2, 3 or 4.

[0065] In some embodiments, the lipid comprises at least one tail of formula (TIII), wherein each R a is methyl, R b is independently H, ethyl or butyl in each case, u1 is 3-5, u2 is 0-3, and u3 is 1-7 (for example, 1-4).

[0066] In some embodiments, the lipid comprises at least one or two tails of formula (TIII), wherein the two tails of formula (TIII) are the same or different. In some embodiments, the lipid comprises at least three tails of formula (TIII), wherein each tail can be the same or different. In some embodiments, the lipid has four tails of formula (TIII), wherein each tail can be the same or different.

[0067] In some embodiments, in each tail of formula (TIII), each R a is methyl, and u1 is 3, u2 is 2, and u3 is 4.

[0068] In some embodiments, the lipid comprises at least one tail of formula (TII), wherein each R a is methyl, u1 is 3-5, u2 is 0-3, u3 is 1-4, and u4 is 1-4.

[0069] In some embodiments, the lipid has at least two tails of formula (TII), wherein the two tails of formula (TII) are the same or different. In some embodiments, the lipid comprises at least three tails of formula (TII), wherein each tail can be the same or different. In some embodiments, the lipid has four tails of formula (TII), wherein each tail can be the same or different.

[0070] In some embodiments, in each tail of formula (TII), each R a is methyl, and the variables u1, u2, u3, and u4 are one of the following:

[0071] (i) u1 is 5, u2 is 3, and u3 and u4 are each 1;

[0072] (ii) u1 is 5, u2 is 0, and u3 and u4 are each 2;

[0073] (iii) u1 is 5, u2 is 0, and u3 and u4 are each 3;

[0074] (iv) u1 is 5, u2 is 0, and u3 and u4 are each 4;

[0075] (v) u1 is 5, u2 is 0, u3 is 4, and u4 is 2; or

[0076] (vi) u1 is 3, u2 is 3, and u3 and u4 are each 1.

[0077] In some embodiments, the lipid comprises at least one tail of formula (TIV), wherein each R a is methyl, u1 is 3 - 5, u2 is 0 - 3, u3 is 1 - 4, and u4 is 1 - 4.

[0078] In some embodiments, the lipid comprises at least two tails of formula (TIV), wherein each tail can be the same or different. In some embodiments, the lipid comprises at least three tails of formula (TIV), wherein each tail can be the same or different. In some embodiments, the lipid comprises at least four tails of formula (TIV), wherein each tail can be the same or different.

[0079] In some embodiments, the lipid comprises at least two tails of formula (TV), wherein each tail can be the same or different. In some embodiments, the lipid comprises at least three tails of formula (TV), wherein each tail can be the same or different. In some embodiments, the lipid comprises at least four tails of formula (TV), wherein each tail can be the same or different.

[0080] In some embodiments, the lipid has at least two tails of formula (TII'), where each tail can be the same or different. In some embodiments, the lipid has at least three tails of formula (TII'), where each tail can be the same or different. In some embodiments, the lipid has at least four tails of formula (TII'), where each tail can be the same or different.

[0081] In some embodiments, the lipid has at least two tails of formula (TIII'), where each tail can be the same or different. In some embodiments, the lipid has at least three tails of formula (TIII'), where each tail can be the same or different. In some embodiments, the lipid has at least four tails of formula (TIII'), where each tail can be the same or different.

[0082] In some embodiments, the lipid has at least one tail selected from the group consisting of formula (TII), (TIII), and (TII').

[0083] In some embodiments, the lipid has at least one tail selected from the group consisting of (TIV), (TV), and (TIII').

[0084] In some embodiments, the lipid has at least one tail selected from the group consisting of formula (TII), (TIII), and (TII'), and at least one tail selected from the group consisting of (TIV), (TV), and (TIII').

[0085] In some embodiments, the lipid has at least two tails selected from the group consisting of (TII), (TIII), and (TII').

[0086] In some embodiments, the lipid has at least two tails selected from the group consisting of (TIV), (TV), and (TIII').

[0087] In some embodiments, the lipid has at least two tails selected from the group consisting of formula (TII), (TIII), and (TII'), and at least one tail selected from the group consisting of (TIV), (TV), and (TIII').

[0088] In some embodiments, the lipid has at least one tail selected from the group consisting of formula (TII), (TIII), and (TII'), and at least two tails selected from the group consisting of (TIV), (TV), and (TIII').

[0089] In some embodiments, the lipid has at least two tails selected from the group consisting of formula (TII), (TIII), and (TII'), and at least two tails selected from the group consisting of (TIV), (TV), and (TIII').

[0090] In some embodiments, the lipid has at least three tails selected from the group consisting of formula (TII), (TIII), and (TII').

[0091] In some embodiments, the lipid has at least three tails selected from the group consisting of (TIV), (TV), and (TIII').

[0092] In some embodiments, the lipid has at least three tails selected from the group consisting of formula (TII), (TIII), and (TII'), and at least one tail selected from the group consisting of (TIV), (TV), and (TIII').

[0093] In some embodiments, the lipid has at least one tail selected from the group consisting of formula (TII), (TIII), and (TII'), and at least three tails selected from the group consisting of (TIV), (TV), and (TIII').

[0094] In some embodiments, the lipid has at least one tail of formula (TII) or (TIII), and at least one tail of formula (TIV) or (TV). In some embodiments, the lipid has at least two tails of formula (TII) or (TIII) and at least two tails of formula (TIV) or (TV).

[0095] In some embodiments, the lipid has at least one tail of formula (TII) or (TIII), and at least one tail of formula (TII') or (TIII'). In some embodiments, the lipid has at least two tails of formula (TII) or (TIII), and at least two tails of formula (TII') or (TIII').

[0096] In some embodiments, the lipid has at least one tail of formula (TIV) or (TV), and at least one tail of formula (TII') or (TIII'). In some embodiments, the lipid has at least two tails of formula (TIV) or (TV), and at least two tails of formula (TII') or (TIII').

[0097] In some embodiments, the lipid has at least one tail of formula (TII) and at least one tail of formula (TIII). In some embodiments, the lipid has at least two tails of formula (TII) and at least two tails of formula (TIII). In some embodiments, in each tail of formula (TII) or formula (TIII), R a is methyl, u1 is 3 - 5, u2 is 0 - 2, u3 is 1 - 4, and u4 is 1 - 4.

[0098] In some embodiments, the lipid has at least one tail of formula (TII) and / or at least one tail of formula (TIII); the lipid further comprises at least one tail that does not have the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TII'), and / or (TIII'). That is, the lipid further comprises at least one tail that does not contain a geminal difunctional group bonded to the same carbon adjacent to E (e.g., -C(O)O-).

[0099] In some embodiments, the lipid further comprises at least one tail that does not have the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'). That is, the lipid further comprises at least one tail that does not contain a geminal difunctional group bonded to the same carbon adjacent to E.

[0100] In some embodiments, the lipid further comprises at least one tail of formula (TNG-I):

[0101] wherein

[0102] E are each independently a biodegradable group as described herein, such as -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -S-S- or -C(O)N(R 7 )-;

[0103] u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7; and

[0104] R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl.

[0105] In some embodiments, the at least one tail of formula (TNG-I) can be represented by

[0106] wherein

[0107] u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; and

[0108] R b is independently H or a C1-C4 alkyl in each case.

[0109] As described above, all embodiments of the definitions of E, R b 、R t 、u1, u2, u3 and u4 relating to a tail group containing a gem-difunctional group of formula (T), (TI), (TII), (TIII), (TIV), (TV), (TII') or (TIII') bonded to the same carbon next to E also apply to a tail group containing a gem-difunctional group of formula (TNG-I), (TNG-II) or (TNG-III) not bonded to the same carbon next to E.

[0110] In some embodiments, the lipid further comprises at least two tails not having the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII') and / or (TIII'). In some embodiments, the lipid comprises two tail groups of formula (TNG-II) or (TNG-III), and each tail group can be the same or different,

[0111] In some embodiments, the lipid further comprises at least three tails not having the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII') and / or (TIII'). In some embodiments, the lipid comprises three tail groups of formula (TNG-II) or (TNG-III), and each tail group can be the same or different,

[0112] Head group

[0113] The head group of the lipid can be any amine-containing head group of a typical ionizable lipid.

[0114] In some embodiments, the head group of the lipid has the structure of formula (HA-I):

[0115]

[0116] Wherein:

[0117] R 20 and R 30 are each independently H, a C1-C5 branched or unbranched alkyl or a C2-C5 branched or unbranched alkenyl, the alkyl or alkenyl being optionally interrupted by one or more heteroatoms or substituted by OH, SH, halogen or cycloalkyl; or

[0118] R 20 and R 30 together with the adjacent N atom form a 3- to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, which heterocyclic or heteroaromatic ring is optionally substituted by one or more OH, SH, halogen, alkyl or cycloalkyl;

[0119] Each of R1 and R2 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, OH, halogen, SH or NR 10 R 11 ; or R1 and R2 together form a ring;

[0120] R 10 and R 11 each of which is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl; or R 10 and R 11 together form a heterocyclic ring;

[0121] n is 0, 1, 2, 3 or 4;

[0122] Z is absent, O, S or NR 12 wherein R 12 is H or a C1-C7 branched or unbranched alkyl; provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 , SH.

[0123] In some embodiments, R 20 and R 30 together with the adjacent N atom form a 3- to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, which heterocyclic or heteroaromatic ring is optionally substituted by one or more OH, SH, halogen, alkyl or cycloalkyl.

[0124] In some embodiments, the head group of the lipid has the structure of formula (HA-IA):

[0125]

[0126] Wherein:

[0127] Each of R1 and R2 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, OH, halogen, SH or NR 10 R 11 ; or R1 and R2 together form a ring;

[0128] R 10 and R 11Each of them is independently H, a C1-C3 branched or unbranched alkyl group, or a C2-C3 branched or unbranched alkenyl group; or R 10 and R 11 together form a heterocycle;

[0129] m is 1, 2, 3, 4, 5, 6, 7, or 8;

[0130] n is 0, 1, 2, 3, or 4;

[0131] Z is absent, O, S, or NR 12 , where R 12 is H or a C1-C7 branched or unbranched alkyl group; provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 , SH; and

[0132] represents a bond connecting the head group and the tail group.

[0133] In some embodiments, m is 1, 2, 3, or 4.

[0134] In some embodiments, the head group of the lipid has a structure of formula (HA-III):

[0135] where Z is absent, O, S, or NR 12 ; and R 12 is a C1-C7 alkyl group. The definitions of the other variables in (HA-III) are the same as those defined in (HA-I) above.

[0136] In some embodiments, in any of the above formulas (such as (HA-I), (HA-IA), or (HA-III)), Z is absent, O, S, or NH.

[0137] In some embodiments, in any of the above formulas (such as (HA-I), (HA-IA), or (HA-III)), each of R1 and R2 is H.

[0138] In some embodiments, in any of the above formulas (such as (HA-I), (HA-IA), or (HA-III)), n is 0, 1, or 2.

[0139] In some embodiments, where the head group has the following structure: where:

[0140] Rc is H or an alkyl group, optionally substituted with OH; and

[0141] m1 is 1, 2, or 3.

[0142] In some embodiments, the head group of the lipid has a structure of formula (HA-V):

[0143]

[0144] Wherein:

[0145] R1 is H, C1-C3 alkyl, OH, halogen, SH or NR 10 R 11 ;

[0146] R2 is OH, halogen, SH or NR 10 R 11 ; or R1 and R2 can combine together to form a ring;

[0147] R 10 and R 11 are each independently H or C1-C3 alkyl; or R 10 and R 11 can combine together to form a heterocycle;

[0148] R 20 and R 30 are each independently H, C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl; or R 20 and R 30 can combine together to form a ring; and

[0149] Each of v and y is independently 1, 2, 3 or 4.

[0150] In some embodiments, the head group of the lipid has a structure of formula (HA-VI):

[0151] All variables in (HA-VI) are defined the same as those defined above in (HA-V).

[0152] In some embodiments, in any one of the above formulas (such as (HA-V) or (HA-VI)), each R 20 and R 30 is independently C1-C3 alkyl. In one embodiment, each R 20 and R 30 is independently methyl.

[0153] In some embodiments, the head group of the lipid has a structure of formula (HA-VII): Wherein u20 is 1, 2, 3, 4 or 5.

[0154] In some embodiments, the head group of the lipid has a structure of formula (HB-I):

[0155]

[0156] wherein W is

[0158] wherein

[0159] R5 is OH, SH, (CH2) s OH or NR 10 R 11 ;

[0160] each R6 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, or a cycloalkyl;

[0161] each R7 and R8 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, a halogen, (CH2) v OH, (CH2) v SH, (CH2) s N(CH3)2 or NR 10 R 11 , wherein each R 10 and R 11 is independently H or a C1-C3 alkyl, or R 10 and R 11 together form a heterocycle; or R7 and R8 together form a ring;

[0162] each R 20 is independently H or a C1-C3 branched or unbranched alkyl;

[0163] R 14 is a heterocycle, NR 10 R 11 、C(O)NR 10 R 11 、NR 10 C(O)NR 10 R 11 or NR 10 C(S)NR 10 R 11， wherein each R 10 and R 11 is independently H, a C1-C3 alkyl, a C3-C7 cycloalkyl, a C3-C7 cycloalkenyl, the alkyl, cycloalkyl, cycloalkenyl being optionally substituted with one or more NH and / or oxo groups, or R 10 and R 11 together form a heterocycle;

[0164] R 16 is H, =O, =S or CN;

[0165] Each of s, u and t is independently 1, 2, 3, 4 or 5;

[0166] Each v is independently 0, 1, 2, 3, 4 or 5;

[0167] Each Y is a divalent heterocycle;

[0168] Each Z independently does not exist, is O, S or NR 12 , where R 12 is H, a C1-C7 branched or unbranched alkyl or a C2-C7 branched or unbranched alkenyl;

[0169] Q is O, S, CH2 or NR 13 , where each R 13 is H, a C1-C5 alkyl;

[0170] V is a branched or unbranched C2-C 10 alkylene, C2-C 10 alkenylene, C2-C 10 alkynylene or C2-C 10 heteroalkylene, and the alkylene, alkenylene, alkynylene or heteroalkylene is optionally substituted by one or more OH, SH and / or halogen groups; and

[0171] T is –NHC(O)O-, –OC(O)NH- or a divalent heterocycle.

[0172] In some embodiments, R5 is OH or (CH2) s OH; and s is 1 or 2.

[0173] In some embodiments, each of R6, R7 and R8 is independently H or a C1-C3 alkyl.

[0174] In some embodiments, each of u and t is independently 1, 2 or 3.

[0175] In some embodiments, each v is independently 0, 1, 2 or 3.

[0176] In some embodiments, R 16 is H or =O.

[0177] In some embodiments, each Z independently does not exist, is O or NR 12 , where R 12 is H or a C1-C3 alkyl.

[0178] In some embodiments, T is a divalent heterocycle.

[0179] In some embodiments, Q is O or CH2.

[0180] In some embodiments, V is a C2-C6 alkylene or a C2-C6 alkenylene.

[0181] In some embodiments, the heterocycle (or divalent heterocycle) is piperazine, piperazinedione, piperazine-2,5-dione, piperidine, pyrrolidine, piperidinol, dioxopiperazine, bispiperazine, aromatic or heteroaromatic.

[0182] In some embodiments, in formula (HB-I), W is wherein:

[0183] each of R6, R7, and R8 is independently H or methyl; and

[0184] each of u and t is independently 1, 2, or 3.

[0185] In some embodiments, in formula (HB-I), W is wherein:

[0186] R 16 is H or ═O;

[0187] R 14 is a 5- or 6-membered nitrogen-containing heterocycle, NR 10 R 11 、C(O)NR 10 R 11 、NR 10 C(O)NR 10 R 11 or NR 10 C(S)NR 10 R 11， where each R 10 and R 11 is independently H or a C1-C3 alkyl; and

[0188] each of u and v is independently 1, 2, or 3.

[0189] In some embodiments, in formula (HB-I), W is wherein:

[0190] each R6 is independently H or methyl;

[0191] each u is independently 1, 2, or 3; and

[0192] V is a C2-C6 alkylene or a C2-C6 alkenylene.

[0193] In some embodiments, in formula (HB-I), W is Wherein:

[0194] Each R6 is independently H or methyl;

[0195] Each R7 is independently H;

[0196] Each R8 is methyl;

[0197] Each u is independently 1, 2 or 3; and

[0198] V is a C2-C6 alkylene or a C2-C6 alkenylene.

[0199] In some embodiments, in formula (HB-I), W is Wherein:

[0200] Each u is independently 1, 2 or 3; and

[0201] T is a divalent nitrogen-containing 5- or 6-membered heterocycle.

[0202] In some embodiments, in formula (HB-I), W is

[0203] Wherein:

[0204] Each u is independently 1, 2 or 3;

[0205] Q is O;

[0206] Each Z is independently NR 12； And

[0207] R 12 is H or a C1-C3 alkyl.

[0208] In some embodiments, the head group has the following structure:

[0209] Where each of u and t is independently 1 or 2.

[0210] In some embodiments, the head group of the lipid has the structure of formula (HC-I):

[0211]

[0212] Where

[0213] is a cyclic or heterocyclic moiety;

[0214] Y is alkyl, hydroxy, hydroxyalkyl,

[0215] A is absent, is -O-, -N(R 7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -S-S-;

[0216] Each of X and Z independently is absent, is -O-, -C(O)-, -N(R 7 )-, alkylene, -O-alkylene-; -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-;

[0217] Each R 7 independently is H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl or aminoalkyl;

[0218] t is 0, 1, 2 or 3;

[0219] t1 is an integer from 0 to 10; and

[0220] W is hydroxy, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl or substituted or unsubstituted heterocyclic group or heteroaryl.

[0221] In some embodiments, W is hydroxy, substituted or unsubstituted hydroxyalkyl, or one of the following moieties:

[0222]

[0223] Wherein

[0224] Each Q independently is absent, is -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2) q -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 );

[0225] R 6 independently is H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, thiol, thiolalkyl or N + (R 7)3-alkylene-Q-;

[0226] Each R 8 is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol or thioalkyl, heterocyclic group, heteroaryl, or two Rs 8 together with the nitrogen atom can form a ring, which is optionally substituted by one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, aminoalkyl;

[0227] q is 0, 1, 2, 3, 4 or 5; and

[0228] p is 0, 1, 2, 3, 4 or 5.

[0229] In some embodiments, the head group of the lipid has the structure of formula (HC-IA):

[0230] All variables in (HC-IA) or (HC-IB) are defined the same as those defined in (HC-V) above.

[0231] In some embodiments, in any of the above formulas (such as (HC-I), (HC-IA) or (HC-IB)), is a 5- to 7-membered monocyclic ring. In some embodiments, is a 5- to 7-membered monocyclic cycloalkane ring. In some embodiments, is a 5- to 7-membered monocyclic heterocycle.

[0232] In some embodiments, in any of the above formulas (such as (HC-I), (HC-IA) or (HC-IB)), is bicyclic or tricyclic, i.e., contains two or more rings, such as fused rings.

[0233] In some embodiments, in any of the above formulas (such as (HC-I), (HC-IA) or (HC-IB)), has the formula structure,

[0234] wherein:

[0235] Each of G1, G2, G3, G4, G5, G6 and G7 is independently C(R')(R”), O or N, provided that no more than two of G1-G7 are O or N;

[0236] R' and R” each independently do not exist, are H, alkyl, or two R's together with two adjacent Gs form a second 5- to 7-membered ring or heterocycle; and

[0237] n1 and n2 are each independently 0 or 1.

[0238] In some embodiments, in any of the above formulas (such as (HC-I), (HC-IA), or (HC-IB)), selected from pyrrolidine, piperidine, piperazine, cyclohexane, cyclopentane, tetrahydrofuran; tetrahydropyran; morpholine, and dioxane.

[0239] In some embodiments, in any of the above formulas (such as (HC-I), (HC-IA), or (HC-IB)), selected from the group consisting of:

[0240]

[0241] In some embodiments, in any of the above formulas (such as (HC-I), (HC-IA), or (HC-IB)), selected from the group consisting of:

[0242]

[0243] In some embodiments, the head group of the lipid has the structure of formula (HC-IIA):

[0244] Each R 7 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a cycloalkyl group, a hydroxyalkyl group, or an aminoalkyl group. The definitions of all other variables in (HC-IIA) are the same as those defined above in (HC-I).

[0245] In some embodiments, the head group of the lipid has the structure of formula (HC-IIA'):

[0246] Each R 7 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a cycloalkyl group, a hydroxyalkyl group, or an aminoalkyl group. The definitions of all other variables in (HC-IIA') are the same as those defined above in (HC-I).

[0247] In some embodiments, the head group of the lipid has the structure of formula (HC-IIC):

[0248] Each R 7 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a cycloalkyl group, a hydroxyalkyl group, or an aminoalkyl group. The definitions of all other variables in (HC-IIC) are the same as those defined above in (HC-I).

[0249] In some embodiments, the head group of the lipid has a structure of formula (HC-IIC'):

[0250] Each R 7 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a cycloalkyl group, a hydroxyalkyl group or an aminoalkyl group. The definitions of all other variables in (HC-IIC') are the same as those defined in (HC-I) above.

[0251] In some embodiments, in any of the above formulas (such as (HC-I), (HC-IA), (HC-IB), (HC-IIA), (HC-IIA'), (HC-IIC) and (HC-IIC')), X is absent, is -O- or –C(O)-.

[0252] In some embodiments, in any of the above formulas (such as (HC-I), (HC-IA), (HC-IB), (HC-IIA), (HC-IIA'), (HC-IIC) and (HC-IIC')), Z is –O-, –C(O)O- or –OC(O)-.

[0253] In some embodiments, the head group of the lipid has a structure of one of the following formulas:

[0254] The definitions of all variables are the same as those defined in (HC-I) above.

[0255] In some embodiments, the head group of the lipid has a structure of one of the following formulas:

[0256] The definitions of all variables are the same as those defined in (HC-I) above.

[0257] In some embodiments, in any of the above formulas (such as (HC-I), (HC-IA), (HC-IB), (HC-IIA), (HC-IIA'), (HC-IIC), (HC-IIC'), (HC-IIIA), (HC-IIIA'), (HC-IIIC) and (HC-IIIC')), A is absent, is -O-, -N(R 7 )-, -C(O)N(R 7 )-, -N(R 7 )C(O)-, -OC(O)- or -C(O)O-. In one embodiment, A is absent. In one embodiment, A is -O-. In one embodiment, A is -N(R 7 )-, where R7 is H or a C1-C3 alkyl group. In one embodiment, A is -OC(O)- or -C(O)O-. In one embodiment, A is -NHC(O)- or -C(O)NH-.

[0258] In some embodiments, the head group of the lipid has the structure of one of the following formulas:

[0259] where t1 is 0, 1, 2, or 3. The definitions of the other variables in these formulas are the same as those defined above in (HC-I).

[0260] In some embodiments, the head group of the lipid has the structure of one of the following formulas:

[0261] where the definitions of the variables in these formulas are the same as those defined above in (HC-I).

[0262] In some embodiments, in any of the above formulas (such as (HC-I), (HC-IA), (HC-IB), (HC-IIA), (HC-IIA'), (HC-IIC), (HC-IIC'), (HC-IIIA), (HC-IIIA'), (HC-IIIC), (HC-IIIC'), (HC-IIID), (HC-IIID'), (HC-IIIE), and (HC-IIIE')), t is 0, 1, or 2.

[0263] In some embodiments, in any of the above formulas (such as (HC-I), (HC-IA), (HC-IB), (HC-IIA), (HC-IIA'), (HC-IIC), (HC-IIC'), (HC-IIIA), (HC-IIIA'), (HC-IIIC), (HC-IIIC'), (HC-IIID), (HC-IIID'), (HC-IIIE), and (HC-IIIE')), W is OH.

[0264] In some embodiments, in any of the above formulas (such as (HC-I), (HC-IA), (HC-IB), (HC-IIA), (HC-IIA'), (HC-IIC), (HC-IIC'), (HC-IIIA), (HC-IIIA'), (HC-IIIC), (HC-IIIC'), (HC-IIID), (HC-IIID'), (HC-IIIE), and (HC-IIIE')), W is OH,

[0265] In some embodiments, W is where Q is absent, is -(CH2) q -C(R 7 )2- or -N(R 7 );q is 0 or 1;R 7 is H or methyl;and each R 8 is independently H or a C1-C3 alkyl. In one embodiment, W is

[0266] In some embodiments, W is where Q is absent, is -(CH2) q -C(R 7 )2- or -N(R 7 );q is 0 or 1;R 7 is H or methyl;and each R 8 is independently H or a C1-C3 alkyl. In one embodiment, W is

[0267] In some embodiments, W is where Q is absent, is -(CH2) q -C(R 7 )2- or -N(R 7 );q is 0 or 1;R 7 is H or methyl;and each R 8 is independently H or a C1-C3 alkyl. In one embodiment, W is

[0268] In some embodiments, W is where Q is -(CH2) q -C(R 7 )2-;q is 0 or 1;R 7 is H or methyl;and each R 8 is independently H or a C1-C3 alkyl. In one embodiment, W is In one embodiment, W is In one embodiment, W is In W, is

[0269] In some embodiments, W is where q is 0, and each R 8 is independently H, a C1-C3 alkyl, a hydroxyalkyl, a heterocyclic group, or a heteroaryl, optionally substituted by one or more alkyls. In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is

[0270] In some embodiments, W is where each R 6 is independently H, C1-C3 alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl or -O-alkylene-N(R 7 )2, and each R 7 is independently H or C1-C3 alkyl. In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is

[0271] In one embodiment, W is In one embodiment, W is

[0272] In one embodiment, W is In one embodiment, W is

[0273] In some embodiments, W is where each R 6 is independently H, C1-C3 alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl or -O-alkylene-N(R 7 )2; Q is -O-, -C(R 7 )2- or N(R 7 ); and R 7 is H, C1-C3 alkyl or hydroxyalkyl. In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is

[0274] In one embodiment, W is In one embodiment, W is In one embodiment, W is

[0275] In some embodiments, W is where q is 0 and each R 8 is independently H, C1-C3 alkyl or hydroxyalkyl. In one embodiment, W is In one embodiment, W is

[0276] In some embodiments, W is where R 6 is independently H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl or -O-alkylene-N(R 7 )2; and each R 7 is independently H or C1-C3 alkyl. In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is

[0277] In some embodiments, W is where each R 8 is independently H, C1-C3 alkyl or hydroxyalkyl; each Q independently is absent, is -O-, -CO-, -C(R 7 )2- or -N(R 7 )-; and each R 7 is independently H, C1-C3 alkyl, alkylamino, alkylaminoalkyl or aminoalkyl. In one embodiment, W is In one embodiment, W is

[0278] In some embodiments, W is where each R 8 is independently H, C1-C3 alkyl or hydroxyalkyl; each Q independently is absent, is -O-, -CO-, -C(R 7)2- or -N(R 7 )-; and each R 7 is independently H, C1-C3 alkyl, alkylamino, alkylaminoalkyl or aminoalkyl. In one embodiment, W is In one embodiment, W is

[0279] In some embodiments, provided herein is a lipid comprising at least one head group and at least one tail group, wherein:

[0280] The tail group has a structure of formula (TI) or (TI')

[0281] its pharmaceutically acceptable salt or stereoisomer of any of the foregoing,

[0282] wherein:

[0283] E are each independently biodegradable groups;

[0284] R a are each independently C1-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl;

[0285] u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7;

[0286] R t are each independently H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, said alkyl or alkenyl optionally interrupted by a heteroatom or substituted by OH, SH or halogen or cycloalkyl or substituted cycloalkyl;

[0287] represents the bond connecting the tail group to the head group; and

[0288] The head group has a structure of one of the following formulas:

[0289]

[0290] wherein:

[0291] R 20 and R 30 are each independently H, C1-C5 branched or unbranched alkyl or C2-C5 branched or unbranched alkenyl, said alkyl or alkenyl optionally interrupted by one or more heteroatoms or substituted by OH, SH, halogen or cycloalkyl; or

[0292] R 20 and R 30Together with an adjacent N atom, it forms a 3- to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, which is optionally substituted by one or more OH, SH, halogen, alkyl or cycloalkyl;

[0293] Each of R1 and R2 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH or NR 10 R 11 ; or R1 and R2 together form a ring;

[0294] R 10 and R 11 Each of them is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl; or R 10 and R 11 together form a heterocyclic ring;

[0295] n is 0, 1, 2, 3 or 4;

[0296] Z is absent, is O, S or NR 12 wherein R 12 is H or C1-C7 branched or unbranched alkyl; provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 R

[0297]

[0298] Wherein:

[0299] R1 is H, C1-C3 alkyl, OH, halogen, SH or NR 10 R 11 ;

[0300] R2 is OH, halogen, SH or NR 10 R 11 ; or R1 and R2 can combine together to form a ring;

[0301] R 10 and R 11 are each independently H or C1-C3 alkyl; or R 10 and R 11 can combine together to form a heterocyclic ring;

[0302] R 20 and R 30 are each independently H, C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl; or R 20 and R 30 can combine together to form a ring; and

[0303] Each of v and y is independently 1, 2, 3, or 4;

[0304]

[0305] wherein W is

[0306]

[0307] wherein

[0308] R5 is OH, SH, (CH2) s OH or NR 10 R 11 ;

[0309] Each R6 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, or a cycloalkyl group;

[0310] Each of R7 and R8 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, (CH2) v OH, (CH2) v SH, (CH2) s N(CH3)2 or NR 10 R 11 , where each R 10 and R 11 is independently H or a C1-C3 alkyl group, or R 10 and R 11 together form a heterocycle; or R7 and R8 together form a ring;

[0311] Each R 20 is independently H or a C1-C3 branched or unbranched alkyl group;

[0312] R 14 is a heterocycle, NR 10 R 11 , C(O)NR 10 R 11 , NR 10 C(O)NR 10 R 11 or NR 10 C(S)NR 10 R 11， where each R 10 and R 11 is independently H, a C1-C3 alkyl group, a C3-C7 cycloalkyl group, a C3-C7 cycloalkenyl group, the alkyl, cycloalkyl, cycloalkenyl group being optionally substituted by one or more NH and / or oxo groups, or R 10 and R 11Combine to form a heterocycle;

[0313] R 16 is H, =O, =S or CN;

[0314] Each of s, u and t is independently 1, 2, 3, 4 or 5;

[0315] Each v is independently 0, 1, 2, 3, 4 or 5;

[0316] Each Y is a divalent heterocycle;

[0317] Each Z independently does not exist, is O, S or NR 12 , where R 12 is H, C1-C7 branched or unbranched alkyl or C2-C7 branched or unbranched alkenyl;

[0318] Q is O, S, CH2 or NR 13 , where each R 13 is H, C1-C5 alkyl;

[0319] V is a branched or unbranched C2-C 10 alkylene, C2-C 10 alkenylene, C2-C 10 alkynylene or C2-C 10 heteroalkylene, and the alkylene, alkenylene, alkynylene or heteroalkylene is optionally substituted by one or more OH, SH and / or halogen groups; and

[0320] T is –NHC(O)O-, –OC(O)NH- or a divalent heterocycle;

[0321]

[0322] Wherein:

[0323] is a cyclic or heterocyclic moiety;

[0324] Y is alkyl, hydroxy, hydroxyalkyl,

[0325] A does not exist, is -O-, -N(R 7 ), -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 ), -N(R 7 )C(O)N(R 7 ), -S- or -S-S-;

[0326] Each of X and Z is independently absent, or is -O-, -C(O)-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-;

[0327] Each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl or aminoalkyl;

[0328] t1 is an integer from 0 to 10; and

[0329] W is hydroxy, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl or substituted or unsubstituted heterocyclic or heteroaryl; and

[0330] wherein the pKa of the lipid is from about 4 to about 8.

[0331] In some embodiments, provided herein is a lipid comprising at least one head group and at least one tail group, wherein:

[0332] At least one tail group has a structure of at least one of the following formulas:

[0333]

[0334] Wherein:

[0335] R 7 are each independently H or methyl;

[0336] R b is independently H or C1-C4 alkyl in each case;

[0337] R a are each independently C1-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl;

[0338] u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7;

[0339] u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6 or 7; and

[0340] The head group has a structure of one of the following formulas:

[0341] where m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0342]

[0343] and

[0344]

[0345] In some embodiments, in the above lipids, at least one tail group has the structure of formula (TII), (TIII), (TIV), (TV), (TII') or (TIII'), wherein each R a is methyl; u1 is 3 - 5, u2 is 0 - 3; and u3 and u4 are each independently 1 - 7.

[0346] In some embodiments, in the above lipids, the head group has the structure of one of the following formulas:

[0347]

[0348] wherein each R 20 and R 30 are independently C1 - C3 alkyl;

[0349] wherein:

[0350] W is wherein:

[0351] each R6, R7 and R8 are independently H or methyl; and

[0352] each of u and t is independently 1, 2 or 3; or

[0353] W is wherein:

[0354] R 16 is H or =O;

[0355] R 14 is a nitrogen - containing 5 - or 6 - membered heterocycle, NR 10 R 11 、C(O)NR 10 R 11 、NR 10 C(O)NR 10 R 11 or NR 10 C(S)NR 10 R 11， wherein each R 10 and R 11 are independently H or C1 - C3 alkyl; and

[0356] each of u and v is independently 1, 2 or 3; or

[0357] W is wherein:

[0358] each R6 is independently H or methyl;

[0359] each R7 is independently H;

[0360] each R8 is methyl;

[0361] each u is independently 1, 2 or 3; and

[0362] V is C2-C6 alkylene or C2-C6 alkenylene; or

[0363] W is wherein:

[0364] each u is independently 1, 2 or 3;

[0365] each Z is independently NR 12； and

[0366] T is a divalent nitrogen-containing 5- or 6-membered heterocycle; and

[0367] wherein:

[0368] W is hydroxy, substituted or unsubstituted hydroxyalkyl, or one of the following moieties:

[0369] where

[0370] each Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2) q -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 );

[0371] R 6 is independently H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, mercapto, mercaptoalkyl or N + (R 7 )3–alkylene-Q-;

[0372] each R 8 is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, mercapto, mercaptoalkyl, heterocyclic group, heteroaryl; or two Rs 8forms a ring together with the nitrogen atom, which ring is optionally substituted by one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino or aminoalkyl;

[0373] q is 0, 1, 2, 3, 4 or 5; and

[0374] p is 0, 1, 2, 3, 4 or 5.

[0375] Another aspect of the present invention relates to a lipid comprising at least two lipophilic tail groups and a head group of formula (G-HC-IIID):

[0376] its pharmaceutically acceptable salts or stereoisomers of any of the foregoing,

[0377] wherein:

[0378] R a each independently is C1-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl;

[0379] t2 is an integer from 0 to 5;

[0380] W is hydroxy, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl or substituted or unsubstituted heterocyclic or heteroaryl; and

[0381] represents a bond connecting the head group to the tail group.

[0382] In some embodiments, each R a is methyl and t2 is 0-3.

[0383] In some embodiments, W is hydroxy, substituted or unsubstituted hydroxyalkyl, or one of the following moieties:

[0384] where

[0385] each Q independently is absent, is -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2) q -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 );

[0386] R 6 independently is H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R7 ) 2. Amino, alkylamino, aminoalkyl, thiol, thioalkyl, or N + (R 7 )3-alkylene-Q-;

[0387] Each R 8 independently is H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, thioalkyl, heterocyclic group, heteroaryl; or two R 8 together with the nitrogen atom form a ring, which is optionally substituted by one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, or aminoalkyl;

[0388] q is 0, 1, 2, 3, 4, or 5; and

[0389] p is 0, 1, 2, 3, 4, or 5.

[0390] In some embodiments, W is

[0391] In one embodiment, the lipid has the following structure:

[0392]

[0393] Also disclosed herein is a nucleic acid-lipid particle, which comprises:

[0394] Nucleic acid;

[0395] One or more lipid compounds, said one or more lipid compounds comprising at least one head group (e.g., HA-I to HA-VII, HB-I, or HC-I to HC-IIIE; or any subgenus or species of these formulas disclosed herein) and at least one tail group of formula (T to TIII, or TI' to TIII', or any subgenus or species of these formulas disclosed herein);

[0396] Helper lipid;

[0397] Sterol; and

[0398] PEG-modified lipid.

[0399] Also disclosed herein is a pharmaceutical composition, which comprises a lipid particle and a pharmaceutically acceptable diluent, wherein the lipid particle comprises:

[0400] Nucleic acid;

[0401] One or more lipid compounds in an amount of 35 - 65 mol%, said one or more lipid compounds comprising at least one head group (e.g., HA-I to HA-VII, HB-I or HC-I to HC-IIIE; or any subgenus or species of these formulas disclosed herein) and at least one tail group of formula (T to TIII, or TI' to TIII', or any subgenus or species of these formulas disclosed herein);

[0402] 3 - 12 mol% of helper lipids;

[0403] 15 - 45 mol% of sterols; and

[0404] 0.5 - 10 mol% of PEGylated lipids.

[0405] Also disclosed herein are pharmaceutical compositions comprising one or more lipid compounds, said one or more lipid compounds comprising at least one head group (e.g., HA-I to HA-VII, HB-I or HC-I to HC-IIIE; or any subgenus or species of these formulas disclosed herein) and at least one tail group of formula (T to TIII, or TI' to TIII', or any subgenus or species of these formulas disclosed herein), and a therapeutic agent. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Such compositions can be used to form lipid nanoparticles for delivering a therapeutic agent.

[0406] In some embodiments, the present disclosure provides a method for delivering a therapeutic agent to a patient in need thereof, the method comprising administering to the patient a lipid nanoparticle composition comprising one or more lipid compounds, said one or more lipid compounds comprising at least one head group (e.g., HA-I to HA-VII, HB-I or HC-I to HC-IIIE; or any subgenus or species of these formulas disclosed herein) and at least one tail group of formula (T to TIII, or TI' to TIII', or any subgenus or species of these formulas disclosed herein), a pharmaceutically acceptable salt thereof, and / or a stereoisomer of any of the foregoing and the therapeutic agent. In some embodiments, the method further includes preparing a lipid nanoparticle composition comprising one or more lipid compounds, said one or more lipid compounds comprising at least one head group (e.g., HA-I to HA-VII, HB-I or HC-I to HC-IIIE; or any subgenus or species of these formulas disclosed herein) and at least one tail group of formula (T to TIII, or TI' to TIII', or any subgenus or species of these formulas disclosed herein), a pharmaceutically acceptable salt thereof, and / or a stereoisomer of any of the foregoing and a therapeutic agent.

[0407] In some embodiments, the spleen-to-liver ratio of the total therapeutic cargo administered to the subject is at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the spleen-to-liver ratio of the total therapeutic cargo administered to the subject is at least 1. In some embodiments, the spleen-to-liver ratio of the total therapeutic cargo administered to the subject is at least 5.

[0408] These and other aspects of the disclosure will be apparent after reference to the following detailed description. Detailed Description

[0409] Definitions

[0410] As used herein, unless otherwise specified, the following terms have the meanings ascribed to them.

[0411] All technical and scientific terms used herein, unless otherwise defined, have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0412] As used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise.

[0413] Unless the context requires otherwise, throughout this specification and the appended claims, the word "comprise" and variations thereof, such as "comprises" and "comprising," are to be construed in an open and inclusive sense, that is, "including but not limited to."

[0414] The phrase "inducing the expression of a desired protein" refers to the ability of a nucleic acid to increase the expression of a desired protein. To examine the degree of protein expression, a test sample (e.g., a cell sample in a culture expressing a desired protein) or a test mammalian (e.g., a mammalian such as a human or an animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, is contacted with a nucleic acid (e.g., a nucleic acid combined with a lipid of the present disclosure). The expression of the desired protein in the test sample or test animal is compared with the expression of the desired protein in a control sample (e.g., a cell sample in a culture expressing a desired protein) or a control mammalian (e.g., a mammalian such as a human or an animal) model (such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) that is not contacted with or not administered the nucleic acid. When the desired protein is present in the control sample or control mammalian, the expression of the desired protein in the control sample or control mammalian can be assigned a value of 1.0. In some embodiments, induction of the expression of the desired protein is achieved when the ratio of the expression level of the desired protein in the test sample or test mammalian to the expression level of the desired protein in the control sample or control mammalian is greater than 1 (e.g., about 1.1, 1.5, 2.0, 5.0, or 10.0). When the desired protein is not present in the control sample or control mammalian, induction of the expression of the desired protein is achieved when any measurable level of the desired protein is detected in the test sample or test mammalian. Those of ordinary skill in the art will understand the assays used to determine the protein expression level in a sample, such as dot blot, northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays or assays based on a reporter protein that can produce fluorescence or luminescence under appropriate conditions.

[0415] The phrase "inhibiting the expression of a target gene" refers to the ability of a nucleic acid to silence, reduce or inhibit the expression of a target gene. To examine the degree of gene silencing, a test sample (e.g., a cell sample in a culture expressing the target gene) or a test mammalian (e.g., a mammalian, such as a human or an animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, is contacted with a nucleic acid that silences, reduces or inhibits the expression of the target gene. The expression of the target gene in the test sample or test animal is compared with the expression of the target gene in a control sample (e.g., a cell sample in a culture expressing the target gene) or a control mammalian (e.g., a mammalian, such as a human or an animal) model (such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) that is not contacted with or not administered the nucleic acid. The expression of the target gene in the control sample or control mammalian can be assigned a value of 100%. In some embodiments, silencing, inhibition or reduction of the expression of the target gene is achieved when the level of expression of the target gene in the test sample or test mammalian is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% relative to the level of expression of the target gene in the control sample or control mammalian. In other words, relative to the level of expression of the target gene in a control sample or control mammalian that is not contacted with or not administered the nucleic acid, the nucleic acid is capable of silencing, reducing or inhibiting the expression of the target gene in the test sample or test mammalian by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. Suitable assays for determining the level of expression of a target gene include, but are not limited to, techniques known to those of skill in the art for examining protein or mRNA levels, such as dot blot, northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those of skill in the art.

[0416] An "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent (such as a therapeutic nucleic acid) is an amount sufficient to produce a desired effect, such as an increase or inhibition of target sequence expression compared to the normal expression level detected in the absence of the nucleic acid. An increase in target sequence expression is achieved when any measurable level is detected in the absence of an expression product that is not present in the absence of the nucleic acid. In the case where an expression product is present at a certain level prior to contact with the nucleic acid, an increase in expression is achieved when the fold change of the value obtained with the nucleic acid (such as mRNA) relative to the control is increased by about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or greater. Inhibition of target gene or target sequence expression is achieved when the value obtained with the nucleic acid (such as an antisense oligonucleotide) relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0%. Suitable assays for measuring the expression of a target gene or target sequence include, for example, examining protein or RNA levels using techniques known to those skilled in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of a suitable reporter protein, and phenotypic assays known to those skilled in the art.

[0417] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, and includes DNA, RNA, and hybrids thereof. DNA can be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA can be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA, or viral RNA (vRNA) and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages (which are synthetic, naturally occurring, and non-naturally occurring), and which have binding properties similar to a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNA). Unless specifically restricted, the term encompasses nucleic acids containing known analogs of natural nucleotides having binding properties similar to a reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell Probes 8:91-98 (1994)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are joined together by phosphate groups.

[0418] "Base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs; and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0419] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that contains a partial-length or full-length coding sequence necessary to produce a polypeptide or precursor polypeptide.

[0420] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0421] The term "lipid" refers to a group of organic compounds that includes, but is not limited to, esters of fatty acids and is generally characterized by being insoluble in water but soluble in many organic solvents. It is typically divided into at least three categories: (1) "simple lipids", which include fats, oils, and waxes; (2) "compound lipids", which include phospholipids and glycolipids; and (3) "derived lipids", such as steroids.

[0422] A "steroid" is a compound that contains the following carbon skeleton: A non-limiting example of a steroid is cholesterol.

[0423] As used herein, the term "compound" means to include all isomers and isotopes of the depicted structure, all pharmaceutically acceptable salts, solvates, or hydrates thereof, and all crystal forms (e.g., crystal polymorphs), crystal form mixtures, or anhydrides or hydrates thereof.

[0424] An "isotope" refers to atoms with the same atomic number but different mass numbers (caused by different numbers of neutrons in the atomic nucleus). For example, isotopes of hydrogen include tritium (3H) and deuterium (2H).

[0425] "Isomers." The compounds or pharmaceutically acceptable salts thereof described herein may include all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc. For example, a compound may contain one or more stereocenters and may thus give rise to geometric isomers (e.g., double bonds give rise to geometric E / Z isomers), enantiomers, diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers), and other stereoisomeric configurations, which can be defined as (R)- or (S)- according to absolute stereochemistry, such as anomeric sugars, or defined as (D)- or (L)-, such as amino acids, etc. The present disclosure is intended to include all such possible isomers and 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 preparing / separating individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of a racemate (or a racemate of a salt or derivative) using, for example, chiral high performance liquid chromatography (HPLC). Mixtures of enantiomers and stereoisomers of a compound and methods for resolving them into their component enantiomers or stereoisomers are well known. When the compounds described herein contain an olefinic double bond or other centers of geometric asymmetry, and unless otherwise specified, it is intended that the compounds include both E geometric isomers and Z geometric isomers. Similarly, all tautomeric forms are also intended to be included.

[0426] The terms "crystalline polymorph", "polymorph", or "crystalline form" mean crystal structures in which a compound (or its salt or solvate) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optical and electrical properties, stability, and solubility. The recrystallization solvent, crystallization rate, storage temperature, and other factors may cause one crystal form to predominate. The crystalline polymorphs of a compound can be prepared by crystallization under different conditions. The crystallization of the compounds disclosed herein can yield solvates.

[0427] As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of an ionizable lipid of the present disclosure and one or more molecules of a solvent. The solvent can be water, in which case the solvate can be a hydrate, including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, etc. Alternatively, the solvent can be an organic solvent.

[0428] As used herein, "ionizable lipid" refers to a lipid capable of carrying a charge. In some embodiments, the ionizable lipid includes one or more positively charged amine groups. In some embodiments, the ionizable lipid is ionizable such that it can exist in a positively charged or neutral form depending on the pH. The ionization of the ionizable lipid affects the surface charge of lipid nanoparticles comprising the ionizable lipid under different pH conditions. The surface charge of the lipid nanoparticles in turn can affect their plasma protein uptake, blood clearance, and tissue distribution (Semple, S.C. et al., Adv. Drug Deliv Rev 32:3-17 (1998)) and their ability to form non-bilayer structures that dissolve endosomes (Hafez, I.M. et al., Gene Ther 8:1188-1196 (2001)), which may affect the intracellular delivery of nucleic acids.

[0429] The term "polymer-conjugated lipid" refers to a molecule comprising both a lipid moiety and a polymer moiety. Non-limiting examples of polymer-conjugated lipids are polyethylene glycolated lipids. The term "polyethylene glycolated lipid" refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety. Polyethylene glycolated lipids are known in the art and include, for example, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), etc. As used herein, the terms "PEG-lipid" or "PEGylated lipid" are interchangeable and refer to a lipid comprising a polyethylene glycol component.

[0430] The term "neutral lipid" refers to any lipid that exists in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphatidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM); ceramides, and steroids such as sterols and their derivatives. Neutral lipids can be synthetic or of natural origin.

[0431] As used herein, "phospholipid" is a lipid that includes a phosphate ester moiety and one or more carbon chains (such as unsaturated fatty acid chains). A phospholipid can include one or more multiple bonds (e.g., double bonds or triple bonds) (one or more unsaturated bonds). Certain phospholipids can facilitate fusion with a membrane. For example, cationic phospholipids can interact with one or more negatively charged phospholipids of a membrane (e.g., a cell membrane or an intracellular membrane). Fusion of a phospholipid with a membrane can allow one or more elements of a lipid-containing composition to cross the membrane, thereby allowing, for example, delivery of one or more elements to a cell.

[0432] As used herein, the term "liposome" refers to a composition comprising an outer lipid bilayer membrane (e.g., a single lipid bilayer called a unilamellar liposome or multiple lipid bilayers called a multilamellar liposome) surrounding an internal aqueous space that may contain cargo. See, for example, Cullis et al., Biochim. Biophys Acta, 559:399-420 (1987), which is incorporated herein by reference in its entirety. The diameter of unilamellar liposomes is typically in the range of about 20 nanometers to about 400 nanometers (nm), about 50 nm to about 300 nm, about 100 nm to about 200 nm, or about 300 nm to about 400 nm. The diameter of multilamellar liposomes is typically in the range of about 1 μm to about 10 μm and can contain 2 to hundreds of concentric lipid bilayers alternating with aqueous layers.

[0433] The term "lipid nanoparticle" refers to a particle that is nanoscale in at least one dimension (e.g., 1 - 1,000 nm) and contains one or more compounds of formula (I). In some embodiments, lipid nanoparticles comprising one or more compounds of formula (I), pharmaceutically acceptable salts thereof, and / or stereoisomers of any of the foregoing are included in a composition that can be used to deliver a therapeutic agent, such as a nucleic acid (e.g., mRNA), to a target site of interest (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticle comprises one or more compounds of formula (I), pharmaceutically acceptable salts thereof, and / or stereoisomers of any of the foregoing and a nucleic acid. In some embodiments, the lipid nanoparticle comprises one or more compounds of formula (I), pharmaceutically acceptable salts thereof, and / or stereoisomers of any of the foregoing, a nucleic acid, and one or more other lipids selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, a therapeutic agent (such as a nucleic acid) can be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space encapsulated by some or all of the lipid portions in the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesired effects, such as an adverse immune response, induced by the mechanisms of the host organism or cell.

[0434] In some embodiments, the average diameter of the lipid nanoparticles is from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 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, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm or 150 nm, and is substantially non-toxic. In some embodiments, when present in the lipid nanoparticles, the nucleic acid is resistant to nuclease degradation in an aqueous solution.Lipid nanoparticles containing nucleic acids and methods for their preparation are disclosed, for example, in U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031, and PCT Publication Nos. WO 2013 / 016058, WO 2013 / 086373, 8,569,256, 5,965,542, and U.S. Patent Publication Nos. 2016 / 0199485, 2016 / 0009637, 2015 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, 2014 / 0308304, 2014 / 0200257, 2013 / 086373, 2013 / 0338210, 2013 / 0323269, 2013 / 0245107, 2013 / 0195920, 2013 / 0123338, 2013 / 0022649, 2013 / 0017223, 2012 / 0295832, 2012 / 0183581, 2012 / 0172411, 2012 / 0027803, 2012 / 0058188, 2011 / 0311583, 2011 / 0311582, 2011 / 0262527, 2011 / 0216622, 2011 / 0117125, 2011 / 0091525, 2011 / 0076335, 2011 / 0060032, 2010 / 0130588, 2007 / 0042031, 2006 / 0240093, 2006 / 0083780, 2006 / 0008910, 2005 / 0175682, 2005 / 017054, 2005 / 0118253, 2005 / 0064595, 2004 / 0142025, 2007 / 0042031, 1999 / 009076, and PCT Publication Nos. WO99 / 39741, WO 2017 / 117528, WO 2017 / 004143, WO 2017 / 075531, WO2015 / 199952, WO 2014 / 008334, WO 2013 / 086373, WO 2013 / 086322, WO2013 / 016058, WO 2013 / 086373, WO2011 / 141705, and WO 2001 / 07548. The entire disclosures of the documents are incorporated herein by reference in their entirety for all purposes.

[0435] As used herein, the term "size" refers to the hydrodynamic diameter of a population of lipid nanoparticles. Measurement of the size of a lipid nanoformulation can be used to indicate the size and population distribution (polydispersity index, PDI) of the composition.

[0436] As used herein, the "polydispersity index" is the ratio between the weight average molar mass and Mn, where Mn is the number average molar mass that describes the uniformity of the particle size distribution of the system. For example, a small value less than 0.3 indicates a narrow particle size distribution.

[0437] The polydispersity index can be used to indicate the uniformity of a lipid composition (e.g., liposome or LNP), such as the particle size distribution of a liposome or LNP. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The polydispersity index of a lipid composition can be from 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 composition can be from about 0.10 to about 0.20.

[0438] As used herein, the term "apparent pKa" refers to the pH at which 50% of the lipid nanoformulation (e.g., LNP) is protonated. This can be used as an indicator of the pH range at which the lipid nanoformulation (e.g., LNP) will be protonated and thus initiate the endosomal escape process in nucleotide delivery.

[0439] As used herein, the term "zeta potential" refers to the electrokinetic potential of the lipids, for example, in a lipid nanoformulation (e.g., LNP composition). The zeta potential can describe the surface charge of the LNP composition. The zeta potential can be used to predict organotropism and potential interactions with serum proteins.

[0440] The ζ potential of a lipid composition (e.g., liposome or LNP) can be used to indicate the electrokinetic potential of the composition. In some embodiments, the ζ potential can describe the surface charge of a liposome or LNP. Lipid compositions (e.g., liposomes or LNPs) with a relatively low charge (positive or negative) are generally desired because more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the ζ potential of a liposome or LNP can be from 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.

[0441] As used herein, being “encapsulated” by a lipid means that a therapeutic agent (such as a nucleic acid (e.g., mRNA)) is completely or partially encapsulated by a lipid nanoparticle. In some embodiments, the nucleic acid (e.g., mRNA) is completely encapsulated within the lipid nanoparticle.

[0442] As used herein, “encapsulation efficiency” or “entrapment efficiency” refers to the percentage of encapsulated cargo (e.g., therapeutic and / or prophylactic agent) that is successfully incorporated into (e.g., encapsulated or otherwise associated with) a lipid composition (e.g., LNP or liposome) relative to the initial total amount of the therapeutic and / or prophylactic agent provided. For example, if 97 mg of a therapeutic and / or prophylactic agent out of a total of 100 mg initially provided is encapsulated in the lipid composition, the encapsulation efficiency can be 97%. Encapsulation efficiency can be used to indicate the efficiency of loading an encapsulated cargo (e.g., nucleic acid molecule) into a lipid composition using a particular formulation method and formulation recipe.

[0443] The encapsulation efficiency of cargoes such as proteins and / or nucleic acids describes the amount of protein and / or nucleic acid that is encapsulated or otherwise associated with a lipid composition (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%, approaching 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 disrupting the liposomes or LNPs with one or more organic solvents or detergents. 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 the liposomes or LNPs described herein, the encapsulation efficiency of the protein and / or nucleic acid can be at least 50%, such as 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 can be at least 80%. In some embodiments, the encapsulation efficiency can be at least 90%. In some embodiments, the encapsulation efficiency can be at least 95%.

[0444] "Serum stable" with respect to a nucleic acid-lipid nanoparticle means that the nucleic acid does not significantly degrade after exposure to serum or a nuclease assay that would significantly degrade free DNA or RNA. Suitable assays include, for example, a standard serum assay, a DNase assay, or an RNase assay.

[0445] Some administration techniques can result in systemic delivery of certain agents but not others. "Systemic delivery" means delivering a useful (e.g., therapeutic) amount of an agent to most parts of the body. Systemic delivery of lipid nanoparticles can be effected by any method known in the art (including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery). In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0446] As used herein, "local delivery" refers to the direct delivery of an agent to a target site within a living organism. For example, an agent can be locally delivered by direct injection into a diseased site (such as a tumor), other target sites (such as an inflamed site), or target organs (such as the liver, heart, pancreas, kidney, etc.). Local delivery can also include local administration or local injection techniques, such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not exclude systemic pharmacological effects.

[0447] As used herein, "administration method" can include systemic delivery and local delivery. "Systemic delivery" means delivering a useful (e.g., therapeutic) amount of an agent to most parts of the body. Systemic delivery of liposomes or LNPs can be carried out by any method known in the art (including, for example, intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, and intraperitoneal delivery). In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery. As used herein, "local delivery" refers to direct delivery of an agent to a target site within a living organism. For example, an agent can be locally delivered by direct injection into the diseased site (such as a tumor), other target sites (such as an inflamed site), or target organs (such as the liver, heart, pancreas, kidney, etc.). Local delivery can also include local application or local injection techniques, such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not exclude systemic pharmacological effects.

[0448] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues that are typically linked by peptide bonds, which can be naturally occurring (e.g., isolated or purified) or synthetically produced.

[0449] "Nucleic acid" is intended to define oligonucleotide or polynucleotide sequences. 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 by phosphodiester bonds in nucleic acids, but the term "nucleic acid" also encompasses nucleic acid analogs having other types of bonds or backbones (e.g., phosphoramidate, phosphorothioate, dithiophosphate, O-methylphosphoramidite, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptide nucleic acid (PNA) bonds or backbones, etc.). 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, as well as any combination of bases, including, for example, adenine, thymine, cytosine, guanine, uracil, and modified or non-canonical bases (including, for example, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine).

[0450] As used herein, "RNA" refers to ribonucleic acid, which may be naturally occurring or non-naturally occurring. For example, RNA may include modified and / or non-naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA may include a cap structure, chain-terminating nucleosides, stem-loops, polyA sequences, and / or polyadenylation signals. 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, such as in vivo translation of mRNA within mammalian cells, can 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.

[0451] "Alkyl" refers to a straight-chain or branched-chain hydrocarbon radical consisting only of carbon and hydrogen atoms, having, for example, from one to twenty-four carbon atoms (C1-C 24 alkyl), from four to twenty carbon atoms (C4-C 20 alkyl), from six to sixteen carbon atoms (C6-C 16 alkyl), from six to nine carbon atoms (C6-C9 alkyl), from one to fifteen carbon atoms (C1-C 15 alkyl), from one to twelve carbon atoms (C1-C 12 alkyl), from one to eight carbon atoms (C1-C8 alkyl), or from one to six carbon atoms (C1-C6 alkyl), and the alkyl is attached to the remainder of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, vinyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Unless specifically stated otherwise in this specification, alkyl is optionally substituted.

[0452] "Alkylene" or "alkylene chain" refers to a straight-chain or branched-chain divalent hydrocarbon chain that attaches the remainder of the molecule to the radical, consisting only of carbon and hydrogen, having, for example, from one to twenty-four carbon atoms (C1-C 24 alkylene), from one to fifteen carbon atoms (C1-C 15 alkylene), from one to twelve carbon atoms (C1-C 12(alkylene), an alkylene having from one to eight carbon atoms (C1-C8 alkylene), an alkylene having from one to six carbon atoms (C1-C6 alkylene), an alkylene having from two to four carbon atoms (C2-C4 alkylene), an alkylene having from one to two carbon atoms (C1-C2 alkylene), for example, methylene, ethylene, propylene, n-butylene, vinyl, propenyl, n-butenyl, propynyl, n-butynyl, etc. The alkylene chain is connected to the rest of the molecule by a single bond or a double bond and is connected to the radical by a single bond or a double bond. The connection points of the alkylene chain to the rest of the molecule and to the radical can be through one carbon or any two carbons within the chain.

[0453] The term "alkenyl" refers to a straight-chain or branched-chain hydrocarbon chain having one or more double bonds. Unless otherwise indicated, "alkenyl" generally refers to a C2-C8 alkenyl (e.g., C2-C6 alkenyl, C2-C4 alkenyl, or C2-C3 alkenyl). Examples of typical alkenyls include, but are not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl, and 3-octenyl. The term "alkynyl" refers to a straight-chain or branched-chain hydrocarbon chain containing 2-8 carbon atoms and characterized by having one or more triple bonds. Unless otherwise indicated, "alkynyl" generally refers to a C2-C8 alkynyl (e.g., C2-C6 alkynyl, C2-C4 alkynyl, or C2-C3 alkynyl). Some examples of typical alkynyls are ethynyl, 2-propynyl, 3-methylbutynyl, and propargyl. sp 2 and sp 3 The carbon can optionally serve as the connection point for the alkenyl and alkynyl, respectively.

[0454] As used herein, the term "cycloalkyl" or "cyclic group" includes saturated and partially unsaturated but not aromatic cyclic hydrocarbon groups having from 3 to 12 carbons, such as from 3 to 8 carbons, and such as from 3 to 6 carbons, wherein the cycloalkyl can additionally be optionally substituted. Cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0455] The term "heteroaryl" or "heteroar-" refers to an aromatic 5- to 8-membered monocyclic system, an 8- to 12-membered bicyclic system, or an 11- to 14-membered tricyclic system, which, if monocyclic, has 1 to 3 heteroatoms, if bicyclic, has 1 to 6 heteroatoms, or if tricyclic, has 1 to 9 heteroatoms, the heteroatoms being selected from O, N, or S (e.g., carbon atoms and having 1 to 3, 1 to 6, or 1 to 9 N, O, or S heteroatoms if monocyclic, bicyclic, or tricyclic, respectively), where 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. The term also includes 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 include pyrrolyl, pyridyl, pyridazinyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, furyl, imidazolyl, benzimidazolyl, pyrimidinyl, pyrazinyl, indolizinyl, phenylthio or thienyl, quinolinyl, indolyl, thiazolyl, isothiazolyl, thiadiazolyl, purinyl, naphthyridinyl, pteridinyl, isoindolyl, benzothienyl, benzofuryl, dibenzofuryl, indazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one, etc.

[0456] The term "heterocyclyl", "heterocycle", "heterocyclic radical", or "heterocyclic ring" refers to a 5- to 8-membered monocyclic system, an 8- to 12-membered bicyclic system, or an 11- to 14-membered tricyclic system, which, if monocyclic, has 1 to 3 heteroatoms, if bicyclic, has 1 to 6 heteroatoms, or if tricyclic, has 1 to 9 heteroatoms, the heteroatoms being selected from O, N, or S (e.g., carbon atoms and having 1 to 3, 1 to 6, or 1 to 9 N, O, or S heteroatoms if monocyclic, bicyclic, or tricyclic, respectively), where 0, 1, 2, or 3 atoms of each ring may be substituted by a substituent. As used herein, the term generally may include both non-aromatic rings and aromatic rings (e.g., generally covered by heteroaryl). The term also includes groups in which a heterocycle is fused to one or more aryl, cycloalkyl, or heterocyclyl rings. When referring to the ring atoms of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl).

[0457] Examples of the heterocyclic group include triazolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, dioxolanyl, diazacyclic group, oxazacyclic group, thiazacyclic group, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrobenzothienyl, pyrrolidinyl, piperidinyl, pyrroline, tetrahydroquinoline, tetrahydroisoquinoline, decahydroquinoline, oxazolidinyl, quinuclidinyl, etc.

[0458] Examples of the heterocyclic group also include those typical heteroaryl groups, such as pyrrolyl, pyridyl, pyridazinyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, furyl, imidazolyl, benzimidazolyl, pyrimidinyl, pyrazinyl, indolizinyl, phenylthio or thienyl, quinolinyl, indolyl, thiazolyl, isothiazolyl, thiadiazolyl, purinyl, naphthyridinyl, pteridinyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinoline, tetrahydroisoquinoline and pyrido[2,3-b]-1,4-oxazin-3(4H)-one, etc.

[0459] The divalent radicals of alkyl, alkenyl, aryl, heteroaryl, cycloalkyl and heterocyclic group are formed by removing one hydrogen atom from alkyl, alkenyl, aryl, heteroaryl, cycloalkyl and heterocyclic group respectively (or by removing two hydrogen atoms from alkane, alkene, arene, heteroarene, cycloalkane or heterocycle respectively).

[0460] The term "alkoxy" means -O-alkyl.

[0461] The term "aminoalkyl" means an alkyl group substituted by an amino group. The term "alkylamino" means an amino group substituted by an alkyl group.

[0462] The term "aminocarbonyl" means -C(O)-amino.

[0463] As used herein, the term "substituted" means any one of the above groups (e.g., alkyl, hydroxyalkyl, alkylene, cycloalkyl, cycloalkylene, amino, aminocarbonyl, heterocyclic group or heteroaryl), wherein one or more hydrogen atoms are replaced by a non-hydrogen atom through a bond, such as but not limited to: halogen atoms, such as F, Cl, Br or I; oxo group (=O); hydroxy group (-OH); alkoxy, alkoxyalkyl, aralkoxy, alkyl such as C1-C 12alkyl; cycloalkyl; alkenyl, alkynyl, aryl, aralkyl heterocyclic group, heterocyclic group, heteroaryl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, aryloxy, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, aralkyloxycarbonyl, sulfonyl, alkylaminolactam, alkylaminoheteroaryl, alkylaminoheterocyclic group and aminosulfonamide. Exemplary substituents also include: -(C=O)OR; -O(C=O)R; -C(=O)R; -OR; -S(O) x R; -S-SR; -C(=O)SR; -SC(=O)R; -NRR'; -R'C(=O)R; -C(=O)RR'; -RC(=O)R'R”; -OC(=O)RR'; -RC(=O)OR'; -R'S(O) X R”R; -R'S(O) X R; and -S(O) x RR', where: R, R' and R” are each independently H, C1-C 15 alkyl or cycloalkyl, heterocyclic group or heteroaryl, which may be optionally substituted, and x is 0, 1 or 2. In some embodiments, the substituent is C1-C 12 alkyl. In some embodiments, the substituent is cycloalkyl. In some embodiments, the substituent is a halogen group, such as fluorine. 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-OH). In some embodiments, the substituent is an alkoxy group (-OR). In some embodiments, the substituent is a carboxyl group. In some embodiments, the substituent is an amine group (-NRR'). 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))2-3O- or -S(C(R*2))2-3S-, where R* is independently selected from hydrogen, substituted or unsubstituted C1-6 alkyl or unsubstituted 5-6 membered saturated or partially unsaturated ring, or an aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur each time it appears.

[0464] "halo" or "halogen" refers to any radical of fluorine, chlorine, bromine or iodine.

[0465] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described circumstance or event may or may not occur, and the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" means that the alkyl may or may not be substituted, and the description includes both substituted alkyl and unsubstituted alkyl.

[0466] The present disclosure also contemplates all pharmaceutically acceptable compounds of all formulas identified herein that are isotopically labeled by replacing one or more atoms with atoms having different atomic masses or mass numbers. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 C1, 123 I and 125 I. These isotopically labeled compounds can be used to help determine or measure the effectiveness of a compound by characterization, such as site or mode of action or binding affinity for a pharmacologically important site of action. Certain isotopically labeled compounds can be used in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium (i.e., 3 H) and carbon-14 (i.e., 14 C) are useful for this purpose in view of their ease of incorporation and ready means of detection.

[0467] Replacement with heavier isotopes such as deuterium (i.e., 2 H) can provide certain therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements, and can thus be useful in some circumstances.

[0468] Replacement with, for example, 11 C, 18 F, 15 O and 13Substitution with a positron-emitting isotope such as N can be useful in positron emission tomography (PET) studies for examining substrate receptor occupancy. Isotope-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the preparation and examples herein, using appropriate isotope-labeled reagents in place of the previously employed unlabeled reagents.

[0469] The present disclosure also intends to cover the in vivo metabolites of the disclosed compounds. Such products may be produced mainly due to enzymatic processes, such as oxidation, reduction, hydrolysis, amidation, esterification, etc. of, for example, the administered compound. Accordingly, embodiments of the present disclosure include compounds produced by a method comprising administering to a mammal an ionizable lipid of the present disclosure for a period of time sufficient to produce its metabolites. Such products are generally identified by administering a radiolabeled compound of the present disclosure to an animal (such as a rat, mouse, guinea pig, monkey, or human) in a detectable dose, allowing sufficient time for metabolism to occur, and separating its conversion products from urine, blood, or other biological samples.

[0470] "Pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent, or emulsifying agent that has been approved by the United States Food and Drug Administration for use in humans or livestock and is acceptable.

[0471] "Pharmaceutically acceptable salt" includes both acid addition salts and base addition salts.

[0472] "Pharmaceutically acceptable acid addition salts" refer to those salts that retain the biological effectiveness and properties of the free base, which salts are not biologically or otherwise undesirable, and which salts are formed from inorganic acids and organic acids, such as but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. for inorganic acids, and such as but not limited to acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

[0473] "Pharmaceutically acceptable base addition salts" refer to those salts that retain the biological effectiveness and properties of the free acid, which salts are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include but are not limited to sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc. Non-limiting examples of inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. Salts derived from organic bases include but are not limited to primary amine salts, secondary amine salts, and tertiary amine salts; substituted amines (including naturally occurring substituted amines); cyclic amines; and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, meglumine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, phenethylbenzylamine, benzathine penicillin, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Non-limiting examples of organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0474] Crystallization of the ionizable lipids disclosed herein can result in solvates. As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of an ionizable lipid of the present disclosure and one or more molecules of a solvent. The solvent can be water, in which case the solvate can be a hydrate. Alternatively, the solvent can be an organic solvent. Thus, the compounds of the present disclosure can exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., as well as corresponding solvated forms. The solvates of the compounds of the present disclosure can be true solvates, while in other cases, the compounds of the present disclosure can merely retain exogenous water or be a mixture of water plus some exogenous solvent.

[0475] "Pharmaceutical composition" refers to a composition that can comprise an ionizable lipid of the present disclosure and a medium generally accepted in the art for delivering a bioactive compound to a mammal (e.g., a human). Such a medium thus includes pharmaceutically acceptable carriers, diluents, or excipients.

[0476] "Effective amount" or "therapeutically effective amount" refers to an amount of an ionizable lipid of the present disclosure that is sufficient to effect treatment of a mammal (such as a human) when administered to the mammal (such as a human). The amount of the lipid nanoparticles of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art based on their own knowledge and the present disclosure.

[0477] As used herein, "treating" or "treatment" encompasses treatment of a disease or condition of concern in a mammal (such as a human) suffering from the disease or condition of concern and includes:

[0478] (i) preventing the occurrence of the disease or condition in a mammal, particularly when such a mammal is predisposed to the condition but has not been diagnosed as having the condition;

[0479] (ii) inhibiting the disease or condition, i.e., preventing its progression;

[0480] (iii) alleviating the disease or condition, i.e., causing the disease or condition to regress; or

[0481] (iv) alleviating the symptoms caused by the disease or condition, i.e., alleviating pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably or may be different, since a particular malady or condition may not have a known pathogen (and thus the etiology has not been determined), and thus the particular disease or condition has not been identified as a disease but only as an undesirable condition or syndrome, in which a clinician has identified a more or less specific group of symptoms.

[0482] The compounds of the present disclosure or pharmaceutically acceptable salts thereof may contain one or more stereocenters and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined as (R)- or (S)- according to absolute stereochemistry, or as (D)- or (L)- for amino acids. The present disclosure is intended to embrace all such possible isomers as well as their racemic and optically pure forms. The 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 / separation 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 performance liquid chromatography (HPLC). When the compounds described herein contain an olefinic double bond or other centers of geometric asymmetry, and unless otherwise specified, it is intended that the compounds include both the E geometric isomers and the Z geometric isomers. Similarly, all tautomeric forms are also intended to be included.

[0483] "Stereoisomers" refer to compounds of the same atoms bonded by the same bonds but having different three-dimensional structures, and the stereoisomers are non-interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof, and includes "enantiomers", which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other.

[0484] In the following description, certain specific details are set forth to provide a thorough understanding of the various embodiments of the present disclosure. However, one of ordinary skill in the art will understand that the present disclosure may be practiced without these details.

[0485] Exemplary lipid compounds

[0486] In some embodiments, ionizable lipids of formula (LA-I) are disclosed:

[0487]

[0488] their pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0489] Wherein

[0490] Each of R1 and R2 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, OH, a halogen, SH or NR 10 R 11 ; or R1 and R2 together form a ring;

[0491] R 10 and R 11 each is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group; or R 10 and R 11 together form a heterocycle;

[0492] m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0493] n is 0, 1, 2, 3 or 4;

[0494] Z is absent, O, S or NR 12 , wherein R 12 is H or a C1-C7 branched or unbranched alkyl group; provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 , SH;

[0495] Each A is independently a C1-C 16 branched or unbranched alkyl group, optionally substituted with a heteroatom or substituted with OH, SH or a halogen;

[0496] Each B is independently a C1-C 16 branched or unbranched alkyl group, optionally substituted with a heteroatom or substituted with OH, SH or a halogen;

[0497] Each X is independently X', provided that at least one X in the formula is and

[0498] X' is a biodegradable moiety.

[0499] In some embodiments, each X is

[0500] In some embodiments, X' is -OCO-, -COO-, -NR 7 CO-, -CONR 7 -, -C(O-R 13 )-O-(acetal), -COO(CH2) s -, -CONH(CH2) s -, -C(O-R13 )-O-(CH2) s -; wherein R 7 is H or C1-C3 alkyl; and R 13 is C3-C 10 alkyl.

[0501] In some embodiments, X in at least one of the formulas is wherein R 7 is H or methyl. In one embodiment, each X is In one embodiment, each X is wherein R 7 is H or methyl.

[0502] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LA-II):

[0503]

[0504] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0505] wherein

[0506] R1 is independently H, C1-C3 alkyl, OH, halogen, SH or NR 10 R 11 ; R1 and R2 may combine together to form a ring; R 10 and R 11 are independently H, C1-C3 alkyl, and R 10 and R 11 may combine together to form a heterocycle;

[0507] R2 is independently H, C1-C3 alkyl, OH, halogen, SH or NR 10 R 11 ; R1 and R2 may combine together to form a ring; R 10 and R 11 are independently H, C1-C3 alkyl, and R 10 and R 11 may combine together to form a heterocycle;

[0508] m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0509] n is 0, 1, 2, 3 or 4;

[0510] r is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0511] R3 is independently H or C3-C 10Alkyl;

[0512] Each R4 is independently H or C3-C 10 alkyl; provided that at least one of R3 and R4 is not H;

[0513] Z is absent, is O, S or NR 12 ; wherein R 12 is C1-C7 alkyl;

[0514] Each X is independently X', provided that at least one X in one of the formulas is and

[0515] X' is a biodegradable moiety.

[0516] In some embodiments, each X is

[0517] In some embodiments, X' is -OCO-, -COO-, -NR 7 CO-, -CONR 7 -, -C(O-R 13 )-O-(acetal), -COO(CH2) s -, -CONH(CH2) s -, -C(O-R 13 )-O-(CH2) s -; wherein R 7 is H or C1-C3 alkyl; and R 13 is C3-C10 alkyl.

[0518] In some embodiments, at least one X in one of the formulas is wherein R 7 is H or methyl. In one embodiment, each X is In one embodiment, each X is wherein R 7 is H or methyl.

[0519] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LA-III):

[0520]

[0521] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0522] wherein

[0523] Each of R1 and R2 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, OH, halogen, SH, or NR 10 R 11 ; or R1 and R2 together form a ring;

[0524] R 10 and R 11 each is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group; or R 10 and R 11 together form a heterocycle;

[0525] Each R2 is independently H, a C1-C3 alkyl group, OH, halogen, SH, or NR 10 R 11 ; R1 and R2 can together form a ring; R 10 and R 11 are each independently H, a C1-C3 alkyl group, and R 10 and R 11 can together form a heterocycle;

[0526] n is 0, 1, 2, 3, or 4;

[0527] Each r is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0528] Each R3 is independently H or a C3-C 10 alkyl group;

[0529] Each R4 is independently H or a C3-C 10 alkyl group; provided that at least one of R3 and R4 is not H;

[0530] Z is absent, O, S, or NR 12 ; where R 12 is a C1-C7 alkyl group;

[0531] Each X is independently X', provided that at least one X in the formula is and

[0532] X' is a biodegradable moiety.

[0533] In some embodiments, each X is

[0534] In some embodiments, X' is -OCO-, -COO-, -NR 7 CO-, -CONR 7 -, -C(O-R 13 )-O-(acetal), -COO(CH2)s -, -CONH(CH2) s -, -C(O-R 13 )-O-(CH2) s -; wherein R 7 is H or a C1-C3 alkyl; and R 13 is a C3-C10 alkyl.

[0535] In some embodiments, X in at least one of the formulas is wherein R 7 is H or methyl. In one embodiment, each X is In one embodiment, each X is wherein R 7 is H or methyl.

[0536] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LA-IV):

[0537]

[0538] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0539] wherein

[0540] r is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0541] q is independently a C1-C 10 alkyl; and

[0542] Z is absent, is O, S, or NR 12 wherein R 12 is a C1-C7 alkyl.

[0543] In some embodiments, Z is absent.

[0544] In some embodiments, Z is S.

[0545] In some embodiments, Z is O.

[0546] In some embodiments, Z is NH.

[0547] In some embodiments, r is 3.

[0548] In some embodiments, r is 4.

[0549] In some embodiments, q is 3.

[0550] In some embodiments, q is 4.

[0551] In some embodiments, Z is absent, r is 4 and q is 4.

[0552] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LA-V):

[0553]

[0554] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0555] wherein

[0556] R1 is H, C1-C3 alkyl, OH, halogen, SH or NR 10 R 11 ;

[0557] R2 is OH, halogen, SH or NR 10 R 11 ; or R1 and R2 can combine together to form a ring;

[0558] R 10 and R 11 are each independently H or C1-C3 alkyl; or R 10 and R 11 can combine together to form a heterocycle;

[0559] R 20 and R 30 are each independently H, C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl; or R 20 and R 30 can combine together to form a ring;

[0560] Each of v and y is independently 1, 2, 3 or 4;

[0561] Each of A and B is independently C1-C 16 branched or unbranched alkyl or C2-C 16 branched or unbranched alkenyl; the alkyl or alkenyl is optionally interrupted by a heteroatom or substituted by OH, SH or halogen;

[0562] Each X is independently X', provided that at least one X in the formula is and

[0563] X' is a biodegradable moiety.

[0564] In some embodiments, each X is

[0565] In some embodiments, X' is -OCO-, -COO-, -NR 7 CO-, -CONR7 -, -C(O-R 13 )-O-(acetal), -COO(CH2) s -, -CONH(CH2) s -, -C(O-R 13 )-O-(CH2) s -; wherein R 7 is H or C1-C3 alkyl; and R 13 is C3-C10 alkyl.

[0566] In some embodiments, X in at least one formula is wherein R 7 is H or methyl. In one embodiment, each X is In one embodiment, each X is wherein R 7 is H or methyl.

[0567] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LA-VI):

[0568]

[0569] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0570] wherein

[0571] R 20 and R 30 are each independently H, C1-C5 alkyl; R 20 and R 30 may combine together to form a ring;

[0572] v is 1, 2, 3 or 4;

[0573] y is 1, 2, 3 or 4;

[0574] R3 are each independently H or C3-C 10 alkyl;

[0575] R4 are each independently H or C3-C 10 alkyl; provided that at least one of R3 and R4 is not H;

[0576] r are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8; and

[0577] each X is independently X', provided that X in at least one formula is and

[0578] X' is -OCO-, -COO-, -NR 7 CO-, -CONR 7 -, -C(O-R 13 )-O-(acetal), -COO(CH2) s -, -CONH(CH2) s - or -C(O-R 13 )-O-(CH2) s -; wherein R 7 is H or C1-C3 alkyl; and R 13 is C3-C10 alkyl.

[0579] In some embodiments, each X is

[0580] In some embodiments, at least one X in the formula is wherein R 7 is H or methyl. In one embodiment, each X is In one embodiment, each X is wherein R 7 is H or methyl.

[0581] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LA-VII):

[0582]

[0583] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0584] wherein

[0585] R 20 and R 30 are each independently H, C1-C5 alkyl; R 20 and R 30 may combine together to form a ring;

[0586] v is 1, 2, 3 or 4;

[0587] y is 1, 2, 3 or 4;

[0588] r are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8; and

[0589] q are each independently C1-C 10 alkyl.

[0590] In some embodiments, r is 3.

[0591] In some embodiments, r is 4.

[0592] In some embodiments, q is 3.

[0593] In some embodiments, q is 4.

[0594] In some embodiments, r is 4 and q is 4.

[0595] In some embodiments, B or is selected from:

[0596]

[0597] wherein t is 0, 1, 2, 3, 4 or 5.

[0598] In some embodiments, an ionizable lipid of formula (LB-I) is disclosed:

[0599]

[0600] its pharmaceutically acceptable salt or a stereoisomer of any of the foregoing,

[0601] wherein

[0602] each A is independently a C1-C 16 branched or unbranched alkylene or a C1-C 16 branched or unbranched alkenylene, optionally substituted with a heteroatom or substituted with OH, SH or a halogen;

[0603] each B is independently a C1-C 20 branched or unbranched alkyl or a C1-C 20 branched or unbranched alkenyl, optionally substituted with a heteroatom or substituted with OH, SH or a halogen;

[0604] each X is independently X', provided that at least one X in the formula is and

[0605] X' is a biodegradable moiety, and

[0606] W is

[0607]

[0608]

[0609] wherein

[0610] R5 is (CH2) s OH, OH, SH, NR 10 R 11 ;

[0611] Each R6 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, or a cycloalkyl group;

[0612] Each R7 and R8 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, (CH2) v OH, (CH2) v SH, (CH2) s N(CH3)2 or NR 10 R 11 , where each R 10 and R 11 is independently H or a C1-C3 alkyl group, or R 10 and R 11 together form a heterocycle;

[0613] Each R 20 is independently H or a C1-C3 branched or unbranched alkyl group;

[0614] R 14 is a heterocycle, NR 10 R 11 , C(O)NR 10 R 11 , NR 10 C(O)NR 10 R 11 or NR 10 C(S)NR 10 R 11， where each R 10 and R 11 is independently H, a C1-C3 alkyl group, a C3-C7 cycloalkyl group, a C3-C7 cycloalkenyl group, the alkyl, cycloalkyl, cycloalkenyl group being optionally substituted with one or more NH and / or oxo groups, or R 10 and R 11 together form a heterocycle;

[0615] R 16 is H, =O, =S or CN;

[0616] Each of s, u and t is independently 1, 2, 3, 4 or 5;

[0617] Each v is independently 0, 1, 2, 3, 4 or 5;

[0618] Each Z independently does not exist, is O, S or NR 12 , where R 12 is H, a C1-C7 branched or unbranched alkyl group or a C2-C7 branched or unbranched alkenyl group;

[0619] Each Y is a divalent heterocycle;

[0620] Q is O, S, CH2 or NR 13 , where each R 13 is H, C1-C5 alkyl; and

[0621] V is branched or unbranched C2-C 10 alkylene, C2-C 10 alkenylene, C2-C 10 alkynylene or C2-C 10 heteroalkylene, and the alkylene, alkenylene, alkynylene or heteroalkylene is optionally substituted by one or more OH, SH and / or halogen groups.

[0622] In some embodiments, each X is

[0623] In some embodiments, X' is -OCO-, -COO-, -NR 7 CO-, -CONR 7 -, -C(O-R 13 )-O-, -COO(CH2) r -, -CONH(CH2) r -, or -C(O-R 13 )-O-(CH2) r -, -O(CO)O-, where R 7 is H or C1-C3 alkyl; and R 13 is branched or unbranched C3-C 10 alkyl, and r is 1, 2, 3, 4 or 5.

[0624] In some embodiments, at least one X in the formula is where R 7 is H or methyl. In one embodiment, each X is In one embodiment, each X is where R 7 is H or methyl.

[0625] In some embodiments, the heterocycle is piperazine, piperazinedione, piperazine-2,5-dione, piperidine, pyrrolidine, piperidinol, dioxopiperazine, bipiperazine, aromatic or heteroaromatic.

[0626] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LB-II):

[0627]

[0628] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0629] where

[0630] Each R1 and each R2 are independently H, a C1-C3 branched or unbranched alkyl group, OH, halogen, SH, or NR 10 R 11 , or

[0631] each R1 and each R2 together with the carbon atom to which they are attached independently form a ring;

[0632] each R 10 and R 11 are independently H, a C1-C3 branched or unbranched alkyl group, or R 10 and R 11 together form a heterocyclic ring;

[0633] m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0634] each X is independently X', provided that at least one X in the formula is and

[0635] X' is independently a biodegradable moiety;

[0636] each R3 and each R4 are independently H, a C3-C 10 branched or unbranched alkyl group, or a C3-C 10 branched or unbranched alkenyl group; provided that at least one of R3 and R4 is not H;

[0637] W is

[0638]

[0639] wherein

[0640] R5 is OH, SH, NR 10 R 11 ;

[0641] each R6 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, or a cycloalkyl group;

[0642] each R7 and each R8 are independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, halogen, OH, SH, NR 10 R 11 , wherein each R 10 and R 11 are independently H, a C1-C3 alkyl group, or each R 10 and each R 11 together with the carbon atom to which they are attached form a heterocyclic ring;

[0643] Each s is independently 1, 2, 3, 4 or 5;

[0644] Each u is independently 1, 2, 3, 4 or 5;

[0645] t is 1, 2, 3, 4 or 5;

[0646] Each Z is independently absent, O, S or NR 12 , where R 12 is H, a C1-C7 branched or unbranched alkyl or a C2-C7 branched or unbranched alkenyl, provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 or SH; and

[0647] Q is O, S, CH2 or NR 13 , where each R 13 is H, a C1-C5 alkyl.

[0648] In some embodiments, each X is

[0649] In some embodiments, X' is –OC(O)-, -C(O)O-, -NR 7 C(O)-, -C(O)NR 7 -, -C(O-R 13 )-O-, -C(O)O(CH2) r -, -C(O)NH(CH2) r -, -CON(R 13 )-, or -C(O-R 13 )-O-(CH2) r -, -OC(O)O-, where R 7 is H or a C1-C3 alkyl; and R 13 is a branched or unbranched C1-C 10 alkyl, and r is 1, 2, 3, 4 or 5.

[0650] In some embodiments, at least one X in the formula is where R 7 is H or methyl. In one embodiment, each X is In one embodiment, each X is where R 7 is H or methyl.

[0651] In some embodiments, W is

[0652] where

[0653] V is a C2-C6 alkylene, C2-C 10 alkenylene, C2-C 10 alkynylene or C2-C 10 heteroalkylene;

[0654] Each R6 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, or a cycloalkyl; and

[0655] Each u is independently 2, 3, 4 or 5.

[0656] In some embodiments, in formula (HB-I), W is wherein:

[0657] Each R6 is independently H or methyl;

[0658] Each R7 is independently H;

[0659] Each R8 is methyl;

[0660] Each u is independently 1, 2 or 3; and

[0661] V is a C2-C6 alkenylene.

[0662] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LB-III):

[0663]

[0664] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0665] wherein

[0666] Each R1 and each R2 are independently H, a C1-C3 branched or unbranched alkyl, OH, halogen, SH or NR 10 R 11 , or

[0667] Each R1 and each R2 independently together with the carbon atom to which they are attached form a ring;

[0668] Each R 10 and R 11 are independently H, a C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle;

[0669] Each R3 and each R4 are independently H, a C3-C 10 branched or unbranched alkyl or a C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H;

[0670] Each X is independently X', provided that at least one X in the formula is and

[0671] X' is independently a biodegradable moiety;

[0672] Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and

[0673] Each s is independently 1, 2, 3, 4 or 5.

[0674] In some embodiments, each X is

[0675] In some embodiments, X' is –OC(O)-, -C(O)O-, -NR 7 C(O)-, -C(O)NR 7 -, -C(O-R 13 )-O-, -C(O)O(CH2) r -, -C(O)NH(CH2) r -, -CON(R 13 )-, or -C(O-R 13 )-O-(CH2) r -, -OC(O)O-, where R 7 is H or C1-C3 alkyl; and R 13 is branched or unbranched C1-C 10 alkyl, and r is 1, 2, 3, 4 or 5.

[0676] In some embodiments, at least one X in the formula is where R 7 is H or methyl. In one embodiment, each X is In one embodiment, each X is where R 7 is H or methyl.

[0677] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LB-IV):

[0678]

[0679] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0680] wherein

[0681] each R1 and each R2 are independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH or NR 10 R11 , or

[0682] Each R1 and each R2, independently of one another, together with the carbon atom to which they are attached, form a ring;

[0683] Each R 10 and R 11 are independently H, a C1-C3 branched or unbranched alkyl group, or R 10 and R 11 together form a heterocyclic ring;

[0684] Each R3 and each R4 are independently H, a C3-C 10 branched or unbranched alkyl group or a C3-C 10 branched or unbranched alkenyl group, provided that at least one of R3 and R4 is not H;

[0685] Each X is independently X', provided that at least one X in the formula is and

[0686] X' is independently a biodegradable moiety;

[0687] Each q is independently 2, 3, 4 or 5; and

[0688] Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0689] In some embodiments, each X is

[0690] In some embodiments, X' is –OC(O)-, -C(O)O-, -NR 7 C(O)-, -C(O)R 7 H-, -C(O-R 13 )-O-, -C(O)O(CH2) r -, -C(O)NH(CH2) r -, -CON(R 13 )-, or -C(O-R 13 )-O-(CH2) r -, -OC(O)O-, where R 7 is H or a C1-C3 alkyl group; and R 13 is a branched or unbranched C1-C 10 alkyl group, and r is 1, 2, 3, 4 or 5.

[0691] In some embodiments, at least one X in the formula is where R 7 is H or methyl. In one embodiment, each X is In one embodiment, each X is wherein R 7 is H or methyl.

[0692] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LB-V):

[0693]

[0694] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0695] wherein

[0696] each R1 and each R2 are independently H, a C1-C3 branched or unbranched alkyl, OH, halogen, SH or NR 10 R 11 , or

[0697] each R1 and each R2 independently together with the carbon atom to which it is attached form a ring;

[0698] each R 10 and R 11 are independently H, a C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle;

[0699] each R3 and each R4 are independently H, a C3-C 10 branched or unbranched alkyl or a C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H;

[0700] each X is independently X', provided that at least one X in the formula is and

[0701] X' is independently a biodegradable moiety;

[0702] each q is independently 2, 3, 4 or 5; and

[0703] each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0704] In some embodiments, each X is

[0705] In some embodiments, X' is –OC(O)-, -C(O)O-, -NR 7 C(O)-, -C(O)NR 7 -, -C(O-R 13 )-O-, -C(O)O(CH2)r -, -C(O)NH(CH2) r -, -CON(R 13 )- or -C(O-R 13 )-O-(CH2) r -, -OC(O)O-, wherein R 7 is H or C1-C3 alkyl; and R 13 is branched or unbranched C1-C 10 alkyl, and r is 1, 2, 3, 4 or 5.

[0706] In some embodiments, at least one X in the formula is wherein R 7 is H or methyl. In one embodiment, each X is In one embodiment, each X is wherein R 7 is H or methyl.

[0707] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LB-VI):

[0708]

[0709] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0710] wherein

[0711] each R1 and each R2 are independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH or NR 10 R 11 , or

[0712] each R1 and each R2 independently combine with the carbon atom to which they are attached to form a ring;

[0713] each R 10 and R 11 are independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 combine to form a heterocycle;

[0714] each R3 and each R4 are independently H, C3-C 10 branched or unbranched alkyl or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H;

[0715] each X is independently X', provided that at least one X in the formula is and

[0716] X' is independently a biodegradable moiety;

[0717] each q is independently 2, 3, 4, or 5; and

[0718] each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0719] In some embodiments, each X is

[0720] In some embodiments, X' is –OC(O)-, -C(O)O-, -NR 7 C(O)-, -C(O)NR 7 -, -C(O-R 13 )-O-, -C(O)O(CH2) r -, -C(O)NH(CH2) r -, -CON(R 13 )-, or -C(O-R 13 )-O-(CH2) r -, -OC(O)O-, where R 7 is H or C1-C3 alkyl; and R 13 is branched or unbranched C1-C 10 alkyl, and r is 1, 2, 3, 4, or 5.

[0721] In some embodiments, at least one X in the formula is where R 7 is H or methyl. In one embodiment, each X is In one embodiment, each X is where R 7 is H or methyl.

[0722] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LB-VII):

[0723]

[0724] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0725] wherein

[0726] each R1 and each R2 are independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 , or

[0727] each R1 and each R2 independently combine with the carbon atom to which they are attached to form a ring;

[0728] Each R 10 and R 11 is independently H, a C1-C3 branched or unbranched alkyl group, or R 10 and R 11 together form a heterocycle;

[0729] Each R3 and each R4 is independently H, a C3-C 10 branched or unbranched alkyl group or a C3-C 10 branched or unbranched alkenyl group, provided that at least one of R3 and R4 is not H;

[0730] Each X is independently X', provided that at least one X in the formula is and

[0731] X' is independently a biodegradable moiety;

[0732] Each q is independently 2, 3, 4 or 5; and

[0733] Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0734] In some embodiments, each X is

[0735] In some embodiments, X' is –OC(O)-, -C(O)O-, -NR 7 C(O)-, -C(O)NR 7 -, -C(O-R 13 )-O-, -C(O)O(CH2) r -, -C(O)NH(CH2) r -, -CON(R 13 )-, or -C(O-R 13 )-O-(CH2) r -, -OC(O)O-, where R 7 is H or a C1-C3 alkyl group; and R 13 is a branched or unbranched C1-C 10 alkyl group, and r is 1, 2, 3, 4 or 5.

[0736] In some embodiments, at least one X in the formula is where R 7 is H or methyl. In one embodiment, each X is In one embodiment, each X is where R 7 is H or methyl.

[0737] In some embodiments, B or is selected from:

[0738]

[0739]

[0740] where t is 0, 1, 2, 3, 4 or 5.

[0741] In some embodiments, an ionizable lipid of formula (LC-I) is disclosed:

[0742] its pharmaceutically acceptable salt or a stereoisomer of any of the foregoing,

[0743] wherein:

[0744] is a cyclic or heterocyclic moiety;

[0745] Y is alkyl, hydroxy, hydroxyalkyl,

[0746] A is absent, is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -S-S- or a divalent heterocycle;

[0747] Each of X and Z independently is absent, is -O-, -N(R 7 )-, -O-alkylene; -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-;

[0748] Each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl or aminoalkyl;

[0749] Each M is independently M', provided that at least one M in the formula is

[0750] Each M' is independently a biodegradable moiety;

[0751] R 30 、R 40 、R 50, R 60 , R 70 , R 80 , R 90 , R 100 , R 110 and R 120 Each of which is independently H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, wherein the alkyl or alkenyl is optionally interrupted by a heteroatom or substituted by OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl;

[0752] Each of l and m is an integer from 1 to 10;

[0753] t is 0, 1, 2, or 3;

[0754] t1 is an integer from 0 to 10; and

[0755] W is hydroxy, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, substituted or unsubstituted heterocyclic group or heteroaryl, or one of the following moieties:

[0756]

[0757]

[0758] Wherein

[0759] Each Q independently does not exist, is -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2) q -C(R 7 )2, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 );

[0760] R 6 is independently H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, N + (R 7 )3-alkylene-Q-, thiol, or thiolalkyl;

[0761] Each R 8 is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, heterocyclic group, heteroaryl, thiol, or thiolalkyl, or two R 8Together with a nitrogen atom, it can form a ring, which is optionally substituted by one or more alkyl groups, hydroxyl groups, hydroxyalkyl groups, alkoxy groups, alkylaminoalkyl groups, alkylamino groups or aminoalkyl groups;

[0762] q is 0, 1, 2, 3, 4 or 5; and

[0763] p is 0, 1, 2, 3, 4 or 5.

[0764] In some embodiments, Y is a hydroxyl group,

[0765] In some embodiments, R 70 and R 80 each is H; and R 90 is a C1-C 15 branched or unbranched alkyl group, a C1-C 15 branched or unbranched alkenyl group, a cycloalkyl group or a substituted cycloalkyl group. In some embodiments, R 90 is a C1-C 15 branched or unbranched alkyl group, a C1-C 15 branched or unbranched alkenyl group. In some embodiments, R 90 is a C1-C 15 branched or unbranched alkyl group. In some embodiments, R 90 is a C1-C 12 branched or unbranched alkyl group.

[0766] In some embodiments, R 70 is H; and R 80 and R 90 each independently is a C1-C 15 branched or unbranched alkyl group, a C1-C 15 branched or unbranched alkenyl group or a cycloalkyl group or a substituted cycloalkyl group. In some embodiments, R 80 and R 90 each independently is a C1-C 15 branched or unbranched alkyl group, a C1-C 15 branched or unbranched alkenyl group. In some embodiments, R 80 and R 90 each independently is a C1-C 15 branched or unbranched alkyl group. In some embodiments, R 80 and R 90 each independently is a C1-C 12 branched or unbranched alkyl group. In some embodiments, R 80 and R 90 each independently is a C1-C8 branched or unbranched alkyl group.

[0767] In some embodiments, R 100 is H; and each of R 110 and R 120 is independently a C1-C 15 branched or unbranched alkyl, C1-C 15 branched or unbranched alkenyl or cycloalkyl or substituted cycloalkyl. In some embodiments, each of R 110 and R 120 is independently a C1-C 15 branched or unbranched alkyl, C1-C 15 branched or unbranched alkenyl. In some embodiments, each of R 110 and R 120 is independently a C1-C 15 branched or unbranched alkyl. In some embodiments, each of R 110 and R 120 is independently a C1-C 12 branched or unbranched alkyl. In some embodiments, each of R 110 and R 120 is independently a C1-C8 branched or unbranched alkyl.

[0768] In some embodiments, an ionizable lipid of formula (LC-IA) or (LC-IA-2) is disclosed:

[0769]

[0770] its pharmaceutically acceptable salt or a stereoisomer of any of the foregoing,

[0771] wherein:

[0772] is a cyclic or heterocyclic moiety;

[0773] A is absent, is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -S-S- or a divalent heterocycle;

[0774] X is absent, is -O-, -CO-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-;

[0775] Z is absent and is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-;

[0776] Each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl or aminoalkyl;

[0777] Each M is independently M', provided that at least one M in the formula is

[0778] Each M' is independently a biodegradable moiety;

[0779] R 30 , R 40 , R 50 , R 60 , R 100 , R 110 and R 120 each of which is independently H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, said alkyl or alkenyl optionally being interrupted by a heteroatom or being substituted by OH, SH or halogen;

[0780] R 90 is C1-C 15 branched or unbranched alkyl, C1-C 15 branched or unbranched alkenyl or cycloalkyl or a substituted cycloalkyl;

[0781] t is 0, 1, 2 or 3;

[0782] t1 is an integer from 0 to 10;

[0783] l is an integer from 1 to 10;

[0784] m is an integer from 1 to 10; and

[0785] W is hydroxy or a divalent heterocyclic hydroxyalkyl, a substituted or unsubstituted amino, a substituted or unsubstituted aminocarbonyl or a substituted or unsubstituted heterocyclic group or heteroaryl or one of the following moieties:

[0786]

[0787]

[0788] wherein

[0789] each Q independently is absent, or is -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2) q -C(R 7 )2, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 );

[0790] R 6 is independently H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, N + (R 7 )3-alkylene-Q-, mercapto or mercaptoalkyl;

[0791] each R 8 is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, heterocyclic group, heteroaryl, mercapto or mercaptoalkyl, or two Rs 8 together with the nitrogen atom can form a ring, which ring is optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino or aminoalkyl;

[0792] q is 0, 1, 2, 3, 4 or 5; and

[0793] p is 0, 1, 2, 3, 4 or 5.

[0794] In some embodiments, A is absent, or is -O-, -N(R 7 )-, -C(O)N(R 7 )-, -N(R 7 )C(O)-, -OC(O)- or -C(O)O-. In one embodiment, A is absent. In one embodiment, A is -O-. In one embodiment, A is -N(R 7 ), where R 7 is H or C1-C3 alkyl. In one embodiment, A is -OC(O)- or -C(O)O-. In one embodiment, A is -NHC(O)- or -C(O)NH-.

[0795] Embodiments regarding the various variables in formulas (LC-I) and (LC-IA) are further discussed below.

[0796] In some embodiments, X is absent, or is -O- or -C(O)-.

[0797] In some embodiments, Z is –O–, –C(O)O– or –OC(O)–.

[0798] In some embodiments, R 30 , R 40 , R 50 and R 60 each is H or a C1-C4 branched or unbranched alkyl group.

[0799] In some embodiments, R 30 , R 40 , R 50 and R 60 each is H.

[0800] In some embodiments, R 90 is a C1-C 15 branched or unbranched alkyl group, a C1-C 15 branched or unbranched alkenyl group. In some embodiments, R 90 is a C1-C 15 branched or unbranched alkyl group. In some embodiments, R 90 is a C1-C 12 branched or unbranched alkyl group. In some embodiments, R 90 is a C1-C8 branched or unbranched alkyl group.

[0801] In some embodiments, R 100 is H; and each of R 110 and R 120 is independently a C1-C 15 branched or unbranched alkyl group, a C1-C 15 branched or unbranched alkenyl group or a cycloalkyl group or a substituted cycloalkyl group. In some embodiments, each of R 110 and R 120 is independently a C1-C 15 branched or unbranched alkyl group, a C1-C 15 branched or unbranched alkenyl group. In some embodiments, each of R 110 and R 120 is independently a C1-C 15 branched or unbranched alkyl group. In some embodiments, each of R 110 and R 120 is independently a C1-C 12 branched or unbranched alkyl group. In some embodiments, each of R 110 and R 120 is independently a C1-C8 branched or unbranched alkyl group.

[0802] In some embodiments, l is 3 to 10, 3 to 7 or 4 to 7.

[0803] In some embodiments, m is 4 to 10, 5 to 8, 1 to 7, 3 to 7, or 1 to 5.

[0804] In some embodiments, l is 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 4, 5, 6, 7, 8, 9, or 10.

[0805] In some embodiments, l is 4, 5, 6, or 7. In some embodiments, m is 3, 4, or 5. In some embodiments, m is 5, 6, 7, or 8.

[0806] In some embodiments, each M is

[0807] In some embodiments, M' is -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(O-R 13 )-O-, -C(O)O(CH2) r -, -C(O)N(R 7 )(CH2) r - or -C(O-R 13 )-O-(CH2) r , where each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R 13 is a branched or unbranched C3-C 10 alkyl, and r is 1, 2, 3, 4, or 5.

[0808] In some embodiments, at least one M in the formula is where R 7 is H or methyl. In one embodiment, each M is In one embodiment, each M is where R 7 is H or methyl.

[0809] In some embodiments, is a 5- to 7-membered monocyclic ring. In some embodiments, is a 5- to 7-membered monocyclic cycloalkane ring. In some embodiments, is a 5- to 7-membered monocyclic heterocycle.

[0810] In some embodiments, is bicyclic or tricyclic, i.e., contains two or more rings, such as fused rings.

[0811] In some embodiments, has the formula The structure of

[0812] Wherein:

[0813] Each of G1, G2, G3, G4, G5, and G6 is independently C(R')(R”), O, or N, provided that no more than two of G1-G6 are O or N;

[0814] R' and R” each independently do not exist, are H, an alkyl group, or two R' together with two adjacent Gs form a second 5- to 7-membered ring or heterocycle; and

[0815] n1 and n2 are each independently 0 or 1.

[0816] In some embodiments, Selected from pyrrolidine, piperidine, piperazine, cyclohexane, cyclopentane, tetrahydrofuran, tetrahydropyran, morpholine, and dioxane.

[0817] In some embodiments, Has The structure of. In one embodiment, Has the structure of.

[0818] In some embodiments, Has The structure of. In one embodiment, Has The structure of. In one embodiment, Has The structure of. In one embodiment, Has The structure of.

[0819] In some embodiments, Has The structure of. In one embodiment, Has The structure of.

[0820] In some embodiments, Has The structure of. In one embodiment, Has The structure of.

[0821] In some embodiments, Has The structure of. In one embodiment, Has The structure of.

[0822] In some embodiments, Has The structure. In one embodiment, has the structure.

[0823] In some embodiments, has the structure. In one embodiment, has the structure.

[0824] In some embodiments, the present disclosure relates to ionizable lipids of formula (LC-IIA) or (LC-IIA-2):

[0825]

[0826] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing,

[0827] wherein:

[0828] A is absent, is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -S-S- or a divalent heterocycle;

[0829] X is absent, is -O-, -CO-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)N(R 7 )- or -S-;

[0830] Z is absent, is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH- or -S-;

[0831] Each R 7 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl;

[0832] Each M is independently M', provided that at least one M in the formula is

[0833] Each M' is independently a biodegradable moiety;

[0834] R30 , R 40 , R 50 , R 60 , R 100 , R 110 and R 120 each of which is independently H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, said alkyl or alkenyl being optionally interrupted by heteroatoms or substituted by OH, SH or halogen;

[0835] R 90 is C1-C 15 branched or unbranched alkyl, C1-C 15 branched or unbranched alkenyl or cycloalkyl or substituted cycloalkyl;

[0836] t is 0, 1, 2 or 3;

[0837] l is an integer from 1 to 10;

[0838] m is an integer from 1 to 10; and

[0839] W is hydroxy or divalent heterocyclic hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl or substituted or unsubstituted heterocyclic group or heteroaryl or one of the following moieties:

[0840]

[0841]

[0842] wherein

[0843] each Q independently is absent, -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2) q -C(R 7 )2, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 );

[0844] R 6 is independently H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, N + (R 7 )3-alkylene-Q-, thiol or thiolalkyl;

[0845] each R 8is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, heterocyclic group, heteroaryl, thiol or thioalkyl, or two Rs 8 together with the nitrogen atom can form a ring, which is optionally substituted by one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino or aminoalkyl;

[0846] q is 0, 1, 2, 3, 4 or 5; and

[0847] p is 0, 1, 2, 3, 4 or 5.

[0848] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LC-IIIA):

[0849] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing, wherein the definitions of the variables in (LC-IIIA) are the same as the definitions of the variables in (LC-IIA).

[0850] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LC-IIB):

[0851] its pharmaceutically acceptable salts or stereoisomers of any of the foregoing,

[0852] wherein:

[0853] A is absent, is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-,-S-,-S-S-;

[0854] X is absent, is -O-, -CO-,-N(R 7 )-,-O-alkylene-,-alkylene-O-,-OC(O)-,-C(O)O-,-NHC(O)-,-C(O)N(R 7 )- or -S-;

[0855] Z is absent, is -O-,-N(R 7 )-,-O-alkylene-,-alkylene-O-,-OC(O)-,-C(O)O-,-NHC(O)-,-C(O)NH- or -S-;

[0856] each R 7Independently is H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl;

[0857] Each M is independently M', Provided that at least one M in the formula is

[0858] Each M' is independently a biodegradable moiety;

[0859] R 30 、R 40 、R 50 、R 60 、R 70 、R 80 、R 90 、R 100 、R 110 and R 120 Each of them is independently H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, and the alkyl or alkenyl is optionally interrupted by a heteroatom or substituted by OH, SH or halogen;

[0860] t is 0, 1, 2 or 3;

[0861] l is an integer from 1 to 10;

[0862] m is an integer from 1 to 10; and

[0863] W is hydroxy or divalent heterocyclic hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl or substituted or unsubstituted heterocyclic group or heteroaryl or one of the following moieties:

[0864]

[0865]

[0866] wherein

[0867] Each Q independently does not exist, is -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2) q -C(R 7 )2, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 );

[0868] R 6 is independently H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R7 ) 2. Amino, alkylamino, aminoalkyl, N + (R 7 )3 - alkylene - Q -, thiol or thioalkyl;

[0869] Each R 8 is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, heterocyclic group, heteroaryl, thiol or thioalkyl, or two Rs 8 together with the nitrogen atom can form a ring, which is optionally substituted by one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino or aminoalkyl;

[0870] q is 0, 1, 2, 3, 4 or 5; and

[0871] p is 0, 1, 2, 3, 4 or 5.

[0872] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LC - IIIB):

[0873] Its pharmaceutically acceptable salts and stereoisomers of any of the foregoing, wherein the definitions of the variables in (LC - IIIB) are the same as the definitions of the variables in (LC - IIB).

[0874] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LC - IIC):

[0875] Its pharmaceutically acceptable salts or stereoisomers of any of the foregoing,

[0876] wherein:

[0877] A is absent, is - O -, - N(R 7 ) -, - O - alkylene -, - alkylene - O -, - OC(O) -, - C(O)O -, - N(R 7 )C(O) -, - C(O)N(R') -, N(R 7 )C(O)N(R 7 ) -, - S -, - S - S -;

[0878] R 30 , R 40 , R 50 , R 60 , R 100 , R 110 and R 120 each of which is independently H, C1 - C 16 branched or unbranched alkyl or C1 - C 16A branched or unbranched alkenyl group, wherein the alkyl or alkenyl group is optionally interrupted by a heteroatom or substituted by OH, SH or a halogen;

[0879] R 90 is a C1-C 15 branched or unbranched alkyl group, C1-C 15 branched or unbranched alkenyl group, cycloalkyl group or substituted cycloalkyl group;

[0880] Each R 7 independently is H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a cycloalkyl group, a hydroxyalkyl group or an aminoalkyl group;

[0881] Each M independently is M', provided that at least one M in the formula is

[0882] Each M' independently is a biodegradable moiety;

[0883] t is 0, 1, 2 or 3;

[0884] l is an integer from 1 to 10;

[0885] m is an integer from 1 to 10; and

[0886] W is a hydroxy group or a divalent heterocyclic hydroxyalkyl group, a substituted or unsubstituted amino group, a substituted or unsubstituted aminocarbonyl group, a substituted or unsubstituted heterocyclic group or heteroaryl group or one of the following moieties:

[0887]

[0888]

[0889] wherein

[0890] each Q independently is absent, is -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2) q -C(R 7 )2, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 );

[0891] R 6 independently is H, an alkyl group, a hydroxy group, a hydroxyalkyl group, an alkoxy group, -O-alkylene-O-alkyl group, -O-alkylene-N(R 7 )2, an amino group, an alkylamino group, an aminoalkyl group, N + (R 7 )3-alkylene-Q-, a thiol or a thiolalkyl group;

[0892] Each R 8 is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, heterocyclic group, heteroaryl, thiol or thioalkyl, or two Rs 8 together with the nitrogen atom may form a ring, which is optionally substituted by one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino or aminoalkyl;

[0893] q is 0, 1, 2, 3, 4 or 5; and

[0894] p is 0, 1, 2, 3, 4 or 5.

[0895] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LC-IIIC) or:

[0896] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing, wherein the definitions of the variables in (LC-IIIA) are the same as the definitions of the variables in (LC-IIC).

[0897] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LC-IIID):

[0898] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing, wherein the definitions of the variables in (LC-IID) are the same as the definitions of the variables defined above.

[0899] In some embodiments, the present disclosure relates to an ionizable lipid of formula (LC-IIIE):

[0900] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing, wherein the definitions of the variables in (IID) are the same as the definitions of the variables defined above.

[0901] Embodiments regarding various variables in formulas (LC-IIA), (LC-IIB), (LC-IIC), (LC-IIIA), (LC-IIIB), (LC-IIIC), (LC-IIID) or (LC-IIIE) are further discussed below.

[0902] In some embodiments, X is absent, is -O- or –C(O)-. In one embodiment, X is absent. In one embodiment, X is –O-. In one embodiment, X is –C(O)-.

[0903] In some embodiments, Z is –O-, –C(O)O- or –OC(O)-. In one embodiment, Z is –O-. In one embodiment, Z is –C(O)O- or –OC(O)-.

[0904] In some embodiments, R 30 , R 40 , R 50 and R 60 each is H or a C1-C4 branched or unbranched alkyl group.

[0905] In some embodiments, R 30 , R 40 , R 50 and R 60 each is H.

[0906] In some embodiments, each of R 70 and R 80 is H; and R 90 is a C1-C 15 branched or unbranched alkyl group, a C1-C 15 branched or unbranched alkenyl group. In some embodiments, R 90 is a C1-C 15 branched or unbranched alkyl group. In some embodiments, R 90 is a C1-C 12 branched or unbranched alkyl group.

[0907] In some embodiments, R 70 is H; and each of R 80 and R 90 independently is a C1-C 15 branched or unbranched alkyl group, a C1-C 15 branched or unbranched alkenyl group. In some embodiments, each of R 80 and R 90 independently is a C1-C 15 branched or unbranched alkyl group. In some embodiments, each of R 80 and R 90 independently is a C1-C 12 branched or unbranched alkyl group. In some embodiments, each of R 80 and R 90 independently is a C1-C8 branched or unbranched alkyl group.

[0908] In some embodiments, R 100 is H; and each of R 110 and R 120 independently is a C1-C 15 branched or unbranched alkyl group, a C1-C 15 branched or unbranched alkenyl group. In some embodiments, each of R 110 and R 120 independently is a C1-C15 branched or unbranched alkyl. In some embodiments, R 110 and R 120 each independently is a C1-C 12 branched or unbranched alkyl. In some embodiments, R 110 and R 120 each independently is a C1-C8 branched or unbranched alkyl.

[0909] In some embodiments, l is from 3 to 10, from 3 to 7, or from 4 to 7.

[0910] In some embodiments, m is from 4 to 10, from 5 to 8, from 1 to 7, from 3 to 7, or from 1 to 5.

[0911] In some embodiments, l is 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 4, 5, 6, 7, 8, 9, or 10.

[0912] In some embodiments, l is 4, 5, 6, or 7. In some embodiments, m is 3, 4, or 5. In some embodiments, m is 5, 6, 7, or 8.

[0913] In some embodiments, each M is

[0914] In some embodiments, M' is -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(O-R 13 )-O-, -C(O)O(CH2) r -, -C(O)N(R 7 )(CH2) r - or -C(O-R 13 )-O-(CH2) r -, where each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R 13 is a branched or unbranched C3-C 10 alkyl, and r is 1, 2, 3, 4, or 5.

[0915] In some embodiments, at least one M in the formula is where R 7 is H or methyl. In one embodiment, each M is In one embodiment, each M is where R 7 is H or methyl.

[0916] For all ionizable lipid formulas described above, further discussion is provided below regarding Examples.

[0917] In some embodiments, A is absent and is -O-, -N(R 7 )-, -C(O)N(R 7 )-, -N(R 7 )C(O)-, -OC(O)- or -C(O)O-. In one embodiment, A is absent. In one embodiment, A is -O-. In one embodiment, A is -N(R 7 )-, where R 7 is H or C1-C3 alkyl. In one embodiment, A is -OC(O)- or -C(O)O-. In one embodiment, A is -NHC(O)- or -C(O)NH-.

[0918] In some embodiments, t is 0, 1 or 2.

[0919] In some embodiments, W is OH.

[0920] In some embodiments, W is where Q is absent and is -(CH2) q -C(R 7 )2- or -N(R 7 );q is 0 or 1;R 7 is H or methyl;and each R 8 is independently H or C1-C3 alkyl. In one embodiment, W is

[0921] In some embodiments, W is where Q is absent and is -(CH2) q -C(R 7 )2- or -N(R 7 );q is 0 or 1;R 7 is H or methyl;and each R 8 is independently H or C1-C3 alkyl. In one embodiment, W is

[0922] In some embodiments, W is where Q is absent and is -(CH2) q -C(R 7 )2- or -N(R 7 );q is 0 or 1;R 7 is H or methyl;and each R 8 is independently H or C1-C3 alkyl. In one embodiment, W is

[0923] In some embodiments, W is where Q is -(CH2) q -C(R 7 )2-; q is 0 or 1; R 7 is H or methyl; and each R 8 is independently H or a C1-C3 alkyl. In one embodiment, W is In one embodiment, W is In one embodiment, W is In W, is

[0924] In some embodiments, W is where q is 0, and each R 8 is independently H, a C1-C3 alkyl, a hydroxyalkyl, a heterocyclic group, or a heteroaryl, optionally substituted with one or more alkyl groups. In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is

[0925] In some embodiments, W is where each R 6 is independently H, a C1-C3 alkyl, a hydroxy group, a hydroxyalkyl, an alkoxy group, -O-alkylene-O-alkyl or -O-alkylene-N(R 7 )2 and each R 7 is independently H or a C1-C3 alkyl. In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is

[0926] In one embodiment, W is In one embodiment, W is

[0927] In one embodiment, W is In one embodiment, W is

[0928] In some embodiments, W is where each R 6 is independently H, C1-C3 alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl or -O-alkylene-N(R 7 )2; Q is -O-, -C(R 7 )2- or N(R 7 ); and R 7 is H, C1-C3 alkyl or hydroxyalkyl. In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is

[0929] In one embodiment, W is In one embodiment, W is In one embodiment, W is

[0930] In some embodiments, W is where q is 0 and each R 8 is independently H, C1-C3 alkyl or hydroxyalkyl. In one embodiment, W is In one embodiment, W is

[0931] In some embodiments, W is where R 6 is independently H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl or -O-alkylene-N(R 7 )2; and each R 7 is independently H or C1-C3 alkyl. In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is In one embodiment, W is

[0932] In some embodiments, W is where each R 8 is independently H, C1-C3 alkyl or hydroxyalkyl; each Q is independently absent, -O-, -CO-, -C(R 7 )2- or -N(R 7 )-; and each R 7 is independently H, C1-C3 alkyl, alkylamino, alkylaminoalkyl or aminoalkyl. In one embodiment, W is In one embodiment, W is

[0933] In some embodiments, W is where each R 8 is independently H, C1-C3 alkyl or hydroxyalkyl; each Q is independently absent, -O-, -CO-, -C(R 7 )2- or -N(R 7 )-; and each R 7 is independently H, C1-C3 alkyl, alkylamino, alkylaminoalkyl or aminoalkyl.

[0934] In one embodiment, W is In one embodiment, W is

[0935] For all the ionizable lipid formulas described above, embodiments regarding the variables R 70 , R 80 , R 90 , R 100 , R 110 and R 120 are further discussed below.

[0936] In some embodiments, R 70 is H. In some embodiments, R 100 is H.

[0937] In these embodiments, is independently selected from:

[0938]

[0939]

[0940] where t is 0, 1, 2, 3, 4 or 5.

[0941] In some embodiments, the pKa of the protonated form of the ionizable lipid compounds described herein is from about 4 to about 8, for example, about 4.5 to about 8.0, about 4.6 to about 7.5, about 4.6 to about 7.1, about 4.6 to about 5.5, about 4.8 to about 8.0, about 4.8 to about 7.5, about 4.8 to about 7.1, about 4.6 to about 5.5, about 5.7 to about 6.5, about 5.7 to about 6.4, or about 5.8 to about 6.2. In some embodiments, the pKa of the protonated form of the compound is from about 5.5 to about 6.0. In some embodiments, the pKa of the protonated form of the compound is from about 6.1 to about 6.3. In some embodiments, the pKa of the protonated form of the compound is from about 4.7 to about 5.1. In some embodiments, the pKa of the protonated form of the compound is from about 5.4 to about 7.1.

[0942] Non-limiting examples of the ionizable lipid compounds disclosed herein are set forth in Table 1 below.

[0943] Table 1. Exemplary ionizable lipid compounds.

[0944]

[0945]

[0946]

[0947]

[0948]

[0949]

[0950]

[0951]

[0952]

[0953]

[0954]

[0955] Additional non-limiting examples of the ionizable lipid compounds disclosed herein are set forth in Table 2 below.

[0956] Table 2. Exemplary ionizable lipid compounds.

[0957]

[0958]

[0959]

[0960]

[0961]

[0962]

[0963]

[0964]

[0965]

[0966]

[0967]

[0968]

[0969]

[0970]

[0971]

[0972]

[0973]

[0974]

[0975]

[0976]

[0977]

[0978]

[0979]

[0980]

[0981]

[0982]

[0983]

[0984]

[0985]

[0986]

[0987]

[0988]

[0989]

[0990]

[0991]

[0992]

[0993]

[0994]

[0995]

[0996]

[0997]

[0998]

[0999]

[1000]

[1001]

[1002]

[1003]

[1004] Method for preparing exemplary lipid compounds

[1005] Also disclosed herein are various methods for preparing exemplary lipid compounds.

[1006] In some embodiments, provided herein is a method for preparing a lipid comprising at least one head group and at least one tail group of formula (TI) or (TI')

[1007]

[1008] Wherein:

[1009] Each E is independently a biodegradable group;

[1010] R a Each is independently a C1-C5 alkyl group, a C2-C5 alkenyl group or a C2-C5 alkynyl group;

[1011] u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7;

[1012] R t Each is independently H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, wherein the alkyl or alkenyl is optionally interrupted by a heteroatom or substituted by OH, SH or a halogen or a cycloalkyl or a substituted cycloalkyl;

[1013] represents a bond connecting the tail group to the head group; and

[1014] wherein the pKa of the lipid is from about 4 to about 8.

[1015] The method comprises

[1016] reacting a first precursor compound of a tail group of formula (TI) or (TI') with a precursor compound of the head group, wherein the precursor compound of the head group comprises one or more attachment points for the tail group, each attachment point containing a functional group reactive towards a halogen, whereby a lipid is formed by connecting at least one tail group of formula (TI) or (TI') to the head group at the one or more attachment points.

[1017] In some embodiments, one or more attachment points for the tail group in the precursor compound of the head group contain one or more Ns.

[1018] In some embodiments, one or more attachment points for the tail group in the precursor compound of the head group further comprise non-N functional groups, and the one or more Ns contained at the one or more attachment points of the precursor compound of the head group are protected such that the attachment points containing the non-N functional groups react with the precursor compound of the tail group. Then, the method further comprises:

[1019] deprotecting the one or more Ns contained at the one or more attachment points of the head group of the lipid; and

[1020] reacting a second precursor compound of a tail group of formula (TI) or (TI') React with the lipid having one or more deprotected Ns at the one or more attachment points of the head group, thereby forming a lipid by attaching a second tail group of formula (TI) or (TI') to the head group at the one or more attachment points. In some embodiments, the second precursor compound of the tail group is the same as the first precursor compound of the tail group. Thus, the final lipid contains multiple identical tail groups. In some embodiments, the second precursor compound of the tail group is different from the first precursor compound of the tail group. Thus, the final lipid contains multiple different tail groups.

[1021] In some embodiments, at least one tail group has one of the following formulas:

[1022] R 7 Each independently is H or methyl;

[1023] R b In each case independently is H or a C1-C4 alkyl; and

[1024] u3 and u4 each independently are 0, 1, 2, 3, 4, 5, 6, or 7.

[1025] In some embodiments, each R in the above formula a is methyl.

[1026] In some embodiments, provided herein is a method for preparing a lipid that comprises at least one head group and at least one tail group having the following formula

[1027] wherein:

[1028] the tail group is

[1029] u1 and u2 each independently are 0, 1, 2, 3, 4, 5, 6, or 7,

[1030] u3 and u4 each independently are 0, 1, 2, 3, or 4.

[1031] W is hydroxy, hydroxyalkyl, or one of the following moieties:

[1032]

[1033]

[1034] wherein:

[1035] Each Q independently is absent, or is -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2) q -C(R 7 )2, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 );

[1036] R 6 independently is H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, thiol, thioalkyl or N + (R 7 )3-alkylene-Q-;

[1037] Each R 8 independently is H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, thioalkyl, heterocyclic group, heteroaryl; or two Rs 8 together with the nitrogen atom form a ring, which is optionally substituted by one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino or aminoalkyl;

[1038] q is 0, 1, 2, 3, 4 or 5; and

[1039] p is 0, 1, 2, 3, 4 or 5.

[1040] The method comprises

[1041] reacting a compound with a compound to obtain

[1042] reacting compound 33 with a compound to obtain

[1043] removing the N-protecting group of compound 35 to obtain a compound

[1044] reacting compound 36 with a compound which may be the same as or different from compound 33 to obtain a compound and

[1045] reacting compound 38 with a compound to obtain lipid.

[1046] The above method can be shown in the following general reaction scheme:

[1047]

[1048] In some embodiments, provided herein is a method for preparing a lipid, the lipid comprising at least one head group and at least one tail group having the following formula

[1049] wherein:

[1050] the tail group is

[1051] u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7,

[1052] u3 and u4 are each independently 0, 1, 2, 3, or 4.

[1053] Each of R1 and R2 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, OH, a halogen, SH, or NR 10 R 11 ; or R1 and R2 together form a ring;

[1054] R 10 and R 11 are each independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl; or R 10 and R 11 together form a heterocycle;

[1055] m is 1, 2, 3, 4, 5, 6, 7, or 8;

[1056] n is 0, 1, 2, 3, or 4;

[1057] Z is absent, O, S, or NR 12 , where R 12 is H or a C1-C7 branched or unbranched alkyl; provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 , SH.

[1058] The method comprises:

[1059] reacting compound with compound to obtain compound

[1060] reacting compound 3 with compound N-protecting group-NH2 to obtain compound

[1061] removing the O-protecting group of compound 4 to obtain compound

[1062] React compound 5 with (C(O)-halogen)2 to obtain a compound

[1063] React compound 6 with to obtain a compound

[1064] Remove the N-protecting group of compound 8 to obtain a compound and

[1065] React compound 9 with a compound to obtain lipid.

[1066] The above method can be shown in the following general reaction scheme:

[1067]

[1068] In some embodiments, provided herein is a method for preparing a lipid, the lipid comprising at least one head group and at least one tail group having the following formula

[1069] wherein:

[1070] The tail group is

[1071] u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7,

[1072] u3 and u4 are each independently 0, 1, 2, 3 or 4.

[1073] Each R7 and R8 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, OH, SH, (CH2) s N(CH3)2 or NR 10 R 11 where each R 10 and R 11 is independently H or a C1-C3 alkyl group, or R 10 and R 11 together form a heterocycle; or R7 and R8 together form a ring;

[1074] Each of s, u and t is independently 1, 2, 3, 4 or 5.

[1075] The method comprises:

[1076] React a compound with a compound React to obtain a compound

[1077] React compound 3 with compound N-protecting group -NH2 to obtain a compound

[1078] Remove the O-protecting group of compound 4 to obtain a compound

[1079] React compound 5 with (C(O)-halogen)2 to obtain a compound React compound 6 with to obtain a compound

[1080] Remove the N-protecting group of compound 8 to obtain a compound React compound 9 with compound to obtain a compound

[1081] Remove the N-protecting group of compound 23 to obtain a compound And

[1082] React compound 24 with compound to obtain a lipid having the formula The above method can be shown in the following general reaction scheme:

[1083] The above method can be illustrated in the following general reaction scheme:

[1084]

[1085] Additional methods for preparing the lipid compounds described herein are illustrated in Examples 1-6 and 9.

[1086] Lipid composition

[1087] The ionizable lipids disclosed herein can be used to form lipid nanoparticle compositions. In some embodiments, the lipid nanoparticle compositions further comprise one or more therapeutic agents. In some embodiments, the lipid nanoparticles in the composition encapsulate one or more therapeutic agents or associate with one or more therapeutic agents.

[1088] In some embodiments, the present disclosure relates to a composition comprising (i) one or more lipid compounds described herein, the one or more lipid compounds comprising at least one head group (e.g., HA-I to HA-VII, HB-I or HC-I to HC-IIIE; or any subgenus or species of these formulas disclosed herein) and at least one tail group of formula (TI to TIII, or any subgenus or species of these formulas disclosed herein), pharmaceutically acceptable salts thereof, and stereoisomers and therapeutic agents of any of the foregoing, and (ii) one or more lipid components that are different from the lipid components of the lipid compounds described herein. In some embodiments, the composition comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the one or more lipid compounds.

[1089] In some embodiments, the present disclosure relates to a composition comprising (i) one or more lipid nanoparticles and (ii) one or more lipid components that are different from the lipid compounds described herein.

[1090] In some embodiments, the one or more lipid components that are different from the lipid compounds described herein comprise one or more helper lipids and one or more PEG lipids. In some embodiments, the lipid component that is different from the lipid compounds described herein comprises one or more helper lipids, one or more PEG lipids and one or more neutral lipids.

[1091] In some embodiments, the lipid composition may further comprise sterol and PEG lipid. In some embodiments, the lipid composition may further comprise sterol, PEGylated lipid, phospholipid and / or neutral lipid.

[1092] In some embodiments, one or more naturally occurring and / or synthetic lipid compounds can be used to prepare the lipid composition. The lipid composition can contain negatively charged lipids, positively charged lipids or a combination thereof.

[1093] Non-ionizable lipid components

[1094] Charged lipids and neutral lipids

[1095] Examples of suitable negatively charged (anionic) lipids include, but are not limited to, dimyristoyl-phosphatidylglycerol, dipalmitoyl-phosphatidylglycerol, and distearoyl-phosphatidylglycerol; dimyristoyl-phosphatidic acid, dipalmitoyl-phosphatidic acid, and dipalmitoyl-phosphatidic acid; dimyristoyl-phosphatidylethanolamine, dipalmitoyl-phosphatidylethanolamine, and dipalmitoyl-phosphatidylethanolamine; and their unsaturated diacyl and mixed acyl chain counterparts, as well as cardiolipin.

[1096] Examples of positively charged (cationic) lipids include, but are not limited to, N,N'-dimethyl-N,N'-dioctadecylammonium bromide (DDAB) and N,N'-dimethyl-N,N'-dioctadecylammonium chloride (DDAC), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-chol), 1,2-dioleyloxy-3-[trimethylammonio]-propane (DOTAP), 1,2-distearyloxy-3-[trimethylammonio]-propane (DSTAP), and 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium chloride (DORI), as well as cationic lipids described, for example, by Martin et al., Current Pharmaceutical Design, pages 1-394, which is incorporated herein by reference in its entirety.

[1097] Additional exemplary cationic lipids include, but are not limited to, N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), 1,2-dioleoyl-3-dimethylammonio-propane (DODAP), 1,2-dioleoylcarbamoyl-3-dimethylammonio-propane (DOCDAP), 1,2-dilinoleoyl-3-dimethylammonio-propane (DLINDAP), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β-oxy)-3'-(oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLin DMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), and / or mixtures thereof. Neutral lipids can include dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), and / or mixtures thereof.

[1098] In some embodiments, the lipid component comprises one or more neutral lipids. The neutral lipid can be one or more phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids can assemble into one or more lipid bilayers. Generally, a phospholipid can include a phospholipid moiety and one or more fatty acid moieties. For example, a phospholipid can be a lipid according to the following formula: wherein R p represents the phospholipid moiety, and R A and R BRefers to having or not having unsaturated fatty acid moieties, which may be the same or different. The phospholipid moiety may be phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysolecithin or sphingomyelin. The fatty acid moiety may be lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid or docosahexaenoic acid. Also contemplated are non-natural species, including natural species having modifications and substitutions (including branching, oxidation, cyclization and alkynes). For example, phospholipids may be functionalized or crosslinked with one or more alkynes (e.g., alkenyls in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, alkynes can undergo copper-catalyzed cycloaddition when exposed to azides. Such reactions can be used to functionalize the lipid bilayer of lipid nanoparticles to facilitate membrane penetration or cell recognition, or to conjugate lipid nanoparticles with useful components, such as targeting moieties or imaging moieties (e.g., dyes).

[1099] In some embodiments, the neutral lipid can be a phospholipid, such as distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-docosanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) or a mixture thereof.

[1100] Additional non-limiting examples of neutral lipids also include phospholipids such as lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecyl phosphate, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl-phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), palmitoyl oleoyl-phosphatidylglycerol (POPG), dioleoyl phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyl oleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, and mixtures thereof. Other diacyl phosphatidylcholines and diacyl phosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids can be acyl groups derived from fatty acids having C 10 -C 24 carbon chains, such as, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[1101] Steroids and other non-ionizable lipid components

[1102] In some embodiments, the lipid component in the lipid composition comprises one or more steroids or analogs thereof.

[1103] In some embodiments, the lipid component in the lipid composition comprises sterols such as cholesterol, sitosterol, and derivatives thereof. Non-limiting examples of cholesterol derivatives include: polar analogs such as 5a-cholestanol, 5a-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5a-cholestane, cholestenone, 5a-cholestanone, 5a-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[1104] In some embodiments, the non-ionizable lipid component comprises, consists of, or is a mixture of one or more phospholipids and cholesterol or its derivatives. In some embodiments, the non-ionizable lipid component comprises, consists of, or is one or more phospholipids (e.g., cholesterol-free lipid particle formulations). In some embodiments, the non-ionizable lipid component comprises, consists of, or is cholesterol or its derivatives (e.g., phospholipid-free lipid particle formulations).

[1105] In some embodiments, the lipid component in a lipid composition (e.g., an LNP composition) comprises phytosterols or a combination of phytosterols and cholesterol. In some embodiments, the phytosterols are selected from the group consisting of: β-sitosterol, stigmasterol, β-sitostanol, campesterol, brassicasterol, and combinations thereof. In some embodiments, the phytosterols are selected from the group consisting of: β-sitosterol, β-sitostanol, campesterol, brassicasterol, Compound S-140, Compound S-151, Compound S-156, Compound S-157, Compound S-159, Compound S-160, Compound S-164, Compound S-165, Compound S-170, Compound S-173, Compound S-175, and combinations thereof. In some embodiments, the phytosterols are selected from the group consisting of: Compound S-140, Compound S-151, Compound S-156, Compound S-157, Compound S-159, Compound S-160, Compound S-164, Compound S-165, Compound S-170, Compound S-173, Compound S-175, and combinations thereof. In some embodiments, the phytosterols are a combination of Compound S-141, Compound S-140, Compound S-143, and Compound S-148. In some embodiments, the phytosterols comprise sitosterol or a salt or ester thereof. In some embodiments, the phytosterols comprise stigmasterol or a salt or ester thereof. In some embodiments, the phytosterol is β-sitosterol, its salt, or its ester.

[1106] In some embodiments, the LNP composition comprises a phytosterol or a salt or ester thereof, and cholesterol or a salt thereof.

[1107] In some embodiments, the target cell is a cell described herein (e.g., a liver cell or a spleen cell), and the phytosterol or a salt or ester thereof is selected from the group consisting of: β-sitosterol, β-sitostanol, campesterol, brassicasterol, and combinations thereof. In some embodiments, the phytosterol is β-sitosterol. In some embodiments, the phytosterol is β-sitostanol. In some embodiments, the phytosterol is campesterol. In some embodiments, the phytosterol is brassicasterol.

[1108] In some embodiments, the target cell is a cell described herein (e.g., a liver cell or a spleen cell), and the phytosterol or its salt or ester is selected from the group consisting of: β-sitosterol and stigmasterol and combinations thereof. In some embodiments, the phytosterol is β-sitosterol. In some embodiments, the phytosterol is stigmasterol.

[1109] Other examples of non-ionizable lipids include phosphorus-free lipids such as stearylamine, dodecylamine, cetylamine, acetyl palmitate, glyceryl ricinoleate, cetyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethoxylated fatty acid amides, didodecyldimethylammonium bromide, ceramides, and sphingomyelins.

[1110] In some embodiments, the non-ionizable lipid accounts for 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%, 37 mol% to 42 mol%, or 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol% or 45 mol% (or any fraction or range therein) of the total lipids present in the particle.

[1111] In embodiments where the lipid particle composition contains a mixture of phospholipids and cholesterol or cholesterol derivatives, the mixture can account for up to 40 mol%, 45 mol%, 50 mol%, 55 mol% or 60 mol% of the total lipids present in the particle.

[1112] In some embodiments, the phospholipid component in the mixture can account for 2 mol% to 20 mol%, 2 mol% to 15 mol%, 2 mol% to 12 mol%, 4 mol% to 15 mol% or 4 mol% to 10 mol% (or any fraction or range therein) of the total lipids present in the particle. In some embodiments, the phospholipid component in the mixture accounts for 5 mol% to 10 mol%, 5 mol% to 9 mol%, 5 mol% to 8 mol%, 6 mol% to 9 mol%, 6 mol% to 8 mol% or 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol% or 10 mol% (or any fraction or range therein) of the total lipids present in the particle.

[1113] In some embodiments, the cholesterol component in the mixture can be 25 mol% to 45 mol%, 25 mol% to 40 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 27 mol% to 37 mol%, 25 mol% to 30 mol% or 35 mol% to 40 mol% (or any portion or range therein) of the total lipids present in the particles. In some embodiments, the cholesterol component in the mixture is 25 mol% to 35 mol%, 27 mol% to 35 mol%, 29 mol% to 35 mol%, 30 mol% to 35 mol%, 30 mol% to 34 mol%, 31 mol% to 33 mol% or 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol% or 35 mol% (or any portion or range therein) of the total lipids present in the particles.

[1114] In embodiments where the lipid particle composition is phospholipid-free, cholesterol or its derivative can be up to 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol% or 60 mol% of the total lipids present in the particles.

[1115] In some embodiments, cholesterol or its derivative in the phospholipid-free lipid particle formulation can be 25 mol% to 45 mol%, 25 mol% to 40 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 31 mol% to 39 mol%, 32 mol% to 38 mol%, 33 mol% to 37 mol%, 35 mol% to 45 mol%, 30 mol% to 35 mol%, 35 mol% to 40 mol% or 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol% or 40 mol% (or any portion or range therein) of the total lipids present in the particles.

[1116] In some embodiments, the non-ionizable lipid is 5 mol% to 90 mol%, 10 mol% to 85 mol%, 20 mol% to 80 mol%, 10 mol% (e.g., phospholipids only) or 60 mol% (e.g., phospholipids and cholesterol or its derivative) (or any portion or range therein) of the total lipids present in the particles.

[1117] The percentage of non-ionizable lipid present in the lipid particles is a target amount, and the actual amount of non-ionizable lipid present in the particles can vary, e.g., ±5 mol%.

[1118] Compositions containing ionizable lipid compounds can be 30 - 70% ionizable lipid compounds, 0 - 60% cholesterol, 0 - 30% phospholipids, and 1 - 10% polyethylene glycol (PEG). In some embodiments, the composition is 30 - 40% ionizable lipid compounds, 40 - 50% cholesterol, and 10 - 20% PEG. In some embodiments, the composition is 50 - 75% ionizable lipid compounds, 20 - 40% cholesterol, 5 - 10% phospholipids, and 1 - 10% PEG. The composition can contain 60 - 70% ionizable lipid compounds, 25 - 35% cholesterol, and 5 - 10% PEG. The composition can contain up to 90% ionizable lipid compounds and 2 - 15% co-lipids.

[1119] The composition can be a lipid particle composition, for example, containing 8 - 30% compound, 5 - 30% co-lipids, and 0 - 20% cholesterol; 4 - 25% ionizable lipid, 4 - 25% co-lipids, 2 - 25% cholesterol, 10 - 35% cholesterol-PEG, and 5% cholesterol-amine; or 2 - 30% ionizable lipid, 2 - 30% co-lipids, 1 - 15% cholesterol, 2 - 35% cholesterol-PEG, and 1 - 20% cholesterol-amine; or up to 90% ionizable lipid and 2 - 10% co-lipids, or even 100% ionizable lipid.

[1120] Lipid conjugates

[1121] In addition to one or more ionizable lipids, the lipid particles described herein can further comprise one or more lipid conjugates. The conjugated lipids can prevent aggregation of the particles. Non-limiting examples of conjugated lipids include PEG-lipid conjugates, cationic polymer-lipid conjugates, and mixtures thereof.

[1122] In some embodiments, the lipid conjugate is a PEG-lipid or a PEG-modified lipid (alternatively referred to as a PEGylated lipid). A PEG lipid is a lipid modified with polyethylene glycol. Examples of PEG-lipids include, but are not limited to, PEG coupled to dialkyloxopropyl (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DEG), PEG coupled to a phospholipid (such as phosphatidylethanolamine) (PEG-PE), PEG conjugated to ceramide (PEG-CER), PEG conjugated to cholesterol or its derivatives, and mixtures thereof.

[1123] For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[1124] In some embodiments, the PEG-lipids are selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[1125] In some embodiments, the PEG-lipids are selected from the group consisting of: 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-distearylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearoyl, PEG-diacylglycylamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA).

[1126] PEG is a linear water-soluble polymer of ethylene glycol repeating units having two terminal hydroxyl groups. PEG is classified by its molecular weight; and includes the following: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), and such compounds having terminal hydroxyl groups instead of terminal methoxy groups (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH2).

[1127] The PEG moiety of the PEG-lipid conjugates described herein can have an average molecular weight in the range of 550 daltons to 10,000 daltons. In certain cases, the average molecular weight of the PEG moiety is 750 daltons to 5,000 daltons (e.g., 1,000 daltons to 5,000 daltons, 1,500 daltons to 3,000 daltons, 750 daltons to 3,000 daltons, 750 daltons to 2,000 daltons). In some embodiments, the average molecular weight of the PEG moiety is 2,000 daltons or 750 daltons.

[1128] In some cases, the PEG may optionally be substituted with an alkyl, alkoxy, acyl, or aryl group. The PEG may be conjugated directly to the lipid or may be linked to the lipid through a linker moiety. Any linker moiety suitable for coupling the PEG to the lipid may be used, including, for example, an ester-free linker moiety and an ester-containing linker moiety. In some embodiments, the linker moiety is an ester-free linker moiety. Suitable ester-free linker moieties include, but are not limited to, an amido group (-C(O)NH-), an amino group (-NR-), a carbonyl group (-C(O)-), a carbamate group (-NHC(O)O-), a urea group (-NHC(O)NH-), a disulfide group (-S-S-), an ether group (-O-), a succinyl group (-(O)CCH2CH2C(O)-), a succinamido group (-NHC(O)CH2CH2C(O)NH-), an ether, a disulfide, and combinations thereof (such as a linker containing both a carbamate linker moiety and an amine linker moiety). In some embodiments, a carbamate linker is used to couple the PEG to the lipid.

[1129] In some embodiments, an ester-containing linker moiety is used to couple the PEG to the lipid. Suitable ester-containing linker moieties include, for example, a carbonate group (-OC(O)O-), a succinyl group, a phosphate group (-O-(O)POH-O-), a sulfonate group, and combinations thereof.

[1130] Phosphatidylethanolamines having various acyl chain groups of different chain lengths and degrees of saturation may be conjugated with PEG to form lipid conjugates. Such phosphatidylethanolamines are commercially available or may be isolated or synthesized using conventional techniques known to those skilled in the art.

[1131] In some embodiments, the phosphatidylethanolamine contains a saturated or unsaturated fatty acid having a carbon chain length in the range of C10 to C20. Phosphatidylethanolamines having a mixture of a monounsaturated fatty acid or a diunsaturated fatty acid, as well as a saturated fatty acid and an unsaturated fatty acid, may also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[1132] The term "diacylglycerol" or "DAG" includes compounds having two fatty acyl chains R1 and R2, both of which independently have from 2 to 30 carbons bonded through ester linkages to the 1- and 2-positions of glycerol. The acyl groups can be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C12), myristoyl (C14), palmitoyl (C16), stearoyl (C18), and icosoyl (C20). In some embodiments, R1 and R2 are the same, i.e., both R1 and R2 are myristoyl (i.e., dimyristoyl), both R1 and R2 are stearoyl (i.e., distearoyl).

[1133] The term "dialkoxypropyl" or "DAA" includes compounds having two alkyl chains R and R', both of which independently have from 2 to 30 carbons. The alkyl groups can be saturated or have varying degrees of unsaturation.

[1134] In some embodiments, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C10) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16) conjugate, or a PEG-distearyloxypropyl (C18) conjugate. In some embodiments, the average molecular weight of the PEG is 750 daltons or 2,000 daltons. In some embodiments, the terminal hydroxyl group of the PEG is substituted with a methyl group.

[1135] In addition to the foregoing, other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, poly(methyloxazoline), poly(ethyloxazoline), poly(hydroxypropylmethacrylamide), poly(methacrylamide), and poly(dimethylacrylamide), polylactic acid, polyglycolic acid, and derivatized cellulose (such as hydroxymethylcellulose or hydroxyethylcellulose).

[1136] In some embodiments, the PEG-lipid is a compound of the formula or a salt thereof, wherein:

[1137] R 3PL1 is –OR OPL1 ;

[1138] R OPL1 is hydrogen, optionally substituted alkyl, or an oxygen protecting group;

[1139] r PL1 is an integer between 1 and 100, inclusive;

[1140] L 1 is optionally substituted C 1-10Alkylene, wherein at least one methylene group of the optionally substituted C 1-10 of the alkylene is independently replaced by: optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R NPL1 ), S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), -C(O)O, OC(O), OC(O)O, OC(O)N(R NPL1 ), NR NPL1 C(O)O or NR NPL1 C(O)N(R NPL1 );

[1141] D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m PL1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[1142] A has the following formula:

[1143] L 2 Each instance of is independently a bond or optionally substituted C 1-6 alkylene, wherein one methylene unit of the optionally substituted C 1-6 alkylene is optionally replaced by: O, N(R NPL1 ), S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), C(O)O, OC(O), OC(O)O, -OC(O)N(R NPL1 ), NR NPL1 C(O)O or NR NPL1 C(O)N(R NPL1 );

[1144] R 2SL Each instance of is independently optionally substituted C 1-30 alkyl, optionally substituted C 1-30 alkenyl or optionally substituted C 1-30 alkynyl; optionally wherein one or more methylene units of R 2SL are independently replaced by: optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R NPL1 ), O, S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), -NR NPL1 C(O)N(R NPL1), C(O)O, OC(O), OC(O)O, OC(O)N(R NPL1 ), NR NPL1 C(O)O, C(O)S, -SC(O), C(=NR NPL1 ), C(=NR NPL1 )N(R NPL1 ), NR NPL1 C(=NR NPL1 ),-NR NPL1 C(=NR NPL1 )N(R NPL1 )、C(S)、C(S)N(R NPL1 ), NR NPL1 C(S),NR NPL1 C(S)N(R NPL1 ), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R NPL1 )S(O), S(O)N(R NPL1 )、-N(R NPL1 )S(O)N(R NPL1 )、OS(O)N(R NPL1 )、N(R NPL1 )S(O)O、S(O)2、N(R NPL1 )S(O)2、-S(O)2N(R NPL1 )、N(R NPL1 )S(O)2N(R NPL1 )、OS(O)2N(R NPL1 ) or N(R NPL1 )S(O)2O;

[1145] R NPL1 Each instance of is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

[1146] Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl; and

[1147] p SL Either 1 or 2.

[1148] In some embodiments, the PEG-lipid is of the formula A compound or a salt thereof, wherein r PL1 , L 1 ,D,m PL1 and A are as defined above.

[1149] In some embodiments, the PEG-lipid is of the formula A compound or a salt or isomer thereof, wherein:

[1150] R 3PEG is –OR O ;

[1151] R O is hydrogen, C 1-6 alkyl or an oxygen protecting group;

[1152] r PEG is an integer between 1 and 100 (for example, between 40 and 50, for example, 45);

[1153] R 5PEG is C 10-40 alkyl (for example, C 17 alkyl), C 10-40 alkenyl or C 10-40 alkynyl; and optionally one or more methylene groups of R 5PEG are independently replaced by: C 3-10 subcarbocyclic group, 4- to 10-membered heterocyclic group, C 6-10 arylene, 4- to 10-membered heteroarylene, –N(R NPEG )–, –O–, –S–, –C(O)–, –C(O)N(R NPEG )–, –NR NPEG C(O)–, –NR NPEG C(O)N(R NPEG )–, –C(O)O–, –OC(O)–, –OC(O)O–, –OC(O)N(R NPEG )–, –NR NPEG C(O)O–, –C(O)S–, –SC(O)–, –C(=NR NPEG )–, –C(=NR NPEG )N(R NPEG )–, –NR NPEG C(=NR NPEG )–, –NR NPEG C(=NR NPEG )N(R NPEG )–, –C(S)–, –C(S)N(R NPEG )–, –NR NPEG C(S)–, –NR NPEG C(S)N(R NPEG )–, –S(O)–, –OS(O)–, –S(O)O–, –OS(O)O–, –OS(O)2–, –S(O)2O–, –OS(O)2O–, –N(R NPEG )S(O)–, –S(O)N(R NPEG )–, –N(R NPEG )S(O)N(R NPEG)–, –OS(O)N(R NPEG )–, –N(R NPEG )S(O)O–, –S(O)2–, –N(R NPEG )S(O)2–, –S(O)2N(R NPEG )–, –N(R NPEG )S(O)2N(R NPEG )–, –OS(O)2N(R NPEG )– or –N(R NPEG )S(O)2O–; and

[1154] R NPEG Each instance of is independently hydrogen, C 1-6 alkyl or a nitrogen protecting group.

[1155] In some embodiments, the PEG-lipid is a compound of formula where r PEG is an integer between 1 and 100 (e.g., between 40 and 50, such as 45).

[1156] In some embodiments, the PEG-lipid is a compound of formula or a salt or isomer thereof, where s PL1 is an integer between 1 and 100 (e.g., between 40 and 50, such as 45).

[1157] In some embodiments, the PEG-lipid has the formula or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[1158] R 8 and R 9 are each independently a straight-chain or branched-chain, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds (e.g., R 8 and R 9 are each independently a straight-chain, saturated alkyl chain containing 12 to 16 carbon atoms); and

[1159] w has an average value in the range of 30 to 60 (e.g., the average w is about 49).

[1160] In some embodiments, incorporating any one of the PEG-lipids discussed above into the lipid composition can improve the pharmacokinetics and / or biodistribution of the lipid composition. For example, incorporating any one of the PEG-lipids discussed above into the lipid composition can reduce the accelerated blood clearance (ABC) effect.

[1161] Other ionizable lipids

[1162] In some embodiments, the lipid composition can comprise one or more additional ionizable lipids different from the ionizable lipids described herein. Exemplary ionizable lipids include, but are not limited to,

[1163]

[1164] Acuitas Lipid 9 and Acuitas Lipid 10 (see WO 2017 / 004143A1, which is incorporated herein by reference in its entirety).

[1165] In one embodiment, the additional ionizable lipid is 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid heptadec-9-yl ester (SM-102); for example, as described in Example 1 of U.S. Patent No. 9,867,888 (which is incorporated herein by reference in its entirety).

[1166] 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), for example, as synthesized in Example 13 of WO 2015 / 095340 (which is incorporated herein by reference in its entirety).

[1167] In one embodiment, the additional ionizable lipid is bis((Z)-non-2-en-1-yl) 9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319); for example, as synthesized in Examples 7, 8, or 9 of US 2012 / 0027803 (which is incorporated herein by reference in its entirety).

[1168] 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).

[1169] In one embodiment, the additional ionizable lipid is imidazole cholesterol ester (ICE) lipid 3-(1H-imidazol-4-yl)propanoic acid (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 ester, e.g., Structure (I) from WO 2020 / 106946 (which is incorporated herein by reference in its entirety).

[1170] In one embodiment, the additional ionizable lipid is MC3 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO 2019 / 051289A9 (which is incorporated herein by reference in its entirety).

[1171] In one embodiment, the additional ionizable lipid is lipid ATX-002, e.g., as described in Example 10 of WO 2019 / 051289A9 (which is incorporated herein by reference in its entirety).

[1172] In one embodiment, the additional ionizable lipid is (13Z,16Z)-A,A-dimethyl-3-nonyldocos-13,16-dien-1-amine (Compound 32), e.g., as described in Example 11 of WO 2019 / 051289A9 (which is incorporated herein by reference in its entirety).

[1173] In one embodiment, the additional ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO 2019 / 051289A9 (which is incorporated herein by reference in its entirety).

[1174] Examples of additional ionizable lipids that can be used in the lipid composition include the ionizable lipids listed in Table 1 of WO 2019 / 051289, which is incorporated herein by reference.

[1175] Additional lipid components

[1176] Some non-limiting examples of additional lipid compounds that can be used (e.g., in combination with the ionizable lipid compounds and other lipid components described herein) to form a lipid composition include:

[1177]

[1178]

[1179] In some embodiments, the lipid composition further comprises a lipid of formula (i), (ii), (iii), (iv), (v), (vi), (vii), (viii) or (ix).

[1180] In some embodiments, the lipid composition further comprises the following compounds having the following structures:

[1181]

[1182] Wherein:

[1183] X 1 is O, NR 1 or a direct bond, X 2 is C 2-5 alkylene, and X 3 is C(=O) or a direct bond;

[1184] R 1 is H or Me, R 3 is C 1-3 alkyl, R 2 is C 1-3 alkyl, or R 2 together with the nitrogen atom to which it is attached and 1 - 3 carbon atoms of X 2 forms a 4 - membered, 5 - membered or 6 - membered ring; or

[1185] X 1 is NR 1 , R 1 and R 2 together with the nitrogen atom to which it is attached form a 5 - membered or 6 - membered ring, or R 2 together with R 3 and the nitrogen atom to which it is attached form a 5 - membered, 6 - membered or 7 - membered ring;

[1186] Y 1 is C 2-12 alkylene, and Y 2 is selected from

[1187]

[1188] (in any orientation), (in any orientation), (in any orientation),

[1189] n is from 0 to 3;

[1190] R 4 is C 1-15 alkyl;

[1191] Z 1 is C 1-6 alkylene or a direct bond, and Z2 is (in any orientation) or absent, provided that if Z 1 is a direct bond, then Z 2 is absent;

[1192] R 5 is C 5-9 alkyl or C 6-10 alkoxy, R 6 is C 5-9 alkyl or C 6-10 alkoxy;

[1193] W is methylene or a direct bond; and

[1194] R 7 is H or Me or a salt thereof;

[1195] provided that, if R 3 and R 2 are C2 alkyl, X 1 is O, X 2 is a straight-chain C3 alkylene, X 3 is C(=O), Y 1 is a straight-chain C5 alkylene, (Y 2 )n-R 4 is R 4 is a straight-chain C5 alkyl, Z 1 is a C2 alkylene, Z 2 is absent, W is methylene and R 7 is H, then R 5 and R 6 are not C2 alkoxy.

[1196] In some embodiments, the lipid composition further comprises one or more compounds of formula (x).

[1197] Additional non-limiting examples of lipid compounds that can be further included in the lipid composition further include (e.g., in combination with the lipid compounds and other lipid components described herein):

[1198]

[1199]

[1200]

[1201] In some embodiments, the lipid composition further comprises one or more compounds of formula (xi), (xii), (xiii), (xiv), (xv), (xvi), (xvii), (xviii) (e.g., (xviii)a, (xviii)b) or (xix).

[1202] In some embodiments, the lipid composition further comprises a lipid formed by one of the following reactions:

[1203]

[1204] In some embodiments, the lipid composition further comprises a lipid having formula (xxi) (e.g., in combination with the lipid compounds and other lipid components described herein): Wherein:

[1205] Each n is independently an integer from 2 to 15;

[1206] L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3;

[1207] R1 and R3 are each independently a straight-chain or branched-chain C9-C optionally substituted with one or more substituents selected from the group consisting of: 20 alkyl or C9-C 20 alkenyl: oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocycloalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclic)(alkyl)aminoalkyl, heterocyclic, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkoxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocycloalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocycliccarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl and alkylsulfonylalkyl; and

[1208] R2 is selected from the group consisting of:

[1209]

[1210] In some embodiments, the lipid composition further comprises one or more compounds of formula (xxi). In some embodiments, the compounds of formula (xxi) include lipids described by WO 2021 / 113777 (e.g., the lipid of formula (1), such as the lipids in Table 1 of WO2021 / 113777), which is incorporated herein by reference in its entirety.

[1211] In some embodiments, the lipid composition further comprises a lipid having formula (xxii) (e.g., in combination with the lipid compounds and other lipid components described herein): Wherein:

[1212] Each n is independently an integer from 1 to 15;

[1213] R1 and R2 are each independently selected from the group consisting of:

[1214]

[1215] R3 is selected from the group consisting of:

[1216]

[1217] In some embodiments, the lipid composition further comprises one or more compounds of formula (xxii). In some embodiments, the compounds of formula (xxii) include lipids described by WO 2021 / 113777 (e.g., the lipid of formula (2), such as the lipids in Table 2 of WO 2021 / 113777), which is incorporated herein by reference in its entirety.

[1218] In some embodiments, the lipid composition further comprises a lipid having formula (xxiii) (e.g., in combination with the lipid compounds and other lipid components described herein):

[1219] Wherein:

[1220] X is selected from -O-, -S-, or -OC(O)-*, where * indicates the point of attachment to R1;

[1221] R1 is selected from the group consisting of:

[1222]

[1223] And

[1224] R2 is selected from the group consisting of:

[1225]

[1226] In some embodiments, the lipid composition further comprises one or more compounds of formula (xxiii). In some embodiments, the compounds of formula (xxiii) include lipids described by WO 2021 / 113777 (e.g., the lipid of formula (3), such as the lipids in Table 3 of WO 2021 / 113777), which is incorporated herein by reference in its entirety.

[1227] Examples of additional lipids that can be used in lipid compositions include, but are not limited to, one or more of the following formulas: X of US2016 / 0311759; I of US 20150376115 or US 2016 / 0376224; I, II, or III of US 2016 / 0151284; I, IA, II, or IIA of US 2017 / 0210967; I-c of US 2015 / 0140070; A of US 2013 / 0178541; I of US2013 / 0303587 or US 2013 / 0123338; I of US 2015 / 0141678; II, III, IV, or V of US 2015 / 0239926; I of US 2017 / 0119904; I or II of WO 2017 / 117528; A of US 2012 / 0149894; A of US 2015 / 0057373; A of WO 2013 / 116126; A of US 2013 / 0090372; A of US 2013 / 0274523; A of US 2013 / 0274504; A of US 2013 / 0053572; A of WO 2013 / 016058; A of WO 2012 / 162210; I of US 2008 / 042973; I, II, III, or IV of US 2012 / 01287670; I or II of US 2014 / 0200257; I, II, or III of US 2015 / 0203446; I or III of US 2015 / 0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US 2014 / 0308304; of US 2013 / 0338210; I, II, III, or IV of WO 2009 / 132131; A of US2012 / 01011478; I or XXXV of US 2012 / 0027796; XIV or XVII of US 2012 / 0058144; of US 2013 / 0323269; I of US 2011 / 0117125; I, II, or III of US 2011 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US 2012 / 0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US 2011 / 0076335; I or II of US 2006 / 008378; I of US 2013 / 0123338; I or X-A-Y-Z of US 2015 / 0064242;XVI, XVII, or XVIII of US 2013 / 0022649; I, II, or III of US 2013 / 0116307; I, II, or III of US 2013 / 0116307; I or II of US 2010 / 0062967; I-X of US 2013 / 0189351; I of US2014 / 0039032; V of US 2018 / 0028664; I of US 2016 / 0317458; I of US 2013 / 0195920; 5, 6, or 10 of US10,221,127; III-3 of WO 2018 / 081480; I-5 or I-8 of WO 2020 / 081938; 18 or 25 of US 9,867,888; A of US 2019 / 0136231; II of WO 2020 / 219876; 1 of US 2012 / 0027803; OF-02 of US 2019 / 0240349; 23 of US 10,086,013; cKK-E12 / A6 of Miao et al. (2020); C12-200 of WO 2010 / 053572; 7C1 of Dahlman et al. (2017); 304-O13 or 503-O13 of Whitehead et al.; TS-P4C2 of US9,708,628; I of WO 2020 / 106946; I of WO 2020 / 106946; (1), (2), (3), or (4) of WO 2021 / 113777; and any one of Table 1-16 of WO 2021 / 113777, all of the documents being incorporated herein by reference in their entirety.;

[1228] In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises 0.1 mol% to 2 mol%, 0.5 mol% to 2 mol%, 1 mol% to 2 mol%, 0.6 mol% to 1.9 mol%, 0.7 mol% to 1.8 mol%, 0.8 mol% to 1.7 mol%, 0.9 mol% to 1.6 mol%, 0.9 mol% to 1.8 mol%, 1 mol% to 1.8 mol%, 1 mol% to 1.7 mol%, 1.2 mol% to 1.8 mol%, 1.2 mol% to 1.7 mol%, 1.3 mol% to 1.6 mol%, or 1.4 mol% to 1.5 mol% (or any portion or range therein) of the total lipid present in the particle. In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises 0 mol% to 20 mol%, 0.5 mol% to 20 mol%, 2 mol% to 20 mol%, 1.5 mol% to 18 mol%, 2 mol% to 15 mol%, 4 mol% to 15 mol%, 2 mol% to 12 mol%, 5 mol% to 12 mol%, or 2 mol% (or any portion or range therein) of the total lipid present in the particle.

[1229] In further embodiments, the lipid conjugate (e.g., PEG-lipid) comprises 4 mol% to 10 mol%, 5 mol% to 10 mol%, 5 mol% to 9 mol%, 5 mol% to 8 mol%, 6 mol% to 9 mol%, 6 mol% to 8 mol%, or 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol% or 10 mol% (or any portion or range therein) of the total lipid present in the particle.

[1230] The percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid particles of the present disclosure is a target amount, and the actual amount of lipid conjugate present in the composition can vary, e.g., ± 2 mol%. One of ordinary skill in the art will appreciate that the concentration of the lipid conjugate can vary depending on the lipid conjugate employed and the rate at which the lipid particle becomes fusogenic.

[1231] By controlling the composition and concentration of the lipid conjugates, the rate at which the lipid conjugates are exchanged from the lipid particles can be controlled, and the rate at which the lipid particles become fusogenic can then be controlled. In addition, other variables including, for example, pH, temperature, or ionic strength can be used to change and / or control the rate at which the lipid particles become fusogenic. After reading this disclosure, other methods that can be used to control the rate at which the lipid particles become fusogenic will become apparent to those skilled in the art. Similarly, by controlling the composition and concentration of the lipid conjugates, the lipid particle size can be controlled.

[1232] In some embodiments, the composition further comprises one or more nucleic acids, ionizable lipids, amphiphiles, phospholipids, cholesterol, and / or PEGylated cholesterol.

[1233] Other components of the LNP composition

[1234] In addition to the components described above, the lipid nanoparticle composition may further include one or more components. For example, the LNP composition may include one or more small hydrophobic molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[1235] The lipid nanoparticle composition may also include one or more permeation enhancer molecules, carbohydrates, polymers, surface modifiers, or other components.

[1236] Suitable carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[1237] Polymers can be used to encapsulate or partially encapsulate the nanoparticle composition. The polymers can be biodegradable and / or biocompatible. Suitable polymers include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.For example, the polymer can include poly(ε-caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyanhydride, polyorthoester, poly(ester amide), polyamide, poly(ester ether), polycarbonate, polyolefin (such as polyethylene and polypropylene), polyalkylene glycol (such as poly(ethylene glycol) (PEG)), poly(ethylene oxide) (PEO), polyalkylene terephthalate (such as poly(ethylene terephthalate)), polyvinyl alcohol (PVA), polyvinyl ether, polyvinyl ester (such as poly(vinyl acetate)), polyvinyl halide (such as poly(vinyl chloride) (PVC)), polyvinyl pyrrolidone (PVP), polysiloxane, polystyrene (PS), polyurethane, derivatized cellulose (such as alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose), polymers of acrylic acid (such as poly((meth)acrylate methyl) (PMMA), poly((meth)acrylate ethyl), poly((meth)acrylate butyl), poly((meth)acrylate isobutyl), poly((meth)acrylate hexyl), poly((meth)acrylate isodecyl), poly((meth)acrylate lauryl), poly((meth)acrylate phenyl), poly(acrylate methyl), poly(acrylate isopropyl), poly(acrylate isobutyl), poly(acrylate octadecyl) and their copolymers and mixtures), poly(dioxanone) and its copolymers, polyhydroxyalkanoate, polypropylene fumarate, polyoxymethylene, poloxamer, poly(oxyamine), poly(ortho)ester, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol.

[1238] Suitable surface modifiers include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, artemisia argyi, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, alfa-dornase, neltenexine, and erdosteine), and DNAse (e.g., rhDNAse). The surface modifier can be placed within the lipid nanoparticle and / or on the surface of the lipid nanoparticle (e.g., by coating, adsorption, covalent attachment, or other methods).

[1239] The lipid nanoparticle composition can also comprise one or more functionalized lipids. For example, the lipid can be functionalized with an alkyne that can undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized in this manner with one or more groups useful for promoting membrane penetration, cell recognition, or imaging. The surface of the lipid nanoparticle can also be conjugated with one or more useful antibodies. Functional groups and conjugates useful for targeted cell delivery, imaging, and membrane penetration are well known in the art.

[1240] The lipid nanoparticle composition can include any substance useful for a pharmaceutical composition. For example, the lipid nanoparticle composition can include one or more pharmaceutically acceptable excipients or auxiliary components such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and other species. Excipients such as waxes, butters, colorants, coating agents, flavoring agents, and fragrances can also be included.

[1241] Suitable diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof. Granulating agents and dispersing agents may be selected from the non-limiting list consisting of: potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, sodium cross-linked carboxymethyl cellulose (cross-linked carboxymethyl cellulose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate Sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[1242] Suitable surfactants and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, glyceryl triacetate monostearate, ethylene glycol distearate, glycerol monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate Polyoxyethylene sorbitan Polyoxyethylene sorbitan monooleate Sorbitan monopalmitate Sorbitan monostearate Sorbitan tristearate Glycerol monooleate, sorbitan monooleate ), polyoxyethylene esters (e.g., polyoxyethylene monostearate Polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyformaldehyde stearate, and ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., ), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether ), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium docusate and / or combinations thereof.

[1243] Suitable binders can be starches (e.g., corn starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., gum arabic, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husk, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate and larch arabinogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohols; and combinations thereof or any other suitable binder.

[1244] Suitable preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, α-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimonium bromide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerol, hexetidine, imidurea, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, parabens, and / or phenethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, β-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimonium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium laureth sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, methyl paraben, NEOLONE TM 、KATHON TM and / or

[1245] Suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[1246] Suitable oils include, but are not limited to, almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, black currant seed oil, borage oil, juniper oil, chamomile oil, rapeseed oil, caraway oil, Brazilian palm oil, castor oil, cinnamon oil, cocoa butter oil, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geraniol oil, gourd oil, grape seed oil, hazelnut oil, hyssop oil, isopropyl myristate, jojoba oil, macadamia oil, lavender oil, lavender oil, lemon oil, litsea cubeba oil, macadamia nut oil, mallow oil, mango seed oil, white meadowfoam seed oil, mink oil, nutmeg oil, olive oil, sweet orange oil, deep sea fish oil (orange roughy oil), palm oil, palm kernel oil, peach kernel oil, peanut oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, sasquana oil, savoury oil, sea buckthorn oil, sesame oil, shea butter, ketone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, tsubaki oil, vetiver oil, walnut oil and wheat germ oil, as well as butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, simethicone, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil and / or combinations thereof.

[1247] In some embodiments, the composition further comprises one or more cryoprotectants. Suitable cryoprotectants include, but are not limited to, polyols (e.g., diols or triols, such as propylene glycol (i.e., 1,2-propylene glycol), 1,3-propylene glycol, glycerol, (+ / -)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol or diethylene glycol), non-detergent sulfobetaines (e.g., NDSB-201 (3-(1-pyrido)-1-propanesulfonate)), osmolytes (e.g., L-proline or trimethylamine N-oxide dihydrate), polymers (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 2k- DMG, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 20000, methoxypolyethylene glycol 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K15), pentaerythritol propoxylate or polypropylene glycol P400), organic solvents (e.g., dimethyl sulfoxide (DMSO) or ethanol), sugars (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol, inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate or D-(+)-glucose monohydrate) or salts (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate or any of their hydrates) or any combination thereof.

[1248] In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the cryoprotectant and / or excipient is sucrose. In some embodiments, the cryoprotectant comprises sodium acetate. In some embodiments, the cryoprotectant and / or excipient is sodium acetate. In some embodiments, the cryoprotectant comprises sucrose and sodium acetate.

[1249] In some embodiments, the composition further comprises one or more buffers. Suitable buffers include but are not limited to citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconolactate, calcium glucoheptonate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium pivalate, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, sulfamate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol and / or combinations thereof.

[1250] In some embodiments, the buffer is an acetate buffer, a citrate buffer, a phosphate buffer, a tris buffer or a combination thereof.

[1251] Pharmaceutical composition

[1252] Another aspect of the present disclosure also provides a pharmaceutical composition comprising a lipid composition as described herein, the lipid composition comprising one or more lipid compounds selected from the ionizable lipid compounds described herein and a pharmaceutically acceptable excipient. The pharmaceutical composition may further comprise a therapeutic agent.

[1253] All of the above-described embodiments and exemplary variables and compounds related to the aspects of lipid compounds discussed in the above aspects are applicable to these aspects of the present invention related to pharmaceutical compositions.

[1254] All of the above-described embodiments related to the aspects of lipid compositions discussed in the above aspects, including various other lipid components, are applicable to these aspects of the present invention related to pharmaceutical compositions.

[1255] In a lipid composition containing a therapeutic agent, the ratio of the total lipid component to the cargo (e.g., the encapsulated therapeutic agent, such as a nucleic acid) can vary according to desire. For example, the ratio of the total lipid component to the cargo (by mass or weight) can be from about 10:1 to about 30:1. In some embodiments, the ratio of the total lipid component to the 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 amounts of the total lipid component and the cargo can be adjusted to provide a desired N / P ratio, e.g., an N / P ratio of 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. Generally, the total lipid content of the lipid composition can be in the range of about 5 mg / ml to about 30 mg / mL.

[1256] Therapeutic agent

[1257] Nucleic acid molecules

[1258] In some embodiments, the composition further comprises one or more nucleic acid components. The nucleic acid molecule can be a plasmid, an immunostimulatory oligonucleotide, an antisense oligonucleotide, an antagomir, an aptamer, a deoxyribozyme (DNAzyme), and a ribozyme.

[1259] In some embodiments, the composition further comprises one or more RNA and / or DNA components.

[1260] In some embodiments, the composition further comprises one or more DNA components. In some embodiments, the DNA is linear DNA, circular DNA, single-stranded DNA, or double-stranded DNA.

[1261] In some embodiments, the composition further comprises one or more RNA components. In some embodiments, the RNA is mRNA, miRNA, siRNA, 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.

[1262] In some embodiments, one or more RNA components are selected from mRNA. In some embodiments, the mRNA is modified mRNA.

[1263] In some embodiments, the nucleic acid molecule is an enzymatic nucleic acid molecule. The term "enzymatic nucleic acid molecule" refers to a nucleic acid molecule that has complementarity to a designated gene target in a substrate-binding region and also has enzymatic activity that is active in specifically cleaving the target RNA. That is, the enzymatic nucleic acid molecule is capable of cleaving RNA intermolecularly and thereby inactivating the target RNA molecule. The term enzymatic nucleic acid can be used interchangeably with phrases such as ribozyme, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-bound ribozyme, regulatable ribozyme, catalytic oligonucleotide, nucleozyme, DNA enzyme, RNA enzyme, endoribonuclease, endonuclease, small ribozyme, leadzyme, oligonucleotidase, or DNA enzyme. All of these terms describe nucleic acid molecules having enzymatic activity.

[1264] In some embodiments, the nucleic acid molecule 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 or RNA-DNA or RNA-PNA (peptide nucleic acid) interactions and alters the activity of the target RNA.

[1265] 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 5'-phosphorylated 2'-5'-linked adenylic acid residues.

[1266] 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 helix.

[1267] 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 inhibit or activate a target molecule.

[1268] In some embodiments, the nucleic acid molecule (e.g., RNA or DNA) encodes a therapeutic peptide or polypeptide that is operably linked to a promoter of the DNA. The therapeutic peptide or polypeptide can be, for example, a transcription factor; a chromatin remodeling factor; an antigen; a hormone; an enzyme (such as 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-linked enzyme, such as a base editor or a prime editor; a mobile genetic element protein (e.g., a transposase, a retrotransposase, a recombinase, an integrase); a GeneWriter; 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 blood coagulation factor; a membrane protein; a mitochondrial protein; a nuclear protein; an antibody or other protein scaffold binder, such as centyrin, darpin or adnectin.

[1269] In some embodiments, one or more RNA components comprise a gRNA nucleic acid. In some embodiments, the gRNA nucleic acid is a gRNA.

[1270] In some embodiments, one or more RNA components comprise a type II Cas nuclease mRNA and a gRNA. In some embodiments, the gRNA nucleic acid is or encodes a dual guide RNA (dgRNA). In some embodiments, the gRNA nucleic acid is or encodes a single guide RNA (sgRNA). In some embodiments, the gRNA is a modified gRNA. In some embodiments, the modified gRNA comprises a modification at one or more nucleotides among the first five nucleotides at the 5' end. In some embodiments, the modified gRNA comprises a modification at one or more nucleotides among the last five nucleotides at the 3' end.

[1271] In some embodiments, one or more RNA components comprise an mRNA. In some embodiments, one or more RNA components comprise an RNA-guided DNA binder, such as a Cas nuclease mRNA (such as a type II Cas nuclease mRNA) or a Cas9 nuclease mRNA.

[1272] All nucleic acid molecules described herein can be chemically modified. Various modification strategies for nucleic acid molecules are well known to those skilled in the art. In some embodiments, the nucleic acid molecule comprises 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, glycerol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophore (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotide, biotinylated nucleotide, fluorescent base analog, CpG island, methyl-7-guanosine, methylated nucleotide, inosine, thiouridine, pseudouridine, dihydrouridine, wybutosine, 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 building blocks, in which an additional methylene bridge fixes the ribose moiety in the C3'-endo (β-D-LNA) or C2'-endo (α-L-LNA) conformation (Grunweller A, Hartmann R K, BioDrugs, 21(4):235-243 (2007)).

[1273] In some embodiments, the composition further comprises one or more template nucleic acids.

[1274] Additional examples of nucleic acid molecules (including tumor suppressor genes, antisense oligonucleotides, siRNAs, miRNAs, or shRNAs) can be found in U.S. Published Patent Application No. 2007 / 0065499 and U.S. Patent No. 7,780,882, which are incorporated herein by reference in their entirety.

[1275] In some embodiments, the pharmaceutical composition can include multiple nucleic acid molecules that can be of the same or different types.

[1276] The nucleic acids used in conjunction with the embodiments of the present disclosure can be prepared according to any available technology. For mRNA, the main methods of preparation are, but are not limited to, enzymatic synthesis (also known as in vitro transcription), which currently represents the most efficient method for generating long sequence-specific mRNA. In vitro transcription describes a method for template-directed synthesis of RNA molecules from engineered DNA templates containing an upstream phage promoter sequence (e.g., including but not limited to sequences from T7, T3, and SP6 Escherichia coli phages) linked to a downstream sequence encoding the gene of interest. The template DNA for in vitro transcription can be prepared from many sources using suitable techniques well known in the art, including but not limited to plasmid DNA and polymerase chain reaction amplification (see Linpinsel, J.L and Conn, G.L., General protocols for preparation of plasmid DNA template and Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P. and Williams, L.D. RNA in vitro transcription and RNA purification by denaturing PAGE, Recombinant and in vitro RNA syntheses Methods, Volume 941, Conn G.L. (ed.), Humana Press, New York, N.Y., 2012).

[1277] RNA transcription is carried out in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and ribonucleoside triphosphates (rNTPs) of adenosine, guanosine, uridine, and cytidine under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using a variety of commercially available kits as well as commercially available reagents, the commercially available kits including, but not limited to, the RiboMax Large Scale RNA Production System (Promega), the MegaScript Transcription Kit (Life Technologies), and the reagents including RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are well known in the art. (See, for example, Losick, R., 1972, In vitro transcription, Ann Rev Biochem 41:409-46; Kamakaka, R.T. and Kraus, W.L. 2001. In vitro transcription. Current Protocols in Cell Biology 2:11.6:11.6.1-11.6.17; Beckert, B. and Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA, Methods in Molecular Biology, Vol. 703 (edited by Neilson, H.), Humana Press, New York, NY, 2010; Brunelle, J.L. and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology, Vol. 530, 101-114; all of the documents in the literature are incorporated herein by reference).

[1278] The desired in vitro transcribed mRNA can be purified from unwanted components of the transcription or related reactions, including unincorporated rNTPs, proteases, salts, short RNA oligonucleotides, etc. Techniques for isolating mRNA transcripts are well known in the art. For non-limiting examples, well-known procedures include extraction or precipitation with alcohol (ethanol, isopropanol) or phenol / chloroform in the presence of monovalent cations or lithium chloride.

[1279] Additional non-limiting examples of purification procedures that can be used include size exclusion chromatography (Lukavsky, P.J. and Puglisi, J.D., 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA, Vol. 10, 889-893); silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P. and Williams, L.D. In vitro transcription of RNA and purification of RNA by denaturing PAGE, Methods in Recombinant and In Vitro RNA Synthesis, Vol. 941, Conn G.L. (ed.), Humana Press, New York, NY, 2012). Purification can be carried out using a variety of commercially available kits, including but not limited to the SV Total Isolation System (Promega Corporation) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[1280] In addition, although reverse transcription can generate large amounts of mRNA, the product can contain many aberrant RNA impurities associated with unwanted polymerase activities that may need to be removed from the full-length mRNA preparation. These include short RNAs generated by abortive transcription initiation, double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from an RNA template, and self-complementary 3' extensions. It has been demonstrated that these contaminants with dsRNA structures can generate unwanted immunostimulatory activities by interacting with various innate immune sensors in eukaryotic cells, which are used to recognize specific nucleic acid structures and induce an effective immune response. This in turn can significantly reduce mRNA translation, as protein synthesis is reduced during the innate cellular immune response. Thus, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including but not limited to scalable HPLC purification (see, e.g., Kariko, K., Muramatsu, H., Ludwig, J. and Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucleic Acids Research, Vol. 39, e142; Weissman, D., Pardi, N., Muramatsu, H. and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation, Methods in Molecular Biology, Vol. 969 (edited by Rabinovich, P. H.), 2013). It has been reported that HPLC-purified mRNA translates at much higher levels, particularly in primary cells and in vivo.

[1281] A large variety of modifications have been described in the art that are used to alter specific properties of in vitro transcribed mRNA and can improve its utility. These include, but are not limited to, modifications to the 5' and 3' ends of the mRNA. Endogenous eukaryotic mRNA typically contains a cap structure on the 5' end of the mature molecule, which plays an important role in mediating the binding of mRNA cap-binding proteins (CBPs), which in turn is responsible for enhancing mRNA stability and the efficiency of mRNA translation in cells. Thus, the highest levels of protein expression are achieved with capped mRNA transcripts. The 5'-cap contains a 5'-5'-triphosphate bond between the most 5' nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the last and penultimate most 5' nucleotides at the 2'-hydroxy group.

[1282] Multiple different cap structures can be used to generate the 5'-cap of in vitro transcribed synthetic mRNA. 5'-capping of synthetic mRNA can be performed co-transcriptionally with a chemical cap analogue (i.e., capping during in vitro transcription). For example, the anti-reverse cap analogue (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine bond, where one guanine contains an N7 methyl and a 3'-O-methyl. However, during this co-transcriptional process, up to 20% of the transcripts remain uncapped, and the synthetic cap analogue is different from the 5'-cap structure of authentic cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped post-transcriptionally. These can generate a more authentic 5'-cap structure that more closely mimics the endogenous 5'-cap in structure or function (with enhanced binding of cap-binding proteins, increased half-life, and reduced sensitivity to 5' endonucleases and / or reduced 5' decapping). Many synthetic 5' cap analogues have been developed and are known in the art for enhancing mRNA stability and translatability (see, for example, Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, A. N., Slepenkov, S. V., Darynkiewicz, E., Sahin, U., Jemielity, J. and Rhoads, R. E., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation, Methods in Molecular Biology, Vol. 969 (edited by Rabinovich, P. H.), 2013).

[1283] During RNA processing, at the 3' end, a long chain of adenine nucleotides (poly-A tail) is typically added to the mRNA molecule. The 3' end of the transcript is immediately cleaved after transcription to release a 3' hydroxyl, and poly-A polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. The poly-A tail has been widely shown to enhance both the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A) binding protein and the regulation of mRNA stability, Trends Bio Sci, Vol. 14, 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, Vol. 265, 11-23; Dreyfus, M. and Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, Vol. 111, 611-613).

[1284] Poly(A) tailing of in vitro transcribed mRNA can be achieved using various methods, including but not limited to cloning a poly(T) tract into the DNA template or by post-transcriptional addition using a Poly(A) polymerase. The first case allows for in vitro transcription of mRNA with a defined length of poly(A) tail, depending on the size of the poly(T) tract, but requires additional manipulation of the template. The latter case involves enzymatic addition of a poly(A) tail to in vitro transcribed mRNA using a poly(A) polymerase, which catalyzes the incorporation of adenine residues onto the 3' end of the RNA, does not require additional manipulation of the DNA template, but results in mRNA with poly(A) tails of different lengths. 5'-capping and 3'-poly(A) tailing can be performed using various commercially available kits (including but not limited to Poly(A) Polymerase Tailing Kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra Kit, and Poly(A) Tailing Kit (Life Technologies)) as well as commercially available reagents, various ARCA caps, Poly(A) polymerases, etc.

[1285] In addition to 5' capping and 3' polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits related to translation efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors within eukaryotes and trigger an effective innate immune response. The ability to distinguish pathogenic and self DNA and RNA has been shown to be at least partially based on structure and nucleoside modifications, as most nucleic acids from natural sources contain modified nucleosides. In contrast, in vitro synthesized RNA lacks these modifications, thus making it immunostimulatory, which in turn can inhibit efficient mRNA translation as outlined above.The introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent the recognition and activation of RNA sensors, thereby reducing this undesired immunostimulatory activity and enhancing translational capacity (see, e.g., Kariko, K. and Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discov Devel, Vol. 10, 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation, Methods in Molecular Biology, Vol. 969 (edited by Rabinovich, P.H.), 2013; Kariko, K., Muramatsu, H., Welsh, F.A., Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008, Incorporation of Pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability, Mol Ther, Vol. 16, 1833-1840). The modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored, and utilized using common methods and procedures known in the art. A large variety of nucleoside modifications are available, and the large variety of nucleoside modifications can be incorporated into in vitro transcribed mRNA either alone or to some extent in combination with other modified nucleosides (see, e.g., US2012 / 0251618).It has been reported that in vitro synthesis of nucleoside-modified mRNA has a reduced ability to activate immune sensors, along with enhanced translation ability.

[1286] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of the UTRs (good 5' and 3' UTRs can be obtained from cellular or viral RNA), either both or independently, has been shown to increase mRNA stability and the translation efficiency of in vitro transcribed mRNA (see, for example, Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In Vitro Transcription and Cellular Metabolism Regulation of Long RNAs Containing Modified Nucleosides in Synthetic Messenger RNAs, Methods in Molecular Biology, Vol. 969 (Rabinovich, P. H. Ed), 2013).

[1287] In addition to mRNA, other nucleic acid payloads can be used in the present disclosure. For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis and enzymatic chemical cleavage of longer precursors, such as in vitro transcription as described above. Methods for synthesizing DNA and RNA nucleotides are widely used and well-known in the art (see, for example, Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, IRL Press, Oxford [Oxfordshire], Ishington, D.C., 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, Vol. 288, Humana Press, Totowa, N.J., Clifton, N.J., 2005; both of which are incorporated herein by reference).

[1288] For plasmid DNA, the preparations used in conjunction with the examples of the present disclosure generally utilize, but are not limited to, in vitro amplification and isolation of plasmid DNA in a liquid culture of bacteria containing the plasmid of interest. The presence of genes encoding resistance to specific antibiotics (penicillin, kanamycin, etc.) in the plasmid of interest allows those bacteria containing the plasmid of interest to selectively grow in a culture containing the antibiotic. Methods for isolating plasmid DNA are widely used and well-known in the art (see, for example, Heilig, J., Elbing, K. L., and Brent, R., (2001), Large-Scale Preparation of Plasmid DNA, Current Protocols in Molecular Biology, 41:11:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstrom, S., Bjornestedt, R., and Schmidt, S. R., (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture, Biotechnol. Bioeng., 99:557-566; and US 6,197,553 B1). Plasmid isolation can be performed using a variety of commercially available kits (including, but not limited to, the Plasmid Plus (Qiagen), GenJET Plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits) and commercially available reagents.

[1289] In some embodiments, the lipid nanoparticle composition can be used to express a protein encoded by mRNA. In some embodiments, methods for expressing a protein encoded by mRNA are provided herein.

[1290] In some embodiments, the N / P ratio of the LNP composition is from about 1:1 to about 30:1, such as from about 3:1 to about 20:1, from about 3:1 to about 15:1, from about 3:1 to about 10:1, or from about 3:1 to about 6:1. For example, the N / P ratio of the lipid composition encapsulated with nucleic acid molecules can be about 6 ± 1, or the N / P ratio of the lipid composition encapsulated with nucleic acid molecules can be about 6 ± 0.5. In some embodiments, the N / P ratio of the lipid composition encapsulated with nucleic acid molecules is in the range of from about 3:1 to about 15:1. In some embodiments, the N / P ratio of the lipid composition encapsulated with nucleic acid molecules is about 6. The N:P ratio refers to the molar ratio of the amine (such as the amine in the ionizable lipid) present in the lipid composition or lipid nanoparticle formulation to the phosphate ester present in the nucleic acid molecule. This is a factor for effective packaging and potency.

[1291] Other therapeutic agents

[1292] The therapeutic agent can be a peptide or protein encapsulated in the lipid composition, a small molecule drug. The pharmaceutical composition can contain two or more therapeutic agents different from the nucleic acid molecule, peptide or protein, and small molecule drug.

[1293] 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 (such as a nuclease, such as an endonuclease, such as the nuclease element of the CRISPR system, such as Cas9, dCas9, aCas9-nickase, Cpf / Cas12a); a Crispr-linked enzyme, such as a base editor or a primer editor; a mobile genetic element protein (such as a transposase, a retrotransposase, a recombinase, an 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 blood coagulation factor; a membrane protein; a mitochondrial protein; a nuclear protein; an antibody or other protein scaffold binder, such as centyrin, darpin or adnectin.

[1294] In some embodiments, the pharmaceutical composition can include multiple protein molecules that can be of the same or different types.

[1295] In some embodiments, the therapeutic agent is a small molecule drug, such as a small molecule drug approved for human use by the appropriate regulatory agency.

[1296] In some embodiments, the pharmaceutical composition can include multiple small molecule drugs that can be of the same or different types.

[1297] In some embodiments, the therapeutic agent is a vaccine. In some embodiments, the vaccine is an RNA vaccine, such as an RNA cancer vaccine or an RNA vaccine for an infectious disease (e.g., an influenza virus vaccine or a coronavirus vaccine (e.g., a COVID-19 vaccine)).

[1298] Other ingredients

[1299] The pharmaceutical composition may contain one or more pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients are selected according to the mode and route of administration. Suitable pharmaceutical carriers or excipients for pharmaceutical formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, edited by Gennaro, Lippencott Williams & Wilkins (2005); Handbook of Pharmaceutical Excipients, 6th Edition, edited by Rowe et al., Pharmaceutical Press (2009); and USP / NF (United States Pharmacopeia and the National Formulary), the disclosures of which are incorporated herein by reference in their entirety.

[1300] In some embodiments, the pharmaceutically acceptable excipients include one or more of the following: antioxidants, binders, anti-adhesives, buffers, colorants, diluents (e.g., solid or liquid), disintegrants (e.g., coating disintegration), dispersants, dyes, fillers, emulsifiers, flavoring agents, lubricants, pH regulators, pigments, preservatives, stabilizers, solubilizers, solvents, suspending agents, sweeteners, or wetting agents or combinations thereof.

[1301] Examples of suitable excipients include, but are not limited to, acacia, alginate, calcium phosphate, calcium carbonate, calcium silicate, carbopol gel, carboxymethyl cellulose, carnauba wax, cellulose, crospovidone, dextran, diacetylated monoglyceride, ethyl cellulose, gelatin, glyceryl monostearate 40 - 50, gum acacia, gum arabic, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hypromellose phthalate, hypromellose, lactose, lecithin, magnesium stearate, kaolin, methacrylic acid copolymer type C, mannitol, methyl cellulose, methyl hydroxybenzoate, microcrystalline cellulose, povidone, polyethylene glycol, polysorbate 80, polyvinylpyrrolidone, propyl hydroxybenzoate, sodium carboxymethyl cellulose, sodium hydroxide, sodium stearoyl fumarate, sodium starch glycolate, starch, sorbitan monooleate, sorbitol, sorbic acid, sucrose, talc, tragacanth, talc, triethyl citrate, titanium dioxide, yellow iron oxide, talc, an oil medium (e.g., peanut oil, liquid paraffin, mineral oil, olive oil, almond oil, glycerol, propylene glycol) or water,

[1302] When the excipient serves as a diluent, it can be a solid, semi - solid or liquid material (e.g., normal saline), which serves as a vehicle, carrier or medium for the active ingredient. As is known in the art, the type of diluent can vary according to the intended route of administration.

[1303] The pharmaceutical composition can contain a pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized formulation or an aqueous solution. The acceptable carrier, excipient or stabilizer is non - toxic to the recipient at the dosages and concentrations employed and can include: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl esters of p - hydroxybenzoic acid such as methyl or propyl p - hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3 - pentanol; and m - cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including 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 TM 、PLURONICS TM or polyethylene glycol (PEG).

[1304] Suitable carriers or excipients for the pharmaceutical composition may also include substances that enhance the ability of an individual's body to absorb the LNP or liposome. Suitable carriers and / or excipients also include any substances that can be used to fill the formulation with the LNP or liposome to make the dosage convenient and accurate. In addition, carriers and / or excipients can be used during the manufacturing process to assist in handling the LNP or liposome. Different carriers and / or excipients can be used depending on the route of administration and the pharmaceutical form.

[1305] Carriers and / or excipients may also include vehicles and / or diluents. "Vehicle" refers to any of a variety of media that generally serve as a solvent or carrier; "diluent" refers to a diluent used to dilute the active ingredient of the composition; suitable diluents include any substances that can reduce the viscosity of the drug. The type and amount of the carrier and / or excipient are selected according to the selected pharmaceutical form; suitable pharmaceutical forms are liquid systems such as solutions, infusions, suspensions; semi-solid systems such as colloids, gels, pastes or creams; solid systems such as powders, granules, tablets, capsules, pellets, microparticles, mini-tablets, microcapsules, micro-pellets, suppositories, etc.

[1306] Using techniques well known in the art, each of the above systems can be appropriately formulated for normal, delayed or accelerated release.

[1307] Formulations, dosages, and routes of administration

[1308] The pharmaceutical compositions described herein can be prepared according to standard techniques as well as those described herein. For example, the pharmaceutical composition can be prepared in a conventional manner, e.g., by conventional mixing, dissolving, granulating, sugar coating, milling, emulsifying, encapsulating, entrapping or lyophilization methods. Methods for preparing formulations well known in the art are known in the art. See, for example, Remington: The Science and Practice of Pharmacy, 21st Edition, edited by Gennaro, Lippincott Williams & Wilkins Publishers (2005), and Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick and J. C. Boylan, 1988 - 1999, Marcel Dekker, New York.

[1309] Therapeutic agents can be encapsulated in lipid compositions. For example, the therapeutic agent can be located entirely or partially within the internal 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 an environment that may contain enzymes or chemicals or conditions that degrade the therapeutic agent and / or systems or receptors that cause rapid excretion of the therapeutic agent. In addition, incorporating the therapeutic agent into the LNP can facilitate uptake of the therapeutic agent and thus enhance the therapeutic effect.

[1310] In some embodiments, in the pharmaceutical composition, the ratio of the lipid component to the therapeutic agent (mass / mass ratio; w / w ratio) can be in the range of about 1:1 to about 25:1, 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.

[1311] Based on the weight of the lipid composition or the pharmaceutical composition, the lipid composition or the pharmaceutical composition can contain about 5 wt% to about 95 wt% of the therapeutic agent. In some embodiments, based on the weight of the LNP or the pharmaceutical composition, the lipid composition or the pharmaceutical composition contains about 5 wt%, about 10 wt%, about 20 wt%, about 30 wt%, about 40 wt%, about 50 wt%, about 60 wt%, about 70 wt%, about 80 wt%, about 90 wt%, or about 95 wt% of the therapeutic agent. In some embodiments, based on the weight of the lipid composition or the pharmaceutical composition, the lipid composition or the pharmaceutical composition contains the following amounts of the therapeutic agent: 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 20-40%, 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%.

[1312] Based on the weight of the lipid composition or pharmaceutical composition, the lipid composition or pharmaceutical composition can contain a total lipid in an amount of about 5 wt% to about 95 wt%. In some embodiments, based on the weight of the lipid composition or pharmaceutical composition, the lipid composition or pharmaceutical composition contains the following amounts of total lipid: 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 - 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%.

[1313] The compositions of the present disclosure can be administered by various routes, for example, systemic delivery can be achieved by intravenous, parenteral, intraperitoneal or topical routes. In some embodiments, siRNA can be delivered intracellularly, for example, in cells of a target tissue such as the lung or liver or in an inflamed tissue. In some embodiments, the present disclosure provides methods for in vivo delivery of siRNA. The nucleic acid-lipid composition can be administered intravenously, subcutaneously or intraperitoneally to a subject.

[1314] The compositions and methods of the present disclosure can be administered to a subject by a variety of mucosal administration modalities, including by oral, rectal, vaginal, intranasal, intrapulmonary or transdermal or dermal delivery, or by topical delivery to the eye, ear, skin or other mucosal surfaces. In some aspects of the present disclosure, the mucosal tissue layer includes an epithelial cell layer. The epithelial cells can be of the lung, trachea, bronchus, alveoli, nose, cheek, epidermis or gastrointestinal tract. The compositions of the present disclosure can be administered using conventional actuators such as mechanical spray devices as well as pressurized, electrically activated or other types of actuators.

[1315] The compositions of the present disclosure can be administered in aqueous solution as a nasal spray or a pulmonary spray and can be dispensed in spray form by a variety of methods known to those skilled in the art. Pulmonary delivery of the compositions of the present disclosure is achieved by administering the composition in the form of drops, particles or sprays, which can be, for example, aerosolized, pulverized or atomized. The particles of the composition, spray or aerosol can be in liquid or solid form. Non-limiting examples of systems for dispensing liquids as nasal sprays are disclosed in U.S. Patent No. 4,511,069. Such formulations can be conveniently prepared by dissolving the composition according to the present disclosure in water to produce an aqueous solution and rendering the solution sterile. The formulation can be present in a multi-dose container, such as the sealed dispensing system disclosed in U.S. Patent No. 4,511,069. Other suitable nasal spray delivery systems are described in TRANSDERMAL SYSTEMIC MEDICATION, edited by Y.W. Chien, Elsevier Publishers, New York, 1985; and U.S. Patent No. 4,778,810. Additional aerosol delivery forms can include, for example, compressed air jets, ultrasonic and piezoelectric nebulizers that deliver bioactive agents dissolved or suspended in a pharmaceutical solvent such as water, ethanol or mixtures thereof.

[1316] The nasal and pulmonary spray solutions of the present disclosure generally contain a drug or drugs to be delivered, optionally formulated with a surfactant such as a non-ionic surfactant (e.g., polysorbate-80) and one or more buffers. In some embodiments of the present disclosure, the nasal spray solution further contains a propellant. The pH of the nasal spray solution can be pH 6.8 to 7.2. The pharmaceutical solvent employed can also be a slightly acidic aqueous buffer of pH 4 - 6. Other components can be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants or gases.

[1317] In some embodiments, the present disclosure is a pharmaceutical product that includes a solution containing the composition of the present disclosure and an actuator for pulmonary, mucosal or intranasal spray or aerosol.

[1318] The dosage form of the compositions of the present disclosure can be liquid, in the form of droplets or emulsions or in the form of an aerosol.

[1319] The dosage form of the compositions of the present disclosure can be solid, which can be reconstituted in a liquid prior to administration. The solid can be administered in powder form. The solid can be in the form of a capsule, tablet or gel.

[1320] To prepare a composition for pulmonary delivery within the present disclosure, the bioactive agent can be combined with various pharmaceutically acceptable additives and a substrate or carrier for dispersing the active agent.

[1321] Examples of additives include pH control agents such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid and mixtures thereof. Other additives include local anesthetics (e.g., benzyl alcohol), isotonic agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancers (e.g., cyclodextrins and their derivatives), stabilizers (e.g., serum albumin) and reducing agents (e.g., glutathione). When the composition for mucosal delivery is a liquid, the tonicity of the composition is typically adjusted to a value that will not cause substantial, irreversible tissue damage in the mucosa at the site of administration, as measured with reference to the tonicity of a 0.9% (w / v) saline solution taken as a unity. Generally, the tonicity of the solution is adjusted to a value between 1 / 3 and 3, more typically between 1 / 2 and 2, and most typically between 3 / 4 and 1.7.

[1322] The bioactive agent can be dispersed in a substrate or vehicle, which can comprise a hydrophilic compound having the ability to disperse the active agent and any desired additives. The substrate can be selected from a wide range of suitable carriers, including but not limited to polycarboxylic acids or their salts, copolymers of carboxylic anhydrides (e.g., maleic anhydride) with other monomers (e.g., (meth)acrylate, acrylic acid, etc.); hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone; cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, etc.; and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid and their non-toxic metal salts. Generally, biodegradable polymers are selected as the substrate or carrier, such as polylactic acid, poly(lactic-co-glycolic) copolymer, polyhydroxybutyrate, poly(hydroxybutyrate-co-glycolate) copolymer and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglyceryl fatty acid esters, sucrose fatty acid esters, etc. can be used as carriers. The hydrophilic polymers and other carriers can be used alone or in combination, and the carrier can be given enhanced structural integrity by partial crystallization, ionic bonding, crosslinking, etc. The carrier can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres and membranes for direct application to the nasal mucosa. The use of the selected carrier in this context can facilitate the absorption of the bioactive agent.

[1323] Compositions for mucosal, nasal, or pulmonary delivery can contain hydrophilic low molecular weight compounds as a base or excipient. Such hydrophilic low molecular weight compounds can provide a channel medium through which water-soluble active agents, such as bioactive peptides or proteins, can diffuse through the base to the body surface where the active agent is absorbed. The hydrophilic low molecular weight compounds can optionally absorb moisture from the mucosa or the application atmosphere and can dissolve water-soluble active peptides. In some embodiments, the hydrophilic low molecular weight compound has a molecular weight less than or equal to 10,000, such as not exceeding 3,000. Examples of hydrophilic low molecular weight compounds include polyol compounds, such as oligosaccharides, disaccharides, and monosaccharides, including sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentiobiose, glycerol, polyethylene glycol, and mixtures thereof. Additional examples of hydrophilic low molecular weight compounds include N-methylpyrrolidone, alcohols (e.g., low molecular weight polyvinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.), and mixtures thereof.

[1324] The compositions of the present disclosure can alternatively contain pharmaceutically acceptable carrier substances required for approximate physiological conditions, such as pH regulators and buffers, tonicity regulators, and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof. For solid compositions, conventional non-toxic pharmaceutically acceptable carriers can be used, which include, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc.

[1325] In certain embodiments of the present disclosure, the bioactive agent can be administered in a sustained release formulation, for example, in a composition comprising a slow release polymer. The active agent can be prepared with a carrier that will prevent rapid release (e.g., a controlled release vehicle, such as a polymer, a microencapsulated delivery system, or a bioadhesive gel). In the various compositions of the present disclosure, extended delivery of the active agent can be achieved by including it in a composition formulation that delays absorption (e.g., aluminum monostearate hydrogel and gelatin).

[1326] In some embodiments, the lipid composition, pharmaceutical composition, or dosage unit contains from about 0.01 mg to about 1000 mg of one or more of the lipid compounds described herein. In some embodiments, the lipid composition, pharmaceutical composition, or dosage unit contains about 0.01 mg, about 0.1 mg, about 0.5 mg, about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, 250 mg, about 275 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, or about 1000 mg of one or more of the lipid compounds described herein.In some embodiments, the lipid composition, 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 50 mg, from about 0.1 to about 25, from about 0.1 to about 10 mg, from about 0.1 to about 5 mg, from about 0.1 to about 1 mg, from about 1 to about 1000 mg, from about 1 to about 750 mg, from about 1 to about 500 mg, from about 1 to about 250 mg, from about 1 to about 100 mg, from about 1 to about 50 mg, from about 1 to about 25 mg, from about 1 to about 10 mg, from about 1 to about 5 mg, from about 5 to about 1000 mg, from about 5 to about 750 mg, from about 5 to about 500 mg, from about 5 to about 250 mg, from about 5 to about 100 mg, from about 5 to about 50 mg, from about 5 to about 25 mg, from about 5 to about 10 mg, from about 10 to about 1000 mg, from about 10 to about 750 mg, from about 10 to about 500, from about 10 to about 250 mg, from about 10 to about 100 mg, from about 10 to about 50 mg, from about 10 to about 25 mg, from about 25 to about 1000 mg, from about 25 to about 750 mg, from about 25 to about 500 mg, from about 25 to about 250 mg, from about 25 to about 100 mg, from about 25 to about 50 mg, from about 50 to about 1000 mg, from about 50 to about 750 mg, from about 50 to about 500 mg, from about 50 to about 250 mg, from about 50 to about 100 mg, from about 100 to about 1000 mg, from about 100 to about 750 mg, from about 100 to about 500 mg, from about 100 to about 250 mg, from about 250 to about 1000 mg, from about 250 to about 750 mg, from about 250 to about 500 mg, from about 500 to about 1000 mg, from about 500 to about 750 mg or from about 750 to about 1000 mg of one or more of the lipid compounds described herein.

[1327] Method of using a lipid composition

[1328] Another aspect of the present disclosure provides a method for delivering a therapeutic agent to a subject (e.g., a patient) in need thereof, the method comprising administering to the subject (e.g., a patient) a pharmaceutical composition comprising a lipid nanoparticle composition comprising an ionizable lipid compound described herein, a pharmaceutically acceptable salt thereof, and / or a stereoisomer of any of the foregoing and a therapeutic agent.

[1329] In some embodiments, provided herein is a method of delivering a therapeutic cargo to at least one organ selected from the pancreas, one or both lungs, and the spleen of a subject in need thereof, wherein a minimal amount is delivered to other parts of the subject's body (such as the liver). In some embodiments, the method delivers the therapeutic cargo to the pancreas and / or one or both lungs of a subject in need thereof, wherein a minimal amount is delivered to other parts of the subject's body (such as the liver).

[1330] In some embodiments, less than 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the total therapeutic cargo administered to the subject is delivered to the liver of the subject. In some embodiments, less than 6%, 7%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total therapeutic cargo administered to the subject is delivered to the liver of the subject.

[1331] In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the total therapeutic cargo administered to the subject is delivered to the pancreas, spleen, and / or one or both lungs of the subject. In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the total therapeutic cargo administered to the subject is delivered to the pancreas of the subject. In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the total therapeutic cargo administered to the subject is delivered to the lungs of the subject. In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the total therapeutic cargo administered to the subject is delivered to the spleen of the subject.

[1332] In some embodiments, the spleen-to-liver ratio of the total therapeutic cargo administered to the subject is at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the spleen-to-liver ratio of the total therapeutic cargo administered to the subject is at least 1. In some embodiments, the spleen-to-liver ratio of the total therapeutic cargo administered to the subject is at least 5.

[1333] As used herein, the percentage amount of the total therapeutic cargo administered to and delivered to a location within the subject is measured by protein expression levels or mRNA knockdown levels.

[1334] In some embodiments, the method of delivering the therapeutic cargo disclosed above comprises administering to the subject a lipid nanoparticle composition comprising the therapeutic cargo. In some embodiments, the lipid nanoparticles in the lipid nanoparticle composition are formed from one or more compounds selected from ionizable lipids of formulas (I)-(VII), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formula (I), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formula (II), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formula (III), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formula (IV), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formula (V), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formula (VI), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formula (VII), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing.

[1335] In some embodiments, the lipid nanoparticles and lipid nanoparticle compositions disclosed herein can be used for a variety of purposes, including delivering encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells in vitro and / or in vivo. Thus, in some embodiments, methods of treating or preventing a disease or disorder in a subject in need thereof are provided, the methods comprising administering a lipid nanoparticle to the subject. In some embodiments, the lipid nanoparticle encapsulates or is associated with a suitable therapeutic agent, wherein the lipid nanoparticle comprises one or more of the novel ionizable lipids described herein, pharmaceutically acceptable salts thereof, and / or stereoisomers of any of the foregoing.

[1336] In some embodiments, the lipid nanoparticles of the present disclosure can be used to deliver therapeutic cargo. In some embodiments, the therapeutic cargo is selected from one or more nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antimiRs), messenger RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), and the like. Thus, in some embodiments, methods of inducing the expression of a desired protein in vitro and / or in vivo by contacting a cell with a lipid nanoparticle comprising one or more of the novel ionizable lipids described herein are disclosed, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that is expressed to produce the desired protein (e.g., messenger RNA or plasmid encoding the desired protein) or inhibits the process of terminating mRNA expression (e.g., miRNA inhibitor). In some embodiments, methods of reducing the expression of a target gene and protein in vitro and / or in vivo by contacting a cell with a lipid nanoparticle comprising one or more of the novel ionizable lipids described herein are disclosed, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that reduces the expression of the target gene (e.g., antisense oligonucleotide or small interfering RNA (siRNA)). In some embodiments, methods for co-delivering one or more nucleic acids (e.g., mRNA and plasmid DNA) alone or in combination are disclosed, such as can be used to provide the effect of co-localization of different nucleic acids (e.g., mRNA encoding a suitable gene-modifying enzyme and a DNA fragment for incorporation into the host genome).

[1337] In some embodiments, the lipid nanoparticle composition can be used to upregulate endogenous protein expression by delivering an miRNA inhibitor that targets a specific miRNA or modulates a group of miRNAs that regulate a target mRNA or a plurality of mRNAs. In some embodiments, methods for upregulating endogenous protein expression are provided, the methods comprising delivering an miRNA inhibitor that targets one or more miRNAs that regulate one or more mRNAs.

[1338] In some embodiments, the lipid nanoparticle composition can be used to downregulate (e.g., silence) the protein level and / or mRNA level of a target gene. In some embodiments, provided herein are methods for downregulating (e.g., silencing) the protein and / or mRNA level of a target gene.

[1339] In some embodiments, the lipid nanoparticles can be used to deliver mRNA and plasmids for transgene expression. In some embodiments, provided herein are methods for delivering mRNA and plasmids for transgene expression.

[1340] In some embodiments, the lipid nanoparticle composition can be used to induce a pharmacological effect resulting from protein expression, e.g., increasing the production of red blood cells by delivering a suitable erythropoietin mRNA, or protecting against infection by delivering an mRNA encoding a suitable antigen or antibody. In some embodiments, provided herein are methods for inducing a pharmacological effect resulting from protein expression, e.g., increasing the production of red blood cells by delivering a suitable erythropoietin mRNA, or protecting against infection by delivering an mRNA encoding a suitable antigen or antibody.

[1341] Non-limiting exemplary embodiments of the ionizable lipids of the present disclosure, lipid nanoparticles and compositions comprising the ionizable lipids, and their use for delivering agents (e.g., therapeutic agents such as nucleic acids) and / or regulating gene and / or protein expression will be described in further detail below.

[1342] In some embodiments, the present disclosure relates to a method of gene editing, the method comprising contacting a cell with an LNP. In some embodiments, the present disclosure relates to any method of gene editing described herein, the method comprising cutting DNA.

[1343] In some embodiments, the present disclosure relates to a method of cutting DNA, the method comprising contacting a cell with an LNP composition.

[1344] In some embodiments, the present disclosure relates to any method of cutting DNA described herein, wherein the cutting step comprises introducing a single-stranded DNA nick. In some embodiments, the present disclosure relates to any method of cutting DNA described herein, wherein the cutting step comprises introducing a double-stranded DNA break. In some embodiments, the present disclosure relates to any method of cutting DNA described herein, wherein the LNP composition comprises a class 2 Cas mRNA and a guide RNA nucleic acid. In some embodiments, the present disclosure relates to any method of cutting DNA described herein, the method further comprising introducing at least one template nucleic acid into the cell. In some embodiments, the present disclosure relates to any method of cutting DNA described herein, the method comprising contacting a cell with an LNP composition comprising a template nucleic acid.

[1345] In some embodiments, the present disclosure relates to any of the gene editing methods described herein, wherein the method comprises administering an LNP composition to an animal (e.g., a human). In some embodiments, the present disclosure relates to any of the gene editing methods described herein, wherein the method comprises administering an LNP composition to a cell (such as a eukaryotic cell).

[1346] In some embodiments, the present disclosure relates to any of the gene editing methods described herein, wherein the method comprises administering mRNA formulated in a first LNP composition and a second LNP composition, the first LNP composition and the second LNP composition comprising one or more of mRNA, gRNA, gRNA nucleic acid, and template nucleic acid. In some embodiments, the present disclosure relates to any of the gene editing methods described herein, wherein the first LNP composition and the second LNP composition are administered simultaneously. In some embodiments, the present disclosure relates to any of the gene editing methods described herein, wherein the first LNP composition and the second LNP composition are administered sequentially.

[1347] In some embodiments, the present disclosure relates to any of the gene editing methods described herein, wherein the method comprises administering mRNA and a guide RNA nucleic acid formulated in a single LNP composition.

[1348] In some embodiments, the present disclosure relates to any of the gene editing methods described herein, wherein the gene editing results in a gene knockout.

[1349] In some embodiments, the present disclosure relates to any of the gene editing methods described herein, wherein the gene editing results in gene correction.

[1350] In some embodiments, the present disclosure relates to a method for in vivo delivery of interfering RNA to the lungs of a mammalian subject.

[1351] In some embodiments, methods for treating a disease or disorder in a mammalian subject are provided. In some embodiments, these methods comprise administering a therapeutically effective amount of a composition of the present disclosure to a subject having a disease or disorder associated with the expression or overexpression of a gene that can be reduced, decreased, downregulated, or silenced by the composition.

[1352] Examples

[1353] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[1354] Example 1. Synthesis of Compound 2243

[1355]

[1356] Step A1

[1357] At 0 °C, n-BuLi (2.5 M, 26.31 mL, 2.8 eq) was added dropwise to a solution of methoxymethyl(triphenyl)phosphonium; chloride (24.16 g, 70.47 mmol, 3 eq) in THF (360 mL), and the mixture was stirred at 25 °C for 2 h. At 0 °C, a solution of undecan-6-one (B) (4 g, 23.49 mmol, 1 eq) in THF (120 mL) was added to the mixture, and then the mixture was stirred at 25 °C for 12 h. The mixture was poured into H2O (200 mL) at 0 °C and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine (100 mL × 2), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 0 to 50 / 1) to give 6-(methoxymethylene)undecane (C) as a colorless oil (18 g, 90.8 mmol, 77% yield).

[1358] Step A2:

[1359] A solution of 6-(methoxymethylene)undecane (C) (18 g, 90.75 mmol, 1 eq) in THF (72 mL) and aqueous HCl (3 M, 18.00 mL, 5.95e-1 eq) was stirred at 70 °C for 12 h. The mixture was poured into H2O (100 mL) at 0 °C and extracted with EtOAc (50 mL × 3). The combined organic layers were washed with brine (50 mL × 2), dried over Na2SO4, filtered, and the filtrate was 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 2-pentylheptanal (D) as a colorless oil (15 g, 81.38 mmol, 90% yield).

[1360] Step A3:

[1361] At 0 °C, 2-diethoxyphosphorylacetic acid ethyl ester (22.14 g, 98.74 mmol, 19.59 mL, 1.3 equiv) was added dropwise to a solution of NaH (3.95 g, 98.74 mmol, 7.05 mL, 60% purity, 1.3 equiv) in THF (280 mL). The mixture was stirred at 25 °C for 0.5 h. At 0 °C, a solution of 2-pentylheptanal (D) (14 g, 75.96 mmol, 1 equiv) in THF (70 mL) was added to the mixture, and then the mixture was warmed to 25 °C and stirred at 25 °C for 2 h. The mixture was poured into H2O (200 mL) at 0 °C and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine (50 mL × 2), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 20 / 1) to obtain ethyl 4-pentylnon-2-enoate (E) as a colorless oil (16 g, 62.89 mmol, 82.80% yield).

[1362] Step A4

[1363] A solution of Pd / C (2.5 g, 10% purity) and ethyl 4-pentylnon-2-enoate (E) (5 g, 19.65 mmol, 1 equiv) in EtOH (100 mL) was stirred at 25 °C under H2 (15 Psi) for 1 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain ethyl 4-pentylnonanoate (F) as a colorless oil (15 g, crude).

[1364] Step A5

[1365] At 0 °C, a solution of ethyl 4-pentylnonanoate (F) (5 g, 19.50 mmol, 1 equiv) in THF (10 mL) was added to a solution of LAH (1.48 g, 39.00 mmol, 7.05 mL, 2 equiv) in THF (50 mL), and the mixture was stirred at 0 °C for 1 h. The mixture was poured into H2O (30 mL) at 0 °C, then the mixture was filtered and the filtrate was extracted with EtOAc (50 mL * 3). The combined organic layers were washed with brine (50 mL × 2), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 20 / 1) to obtain 4-pentylnonan-1-ol (Compound A) as a colorless oil (10 g, 46.6 mmol, 80% yield).

[1366] Step 1

[1367] To a solution of 2-methylpropanoyl chloride (2) (25.50 g, 239 mmol, 25 mL, 1 equiv) in DCM (400 mL) was added a solution of 2-methylpropan-2-ol (1) (18.63 g, 251 mmol, 24 mL, 1.05 equiv) in DCM (400 mL), and then TEA (36.33 g, 359 mmol, 50 mL, 1.5 equiv) and DMAP (1.46 g, 11.97 mmol, 0.05 equiv) were added to the mixture. The mixture was stirred at 25 °C for 8 h. The mixture was added to H2O (1000 mL), extracted with DCM (300 mL × 2), the organic layer was washed with brine (200 mL × 2), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The crude product was distilled in vacuo (100 °C, 0.08 MPa / oil pump) to give tert-butyl 2-methylpropanoate (3) as a colorless oil (46 g, 319 mmol, 33% yield).

[1368] Step 2

[1369] Under N2 at -40 °C, to a solution of diisopropylamine (10.5 g, 104 mmol, 14.7 mL, 1.5 equiv) in THF (120 mL) was added n-BuLi (2.5 M, 41.6 mL, 1.5 equiv). The mixture was stirred at -40 °C for 0.5 h and then cooled to -70 °C. The solution was added to a solution of tert-butyl 2-methylpropanoate (3) (10 g, 69.3 mmol, 1 equiv) in THF (100 mL) and stirred at -70 °C for 0.5 h under N2. Then at -70 °C, a solution of 1,6-dibromohexane (4) (30.45 g, 124.82 mmol, 19.15 mL, 1.8 equiv) in THF (50 mL) was added to the mixture and stirred at 25 °C for 8 h under N2. The mixture was added to an aqueous NH4Cl solution (200 mL) and extracted with EtOAc (200 mL × 3). The combined organic phases were washed with brine (100 mL × 2), dried over Na2SO4, and filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 50 / 1) to give tert-butyl 8-bromo-2,2-dimethyloctanoate (5) as a colorless oil (10 g, 32.5 mmol, 47% yield).

[1370] 1 1H NMR (400 MHz, CDCl3), δ 3.41 (t, J = 6.8 Hz, 2H), 1.83 - 1.90 (m, 2H), 1.43 - 1.49 (m, 14H), 1.27 - 1.30 (m, 6H), 1.14 (s, 6H).

[1371] Step 3

[1372] A solution of tert-butyl 8-bromo-2,2-dimethyl-octanoate (5) (10 g, 32.55 mmol, 1 equiv) in DCM (30 mL) and TFA (46.20 g, 405.18 mmol, 30.00 mL, 12.45 equiv) was stirred at 25 °C for 2 h. The mixture was concentrated under reduced pressure to give a residue. And then the residue was dissolved in EtOAc (200 mL), washed with NaHCO3 (200 mL × 3), brine (200 mL × 2), dried over Na2SO4, filtered and the filtrate was concentrated under reduc...

Claims

1. A lipid comprising at least one head group and at least one tail group of formula (TI) or (TI') Its pharmaceutically acceptable salts or stereoisomers of any of the foregoing, wherein: Each E is independently a biodegradable group; R a each independently is a C1-C5 alkyl group, a C2-C5 alkenyl group or a C2-C5 alkynyl group; u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7; R t each independently is H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, said alkyl or alkenyl being optionally interrupted by a heteroatom or being substituted by OH, SH or halogen or cycloalkyl or substituted cycloalkyl; represents a bond connecting the tail group and the head group; and wherein the pKa of the lipid is from about 4 to about 8.

2. The lipid according to claim 1, wherein each E is independently -OC(O)-, -C(O)O-, -N(R 7 )(C(O)-, -C(O)N(R 7 )-, -C(O-R 13 )-O-, -C(O)O(CH2) r -, -C(O)N(R 7 )(CH2) r -, -S-S- or -C(O-R 13 )-O-(CH2) r -, wherein each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl; R 13 is a branched or unbranched C3-C 10 alkyl; and r is 1, 2, 3, 4 or 5.

3. The lipid according to claim 1, wherein each E is independently -OC(O)-, -C(O)O-, -N(R 7 )C(O)- or -C(O)N(R 7 ), wherein R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl.

4. The lipid according to claim 1, wherein the lipid comprises at least one tail group of the following formula: wherein R 7 each independently is H or methyl; R b is independently H or a C1-C4 alkyl in each case; and u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6 or 7, and wherein the pKa of the lipid is from about 4 to about 8.

5. The lipid according to claim 4, wherein the lipid comprises at least one tail group selected from the group consisting of:

6. The lipid according to claim 4, wherein the lipid comprises at least one tail group selected from the group consisting of:

7. The lipid according to claim 4, wherein the lipid comprises two, three, four or more tail groups of formula (TII), (TIII), (TIV), (TV), (TII') or (TIII'), and wherein each tail group may be the same or different.

8. The lipid according to any one of claims 1 to 7, wherein each R a is methyl.

9. The lipid according to any one of claims 1 to 7, wherein u1 is 3 - 5, u2 is 0 - 3, and u3 and u4 are each independently 1 - 7.

10. The lipid according to claim 4, wherein the lipid comprises at least one tail of formula (TIII), wherein each R a is methyl, and R b is independently H, ethyl or butyl in each case, u1 is 3 - 5, u2 is 0 - 3, and u3 is 1 - 7.

11. The lipid according to claim 10, wherein the lipid comprises at least two, three or four tails of formula (TIII), wherein the two, three or four tails of formula (TIII) are the same or different.

12. The lipid according to claim 4, wherein the lipid comprises at least one tail of formula (TII), wherein each R a is methyl, u1 is 3 - 5, u2 is 0 - 3, u3 is 1 - 4, and u4 is 1 - 4.

13. The lipid according to claim 12, wherein the lipid has at least two, three or four tails of formula (TII), wherein the two, three or four tails of formula (TII) are the same or different.

14. The lipid according to claim 13, wherein the lipid has four tails of formula (TII), wherein the four tails of formula (TII) are the same or different.

15. The lipid according to claim 4, wherein the lipid has at least one tail of formula (TII) and at least one tail of formula (TIII).

16. The lipid according to claim 15, wherein the lipid has at least two tails of formula (TII) and at least two tails of formula (TIII).

17. The lipid according to claim 4, wherein the lipid has at least one tail of formula (TIV), where each R a is methyl, u1 is 3 - 5, u2 is 0 - 3, u3 is 1 - 4, and u4 is 1 - 4.

18. The lipid according to claim 4, wherein the lipid has at least two, three or four tails of formula (TIV), wherein the two, three or four tails of formula (TIV) are the same or different.

19. The lipid according to claim 4, wherein the lipid has at least two, three or four tails of formula (TV), wherein the two, three or four tails of formula (TV) are the same or different.

20. The lipid according to claim 4, wherein the lipid has at least two, three or four tails of formula (TII'), wherein the two, three or four tails of formula (TII') are the same or different.

21. The lipid according to claim 4, wherein the lipid has at least two, three or four tails of formula (TIII'), and the two, three or four tails of formula (TIII') are the same or different.

22. The lipid according to claim 4, wherein the lipid has at least one tail selected from the group consisting of formula (TII), (TIII) and (TII'), and at least one tail selected from the group consisting of (TIV), (TV) and (TIII').

23. The lipid according to claim 4, wherein the lipid has at least two tails selected from the group consisting of (TII), (TIII) and (TII').

24. The lipid according to claim 4, wherein the lipid has at least two tails selected from the group consisting of (TIV), (TV) and (TIII').

25. The lipid according to claim 4, wherein the lipid has at least two tails selected from the group consisting of formula (TII), (TIII) and (TII'), and at least one tail selected from the group consisting of (TIV), (TV) and (TIII').

26. The lipid according to claim 4, wherein the lipid has at least one tail selected from the group consisting of formula (TII), (TIII) and (TII'), and at least two tails selected from the group consisting of (TIV), (TV) and (TIII').

27. The lipid according to claim 4, wherein the lipid has at least two tails selected from the group consisting of formula (TII), (TIII) and (TII'), and at least two tails selected from the group consisting of (TIV), (TV) and (TIII').

28. The lipid according to claim 4, wherein the lipid has at least three tails selected from the group consisting of (TII), (TIII) and (TII').

29. The lipid according to claim 4, wherein the lipid has at least three tails selected from the group consisting of (TIV), (TV) and (TIII').

30. The lipid according to claim 4, wherein the lipid has at least three tails selected from the group consisting of formula (TII), (TIII) and (TII'), and at least one tail selected from the group consisting of (TIV), (TV) and (TIII').

31. The lipid according to claim 4, wherein the lipid has at least one tail selected from the group consisting of formula (TII), (TIII) and (TII'), and at least three tails selected from the group consisting of (TIV), (TV) and (TIII').

32. The lipid according to claim 4, wherein the lipid has at least two tails of formula (TII) or (TIII) and at least two tails of formula (TIV) or (TV).

33. The lipid according to claim 4, wherein the lipid has at least two tails of formula (TII) or (TIII) and at least two tails of formula (TII') or (TIII').

34. The lipid according to claim 4, wherein the lipid has at least two tails of formula (TIV) or (TV) and at least two tails of formula (TII') or (TIII').

35. The lipid according to any one of claims 1 to 34, which further comprises at least one tail of formula (TNG-I): wherein E is -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -S-S- or -C(O)N(R 7 )-; u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7; and R 7 independently is H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl.

36. The lipid according to claim 35, wherein the at least one tail of formula (TNG-I) can be represented by wherein u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6 or 7; and R b independently in each case is H or a C1-C4 alkyl group.

37. The lipid according to claim 36, wherein the lipid comprises two or three tail groups of formula (TNG-II) or (TNG-III), and wherein each tail group may be the same or different.

38. The lipid according to any one of claims 1 to 37, wherein the head group is an amine-containing head group.

39. The lipid according to claim 38, wherein the head group has a structure of formula (HA-I): Wherein: R 20 and R 30 each independently is H, a C1-C5 branched or unbranched alkyl group or a C2-C5 branched or unbranched alkenyl group, where the alkyl or alkenyl group is optionally interrupted by one or more heteroatoms or substituted by OH, SH, halogen or cycloalkyl; or R 20 and R 30 together with an adjacent N atom form a 3- to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, which heterocyclic or heteroaromatic ring is optionally substituted by one or more OH, SH, halogen, alkyl or cycloalkyl; Each of R1 and R2 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, OH, halogen, SH or NR 10 R 11 ; or R1 and R2 together form a ring; R 10 and R 11 each independently is H, a C1-C3 branched or unbranched alkyl, or a C2-C3 branched or unbranched alkenyl; or R 10 and R 11 together form a heterocycle; n is 0, 1, 2, 3 or 4; Z is absent, and is O, S or NR 12 , where R 12 is H or a C1-C7 branched or unbranched alkyl; provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 , SH.

40. The lipid according to claim 39, wherein R 20 and R 30 together with the adjacent N atom form a 3- to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, said heterocyclic or heteroaromatic ring being optionally substituted with one or more OH, SH, halogen, alkyl or cycloalkyl groups.

41. The lipid according to claim 39, wherein the head group has a structure of formula (HA-IA): Wherein m is 1, 2, 3, 4, 5, 6, 7 or 8.

42. The lipid according to claim 41, wherein the head group has a structure of formula (HA-III): wherein Z is absent, and is O, S or NR 12 ; and R 12 is H or a C1-C7 branched or unbranched alkyl group.

43. The lipid according to any one of claims 39 to 42, wherein: Z is absent, is O, S or NH; Each R1 and R2 is H; and n is 0, 1 or 2.

44. The lipid according to claim 43, wherein the head group has the following structure: Wherein: Rc is H, alkyl or alkyl substituted by OH; and m1 is 1, 2 or 3.

45. The lipid according to claim 38, wherein the head group has a structure of formula (HA-V): Wherein: R1 is H, C1-C3 alkyl, OH, halogen, SH or NR 10 R 11 ; R2 is OH, halogen, SH or NR 10 R 11 ; or R1 and R2 can combine together to form a ring; R 10 and R 11 each independently is H or a C1-C3 alkyl group; or R 10 and R 11 are able to combine together to form a heterocyclic ring; R 20 and R 30 each independently is H, a C1-C5 branched or unbranched alkyl group, or a C2-C5 branched or unbranched alkenyl group; or R 20 and R 30 are capable of combining together to form a ring; and Each of v and y is independently 1, 2, 3 or 4.

46. The lipid according to claim 45, wherein the head group has a structure of formula (HA-VI):

47. The lipid according to claim 45 or 46, wherein each R 20 and R 30 is independently a C1-C3 alkyl group.

48. The lipid according to claim 38, wherein the head group has a structure of formula (HB-I): where W is Where R5 is OH, SH, (CH2) s OH or NR 10 R 11 ; Each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; Each R7 and R8 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, (CH2) v OH, (CH2) v SH, (CH2) s N(CH3)2 or NR 10 R 11 , where each R 10 and R 11 is independently H or a C1-C3 alkyl group, or R 10 and R 11 together form a heterocycle; or R7 and R8 together form a ring; Each R 20 is independently H or a C1-C3 branched or unbranched alkyl group; R 14 is a heterocycle, NR 10 R 11 、C(O)NR 10 R 11 、NR 10 C(O)NR 10 R 11 or NR 10 C(S)NR 10 R 11， wherein each R 10 and R 11 is independently H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, said alkyl, cycloalkyl, cycloalkenyl being optionally substituted by one or more NH and / or oxo groups, or R 10 and R 11 together form a heterocycle; R 16 is H, =O, =S or CN; Each of s, u and t is independently 1, 2, 3, 4 or 5; Each v is independently 0, 1, 2, 3, 4 or 5; Each Y is a divalent heterocycle; Each Z is independently absent, O, S or NR 12 , where R 12 is H, a C1-C7 branched or unbranched alkyl or a C2-C7 branched or unbranched alkenyl; Q is O, S, CH2 or NR 13 , where each R 13 is H, C1-C5 alkyl; V is a branched or unbranched C2-C 10 alkylene, C2-C 10 alkenylene, C2-C 10 alkynylene or C2-C 10 heteroalkylene, where the alkylene, alkenylene, alkynylene or heteroalkylene is optionally substituted by one or more OH, SH and / or halogen groups; and T is –NHC(O)O-, –OC(O)NH- or a divalent heterocycle.

49. The lipid according to claim 48, wherein: R5 is OH or (CH2) s OH; and s is 1 or 2; Each of R6, R7 and R8 is independently H or C1-C3 alkyl; Each of u and t is independently 1, 2 or 3; Each v is independently 0, 1, 2 or 3; R 16 is H or =O; Each Z is independently absent, O or NR 12 , where R 12 is H or C1-C3 alkyl; T is a divalent heterocycle; Q is O or CH2; and V is C2-C6 alkylene or C2-C6 alkenylene.

50. The lipid according to claim 49, wherein W is wherein: Each of R6, R7, and R8 is independently H or methyl; and Each of u and t is independently 1, 2, or 3.

51. The lipid according to claim 49, wherein W is wherein: R 16 is H or =O; R 14 is a nitrogen-containing 5- or 6-membered heterocycle NR 10 R 11 , C(O)NR 10 R 11 , NR 10 C(O)NR 10 R 11 or NR 10 C(S)NR 10 R 11， where each R 10 and R 11 is independently H or a C1-C3 alkyl; and Each of u and v is independently 1, 2, or 3.

52. The lipid according to claim 49, wherein W is wherein: Each R6 is independently H or methyl; Each R7 is independently H; Each R8 is methyl; Each u is independently 1, 2, or 3; and V is a C2-C6 alkylene or a C2-C6 alkenylene.

53. The lipid according to claim 49, wherein W is and Wherein: Each u is independently 1, 2, or 3; Q is O; Each Z is independently NR 12； R 12 is H or a C1-C3 alkyl; and T is a divalent nitrogen-containing 5- or 6-membered heterocycle.

54. The lipid according to claim 48, wherein the head group has the following structure: Each of u and t is independently 1 or 2.

55. The lipid according to claim 38, wherein the head group has the structure of formula (HC-I): Wherein: is a cyclic or heterocyclic moiety; Y is an alkyl group, a hydroxyl group, a hydroxyalkyl group, A is absent and is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S- or -S-S-; Each of X and Z is independently absent, is -O-, -C(O)-, -N(R 7 )-, alkylene, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-; Each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl or aminoalkyl; t is 0, 1, 2, or 3; t1 is an integer from 0 to 10; and W is a hydroxyl group, a substituted or unsubstituted hydroxyalkyl group, a substituted or unsubstituted amino group, a substituted or unsubstituted aminocarbonyl group, or a substituted or unsubstituted heterocyclic group or heteroaryl group.

56. The lipid according to claim 55, wherein W is a hydroxyl group, a substituted or unsubstituted hydroxyalkyl group, or one of the following moieties: Wherein: Each Q independently does not exist and is -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2) q -C(R 7 )2, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 ); R 6 independently is H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, mercapto, mercaptoalkyl or N + (R 7 )3-alkylene-Q-; Each R 8 is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, thioalkyl, heterocyclic group, heteroaryl; or two Rs 8 together with the nitrogen atom form a ring, which is optionally substituted by one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino or aminoalkyl; q is 0, 1, 2, 3, 4, or 5; and p is 0, 1, 2, 3, 4, or 5.

57. The lipid according to claim 55 or 56, wherein the head group has the structure of formula (HC-IA):

58. The lipid according to claim 55, 56 or 57, wherein has the formula structure of Wherein: Each of G1, G2, G3, G4, G5, G6, and G7 is independently C(R')(R”), O, or N, provided that no more than two of G1-G7 are O or N; R' and R” each independently do not exist, are H, or an alkyl group; or two R' together with two adjacent G variables form a second 5- to 7-membered ring or heterocycle; and n1 and n2 are each independently 0 or 1.

59. The lipid according to claim 58, wherein selected from the group consisting of pyrrolidine, piperidine, piperazine, cyclohexane, cyclopentane, tetrahydrofuran, tetrahydropyran, morpholine and dioxane.

60. The lipid according to claim 58, wherein selected from the group consisting of:

61. The lipid according to claim 58, wherein selected from the group consisting of:

62. The lipid according to claim 55 or 56, wherein the head group has the structure of the following formula 63. The lipid according to claim 62, wherein the head group has the structure of the following formula 64. The lipid according to any one of claims 55 to 63, wherein: A is absent and is -O-, -N(R 7 )-, -OC(O)- or -C(O)O-; X does not exist, is O- or –C(O)-; and Z is –O-, –C(O)O-, or –OC(O)-.

65. The lipid according to claim 63, wherein the head group has a structure of the following formula wherein t1 is 0, 1, 2 or 3.

66. The lipid according to any one of claims 55 to 65, wherein W is one of the following: OH, 67. A lipid that comprises at least one head group and at least one tail group, wherein: At least one tail group has the structure of formula (TI) or (TI') Wherein: Each E is independently a biodegradable group; R a each independently is a C1-C5 alkyl group, a C2-C5 alkenyl group or a C2-C5 alkynyl group; u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; R t each independently is H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, the alkyl or alkenyl optionally being interrupted by a heteroatom or being substituted by OH, SH or a halogen or a cycloalkyl or a substituted cycloalkyl; and represents a bond connecting the tail group and the head group; and The head group has the structure of one of the following formulas: i) Wherein: R 20 and R 30 each independently is H, a C1-C5 branched or unbranched alkyl group or a C2-C5 branched or unbranched alkenyl group, where the alkyl or alkenyl group is optionally interrupted by one or more heteroatoms or substituted by OH, SH, halogen or cycloalkyl; or R 20 and R 30 together with an adjacent N atom form a 3- to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, which heterocyclic or heteroaromatic ring is optionally substituted by one or more OH, SH, halogen, alkyl or cycloalkyl; Each of R1 and R2 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, OH, a halogen, SH or NR 10 R 11 ; or R1 and R2 together form a ring; R 10 and R 11 each independently is H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group; or R 10 and R 11 together form a heterocycle; n is 0, 1, 2, 3, or 4; and Z is absent, and is O, S or NR 12 , wherein R 12 is H or a C1-C7 branched or unbranched alkyl; provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 , SH; ii) Wherein: R1 is H, C1-C3 alkyl, OH, halogen, SH or NR 10 R 11 ; R2 is OH, halogen, SH or NR 10 R 11 ; or R1 and R2 can combine together to form a ring; R 10 and R 11 each independently is H or C1-C3 alkyl; or R 10 and R 11 are capable of combining together to form a heterocycle; R 20 and R 30 each independently is H, a C1-C5 branched or unbranched alkyl group, a C2-C5 branched or unbranched alkenyl group; or R 20 and R 30 are capable of combining together to form a ring; and Each of v and y is independently 1, 2, 3, or 4; iii) where W is Where R5 is OH, SH, (CH2) s OH or NR 10 R 11 ; Each R6 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, or a cycloalkyl group; Each of R7 and R8 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, (CH2) v OH, (CH2) v SH, (CH2) s N(CH3)2 or NR 10 R 11 , where each R 10 and R 11 is independently H or a C1-C3 alkyl group, or R 10 and R 11 together form a heterocycle; or R7 and R8 together form a ring; Each R 20 is independently H or a C1-C3 branched or unbranched alkyl group; R 14 is a heterocycle, NR 10 R 11 、C(O)NR 10 R 11 、NR 10 C(O)NR 10 R 11 or NR 10 C(S)NR 10 R 11， wherein each R 10 and R 11 is independently H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, said alkyl, cycloalkyl, cycloalkenyl optionally substituted by one or more NH and / or oxo groups, or R 10 and R 11 together form a heterocycle; R 16 is H, =O, =S or CN; Each of s, u, and t is independently 1, 2, 3, 4, or 5; Each v is independently 0, 1, 2, 3, 4, or 5; Each Y is a divalent heterocycle; Each Z is independently absent, O, S or NR 12 , where R 12 is H, a C1-C7 branched or unbranched alkyl or a C2-C7 branched or unbranched alkenyl; Q is O, S, CH2 or NR 13 , wherein each R 13 is H, C1-C5 alkyl; V is a branched or unbranched C2-C 10 alkylene, C2-C 10 alkenylene, C2-C 10 alkynylene or C2-C 10 heteroalkylene, wherein the alkylene, alkenylene, alkynylene or heteroalkylene is optionally substituted by one or more OH, SH and / or halogen groups; and T is –NHC(O)O–, –OC(O)NH– or a divalent heterocycle; and iv) Wherein: is a cyclic or heterocyclic moiety; Y is an alkyl group, a hydroxyl group, a hydroxyalkyl group, A is absent and is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S- or -S-S-; Each of X and Z is independently absent, is -O-, -C(O)-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-; Each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl or aminoalkyl; t1 is an integer from 0 to 10; and W is a hydroxyl group, a substituted or unsubstituted hydroxyalkyl group, a substituted or unsubstituted amino group, a substituted or unsubstituted aminocarbonyl group, or a substituted or unsubstituted heterocyclic group or heteroaryl group; and wherein the pKa of the lipid is from about 4 to about 8.

68. The lipid according to claim 67, wherein: at least one tail group has a structure of at least one of the following formulas: wherein: R 7 Each independently is H or methyl; R b is independently H or C1-C4 alkyl in each case; and u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; and the head group has a structure of one of the following formulas: i) where m is 1, 2, 3, 4, 5, 6, 7 or 8; ii) iii) iv) 69. The lipid according to claim 67, wherein at least one tail group has the structure of formula (TII), (TIII), (TIV), (TV), (TII') and / or (TIII'), wherein each R a is methyl; u1 is 3 - 5, u2 is 0 - 3; and u3 and u4 are each independently 1 - 7.

70. The lipid according to claim 67, wherein the head group has a structure of one of the following formulas i) ii) where each R 20 and R 30 is independently a C1-C3 alkyl group; iii) wherein: W is Wherein: each of R6, R7, and R8 is independently H or methyl; and each of u and t is independently 1, 2, or 3; or W is Wherein: R 16 is H or =O; R 14 is a nitrogen-containing 5- or 6-membered heterocycle, NR 10 R 11 、C(O)NR 10 R 11 、NR 10 C(O)NR 10 R 11 or NR 10 C(S)NR 10 R 11， wherein each R 10 and R 11 is independently H or a C1-C3 alkyl; and each of u and v is independently 1, 2, or 3; or W is Wherein: each R6 is independently H or methyl; each R7 is independently H; each R8 is methyl; each u is independently 1, 2, or 3; and V is a C2-C6 alkylene group or a C2-C6 alkenylene group; or W is Wherein: each u is independently 1, 2, or 3; Q is O; Each Z is independently NR 12； and T is a divalent nitrogen-containing 5- or 6-membered heterocycle; and iv) wherein: W is a hydroxyl group, a substituted or unsubstituted hydroxyalkyl group, one of the following moieties: wherein Each Q is independently absent, and is -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2) q -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 ); R 6 is independently H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, mercapto, mercaptoalkyl or N + (R 7 )3-alkylene-Q-; Each R 8 independently is H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, thioalkyl, heterocyclic group, heteroaryl; or two Rs 8 together with the nitrogen atom form a ring, which ring is optionally substituted by one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino or aminoalkyl; q is 0, 1, 2, 3, 4, or 5; and p is 0, 1, 2, 3, 4, or 5.

71. The lipid according to claim 1 or claim 67, having one of the following structures:

72. The lipid according to claim 1 or claim 67, having one of the following structures:

73. A lipid comprising at least two lipophilic tail groups and a head group of formula (G-HC-IIID): its pharmaceutically acceptable salts or stereoisomers of any of the foregoing wherein: R a each independently is a C1-C5 alkyl group, a C2-C5 alkenyl group or a C2-C5 alkynyl group; t2 is an integer from 0 to 5; W is a hydroxyl group, a substituted or unsubstituted hydroxyalkyl group, a substituted or unsubstituted amino group, a substituted or unsubstituted aminocarbonyl group, or a substituted or unsubstituted heterocyclic group or heteroaryl group; and represents a bond connecting the head group and the tail group.

74. The lipid according to claim 73, wherein each R a is methyl and t2 is 0 - 3.

75. The lipid according to claim 73, wherein W is a hydroxyl group, a substituted or unsubstituted hydroxyalkyl group, or one of the following moieties: wherein Each Q independently does not exist and is -O-, -C(O)-, -C(S)-, -C(O)O-, -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 ); R 6 independently is H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, mercapto, mercaptoalkyl or N + (R 7 )3-alkylene-Q-; Each R 8 independently is H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, mercapto, mercaptoalkyl, heterocyclic group, heteroaryl; or two Rs 8 together with the nitrogen atom form a ring, which ring is optionally substituted by one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino or aminoalkyl; q is 0, 1, 2, 3, 4, or 5; and p is 0, 1, 2, 3, 4, or 5.

76. The lipid according to claim 75, wherein W is 77. The lipid according to claim 73, having the following structure:

78. The lipid according to any one of claims 1 to 77, wherein the pKa of the lipid is from about 4.8 to about 7.

1.

79. The lipid according to any one of claims 1 to 77, wherein the pKa of the lipid is from about 5.4 to about 7.

1.

80. A composition comprising the lipid according to any one of claims 1 to 77 and one or more lipid components different from the lipid.

81. The composition according to claim 80, wherein the combination is an LNP composition.

82. The composition according to any one of claims 80 to 81, wherein the lipid component comprises a helper lipid and a PEG lipid.

83. The composition according to any one of claims 80 to 81, wherein the lipid component comprises a helper lipid, a PEG lipid, and a neutral lipid.

84. The composition according to any one of claims 80 to 81, further comprising a cryoprotectant.

85. The composition according to any one of claims 80 to 84, further comprising a buffer.

86. The composition according to any one of claims 80 to 85, further comprising a nucleic acid component.

87. The composition according to claim 86, wherein the nucleic acid component is an RNA or DNA component.

88. The composition according to claim 87, wherein the nucleic acid component is an RNA component, and wherein the RNA component comprises mRNA.

89. The composition according to claim 86, having an N / P ratio of about 3 - 15.

90. The composition according to claim 89, wherein the N / P ratio is about 6.

91. A nucleic acid-lipid particle, the nucleic acid-lipid particle comprising: a nucleic acid; a lipid according to any one of claims 1 to 77; a co-lipid; a sterol; and a PEG-modified lipid, wherein the pKa of the nucleic acid-lipid particle is about 4 to about 8.

92. A pharmaceutical composition comprising a lipid particle and a pharmaceutically acceptable diluent, wherein the lipid particle comprises: (i) a nucleic acid; (ii) 35 - 65 mol% of a lipid according to any one of claims 1 to 77; (iii) 3 - 12 mol% of a co-lipid; (iv) 15 - 45 mol% of a sterol; and (v) 0.5 - 10 mol% of a PEG-modified lipid.

93. A method for preparing a lipid, the lipid comprising at least one head group and at least one tail group of formula (TI) or (TI') Wherein: Each E is independently a biodegradable group; R a each independently is a C1-C5 alkyl group, a C2-C5 alkenyl group or a C2-C5 alkynyl group; u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7; R t each independently is H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, where the alkyl or alkenyl is optionally interrupted by a heteroatom or substituted by OH, SH or a halogen or a cycloalkyl or a substituted cycloalkyl; and wherein the pKa of the lipid is about 4 to about 8, the method comprising: React the first precursor compound of the tail group of the formula (TI) or (TI') with the precursor compound of the head group, wherein the precursor compound of the head group comprises one or more attachment points for the tail group, each attachment point containing a functional group reactive towards a halogen, thereby forming a lipid by attaching at least one tail group of the formula (TI) or (TI') to the head group at the one or more attachment points.

94. The method according to claim 93, wherein one or more attachment points for the tail group in the precursor compound of the head group contain one or more Ns.

95. The method according to claim 94, wherein one or more attachment points for the tail group in the precursor compound of the head group further comprise non-N functional groups, and the one or more Ns contained at the one or more attachment points of the precursor compound of the head group are protected such that the attachment point containing the non-N functional group reacts with the precursor compound of the tail group, and wherein the method further comprises: deprotecting the one or more Ns contained at the one or more attachment points of the head group of the lipid; and react the second precursor compound of the tail group of formula (TI) or (TI') with the lipid containing one or more deprotected Ns at the one or more attachment points of the head group, thereby forming a lipid by attaching the second tail group of formula (TI) or (TI') to the head group at the one or more attachment points, wherein the second precursor compound of the tail group can be the same as or different from the first precursor compound of the tail group.

96. The method according to any one of claims 93 to 95, wherein at least one tail group has one of the following formulas: Among them R 7 each independently is H or methyl; R b is independently H or C1-C4 alkyl in each case; and u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6 or 7.

97. The method according to claim 96, wherein each R a is methyl.

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