Novel ionizable lipids and lipid nanoparticles and methods of use thereof

Abstract

Novel ionizable lipids and lipid nanoparticles and methods for their use are provided. Novel ionizable lipids and lipid nanoparticles that can be used to deliver therapeutic cargo are disclosed.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 402,929, filed August 31, 2022, and U.S. Provisional Patent Application No. 63 / 502,806, filed May 17, 2023, each of which is incorporated herein by reference in its entirety.

[0002] Lipid nanoparticles ("LNPs") formed from ionizable amine-containing lipids can function as therapeutic loading vehicles for intracellular delivery of bioactive agents, such as coding RNA (i.e., messenger RNA (mRNA), guide RNA), and non-coding RNA (i.e., antisense, siRNA). LNPs can facilitate delivery of oligonucleotide agents across cell membranes and can be used to introduce components and compositions into living cells.

[0003] Bioactive agents that are particularly difficult to deliver into cells include proteins, nucleic acid-based drugs, and their derivatives, especially drugs containing relatively large oligonucleotides such as mRNA or guide RNA. Compositions for delivering promising mRNA therapeutics or editing technologies, such as delivery of CRISPR / CAS9 system components, into cells are of particular interest.

[0004] With the emergence of recent pandemics, messenger RNA therapy has become an increasingly important option for the treatment of various diseases, including viral infectious diseases and diseases associated with the deficiency of one or more proteins. Compositions that can stabilize and / or deliver RNA components and have properties useful for in vitro and in vivo delivery are also of particular interest.

[0005] Thus, there remains a need in the art for novel lipid compounds and for the development of lipid nanoparticles or other lipid delivery mechanisms for the delivery of therapeutic agents, which the present invention addresses. Summary of the Invention

[0006] Disclosed herein are novel ionizable lipids that can be used in combination with at least one other lipid component, such as a neutral lipid, cholesterol, and a polymer-conjugated lipid, to form lipid nanoparticle compositions that can be used to facilitate intracellular delivery of therapeutic nucleic acids in vitro and / or in vivo.

[0007] Disclosed herein is an ionizable amine-containing lipid useful for forming lipid nanoparticle compositions.Such LNP compositions can have advantageous properties for the delivery of nucleic acid transporters, such as the delivery of coding RNA and non-coding RNA to cells.Also provided is a method for treating various diseases or conditions, such as those caused by infectious agents and / or protein deficiencies, using the disclosed lipid nanoparticles.

[0008] Lipids, particularly ionizable lipids having specific tail groups (eg, geminal, ie, Gem-di, functional groups attached to the same carbon next to a biodegradable group E), are disclosed below. tail group

[0009] Certain embodiments of the present invention relate to lipids comprising at least one head group and at least one tail group of formula (T). [ka] (T), a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing; During the ceremony, E is a biodegradable group; R a is, independently at each occurrence, optionally interrupted by a heteroatom or is OH, SH, a halogen, or NR 7C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl, or C2-C5 branched or unbranched alkynyl substituted with 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, or cycloalkyl or substituted cycloalkyl; R b However, for each opportunity, H, C1 to C 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, optionally interrupted with a heteroatom or substituted with OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl, C1-C5 alkyl, C2-C5 alkenyl, or C2-C5 alkynyl; u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; R t are independently H, C1 to C 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted by OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; [ka] represents the bond connecting the tail group to the head group, Lipids have a pKa of about 4 to about 8.

[0010] As used herein, the term "biodegradable" refers to a group containing one or more bonds that can undergo bond-breaking reactions in a biological environment, such as an organism, organ, tissue, cell, or organelle. For example, a biodegradable group may be metabolizable (e.g., by hydrolysis) by the body of a mammal, such as a human. Some groups containing biodegradable bonds include, 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-, -SS-, -C(R)=N-, -N=C(R)-, -C(R)=NO-, -ON=C(R)-, -C(O)(NR)-, -N(R)C(O)-, -C(S)(NR)-, -N(R)C(O)-, -N(R)C(O)N(R)-, -OC(O)O-, -OSi(R)O-, -C(O)(CRR)C(O)O-, or -OC(O)(CRR)C(O)-. R5 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R3 and R4 are independently branched or unbranched C1-C 15 Branched or unbranched alkyl, C1-C 15 It is a branched or unbranched alkenyl, or a cycloalkyl or substituted cycloalkyl.

[0011] In some embodiments, R a are each independently a C1 to C5 branched or unbranched alkyl, a C2 to C5 branched or unbranched alkenyl, or a C2 to C5 branched or unbranched alkynyl.

[0012] In some embodiments, R b are each independently H, C1 to C 16 Branched or unbranched alkyl, or C1-C 16 It is a branched or unbranched alkenyl.

[0013] In some embodiments, R aare each independently a C1-C3 branched or unbranched alkyl. In one embodiment, R a are each methyl.

[0014] In some embodiments, R b are each independently H or C1-C3 branched or unbranched alkyl.

[0015] In some embodiments, E is —OC(O)—, —C(O)O—, —N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(OR 13 )-O-, -C(O)O(CH2) r -, -C(O)N(R 7 )(CH2) r -, -SS-, or -C(OR 13 )-O-(CH2) r -, wherein R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R 13 Branched or unbranched C3-C 10 alkyl, and r is 1, 2, 3, 4, or 5.

[0016] In some embodiments, E is —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.

[0017] 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 —C(O)N(R 7 )-.

[0018] In some embodiments, at least one head group and at least one tail group of formula (TI) or (TI′) is: [ka] (TI) or [ka] (TI'), A lipid comprising a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, is provided, During the ceremony, each E is independently a biodegradable group; R a is, independently at each occurrence, optionally interrupted by a heteroatom or is OH, SH, a halogen, or NR 7 C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl, or C2-C5 branched or unbranched alkynyl substituted with 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, or cycloalkyl or substituted cycloalkyl; u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; R t are independently H, C1 to C 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted by OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; [ka] represents the bond connecting the tail group to the head group, Lipids have a pKa of about 4 to about 8.

[0019] In some embodiments, R aare each independently, for each occurrence, C1 to C5 branched or unbranched alkyl, C2 to C5 branched or unbranched alkenyl, or C2 to C5 branched or unbranched alkynyl. a are each independently at each occurrence C1-C3 branched or unbranched alkyl. In one embodiment, R a are each methyl.

[0020] In some embodiments, each E is independently —OC(O)—, —C(O)O—, —N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(OR 13 )-O-, -C(O)O(CH2) r -, -C(O)N(R 7 )(CH2) r -, -SS-, -C(OR 13 )-O-(CH2) r -, wherein R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R 13 is branched or unbranched C3~C 10 alkyl, and r is 1, 2, 3, 4, or 5.

[0021] In some embodiments, each E is independently -OC(O)-, -C(O)O-, -N(R)C(O)-, or -C(O)N(R 7 )-, wherein R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl.

[0022] In some embodiments, each E is independently -C(O)O-. In some embodiments, each E is independently -OC(O)-. In some embodiments, each E is independently -N(R)C(O)-, where R 7 are independently H or methyl. In some embodiments, each E is independently —C(O)N(R 7)-, wherein R 7 are independently H or methyl.

[0023] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TII): [ka] (TII), wherein u3 and u4 are each independently 1 to 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).

[0024] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TIII): [ka] (TIII) (for example, [ka] (TIIIa) wherein u3 is 0, 1, 2, 3, 4, 5, 6, or 7; b is independently at each occurrence H or C1-C4 alkyl. The definitions of the other variables in (TIII) are the same as those defined above in (TI).

[0025] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TIV): [ka] (TIV), wherein u3 and u4 are each independently 1 to 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).

[0026] In some embodiments, the lipid comprises at least one head group and a group of formula (TV) [ka] (TV) (for example, [ka] (TVa) ), where u3 is 0, 1, 2, 3, 4, 5, 6, or 7; and R 7 are each independently H or methyl, and R b is independently at each occurrence H or C1-C4 alkyl. The definitions of the other variables in (TV) are the same as those defined above in (TI).

[0027] In some embodiments, the lipid comprises at least one head group and a group of formula (TII') [ka] (TII') (for example, [ka] (TII'a) ) wherein u3 is 0, 1, 2, 3, 4, 5, 6, or 7; and R b is independently at each occurrence H or C1-C4 alkyl. The definitions of the other variables in (TII') are the same as defined above in (TI').

[0028] In some embodiments, the lipid comprises at least one head group and a group of formula (TII') [ka] (TIII') (for example, [ka] (TIII'a) ), where u3 is 0, 1, 2, 3, 4, 5, 6, or 7; and R 7 are each independently H or methyl, and R b is independently at each occurrence H or C1-C4 alkyl. The definitions of the other variables in (TIII') are the same as those defined above in (TI').

[0029] In some embodiments, the lipid comprises at least one tail group of the following formula: [ka] (TII), [ka] (TIII) [ka] (TIV), [ka] (TV), [ka] (TII'), and [ka] (TIII'), During the ceremony, R 7 are each independently H or methyl; R b is independently at each occurrence H or C1-C4 alkyl; u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; Lipids have a pKa of about 4 to about 8.

[0030] In some embodiments, the lipid comprises two or more tail groups having the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TII'), and / or (TIII'), where each tail group can be the same or different.

[0031] In some embodiments, the lipid comprises three or more tail groups having the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'), where each tail group can be the same or different.

[0032] In some embodiments, the lipid comprises four or more tail groups having the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'), where each tail group can be the same or different.

[0033] In some embodiments, in any of the above formulas (T), (TI), (TII), (TIII), (TIV), (TV), (TI′), (TII′), and (TIII′), R a is methyl.

[0034] In some embodiments, in any of the above formulas (T), (TI), (TII), and (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'), u1 is 3, 4, or 5.

[0035] In some embodiments, in any of the above formulas (T), (TI), (TII), and (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'), u2 is 0, 1, 2, or 3.

[0036] In some embodiments, in any of the above formulas (T), (TI), (TII), and (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'), u3 and u4 are each independently 1 to 7, e.g., u3 and u4 are each independently 1, 2, 3, or 4.

[0037] In some embodiments, the lipid comprises at least one tail of formula (TIII), where R a are methyl, and R b is independently at each occurrence H, ethyl, or butyl, u1 is 3 to 5, u2 is 0 to 3, and u3 is 1 to 7 (e.g., 1 to 4).

[0038] In some embodiments, the lipid comprises at least two tails of formula (TIII), and the two tails of formula (TIII) are the same or different. In some embodiments, the lipid comprises at least three tails of formula (TIII), and each tail can be the same or different. In some embodiments, the lipid has four tails of formula (TIII), and each tail can be the same or different.

[0039] In some embodiments, in each tail of formula (TIII), R a are each methyl, u1 is 3, u2 is 2, and u3 is 4.

[0040] In some embodiments, the lipid comprises at least one tail of formula (TII), where R a are each methyl, u1 is 3 to 5, u2 is 0 to 3, u3 is 1 to 4, and u4 is 1 to 4.

[0041] In some embodiments, the lipid has at least two tails of formula (TII), and the two tails of formula (TII) are the same or different. In some embodiments, the lipid has at least three tails of formula (TII), and each tail can be the same or different. In some embodiments, the lipid has four tails of formula (TII), and each tail can be the same or different.

[0042] In some embodiments, in each tail of formula (TII), R a are each methyl, and the variables u1, u2, u3, and u4 are one of the following: (i) u1 is 5, u2 is 3, u3 and u4 are each 1; (ii) u1 is 5, u2 is 0, u3 and u4 are each 2; (iii) u1 is 5, u2 is 0, u3 and u4 are each 3; (iv) u1 is 5, u2 is 0, and u3 and u4 are each 4; (v) u1 is 5, u2 is 0, u3 is 4, and u4 is 2, or (vi) u1 is 3, u2 is 3, and u3 and u4 are each 1.

[0043] In some embodiments, the lipid comprises at least one tail of formula (TIV), where R a are each methyl, u1 is 3 to 5, u2 is 0 to 3, u3 is 1 to 4, and u4 is 1 to 4.

[0044] In some embodiments, the lipid comprises at least two tails of formula (TIV), each of which may be the same or different. In some embodiments, the lipid comprises at least three tails of formula (TIV), each of which may be the same or different. In some embodiments, the lipid comprises at least four tails of formula (TIV), each of which may be the same or different.

[0045] In some embodiments, the lipid comprises at least two tails of formula (TV), each of which may be the same or different. In some embodiments, the lipid comprises at least three tails of formula (TV), each of which may be the same or different. In some embodiments, the lipid comprises at least four tails of formula (TV), each of which may be the same or different.

[0046] In some embodiments, the lipid has at least two tails of formula (TII'), each of which may be the same or different. In some embodiments, the lipid has at least three tails of formula (TII'), each of which may be the same or different. In some embodiments, the lipid has at least four tails of formula (TII'), each of which may be the same or different.

[0047] In some embodiments, the lipid has at least two tails of formula (TIII'), each of which may be the same or different. In some embodiments, the lipid has at least three tails of formula (TIII'), each of which may be the same or different. In some embodiments, the lipid has at least four tails of formula (TIII'), each of which may be the same or different.

[0048] In some embodiments, the lipid has at least one tail selected from the group consisting of formula (TII), (TIII), and (TII').

[0049] In some embodiments, the lipid has at least one tail selected from the group consisting of (TIV), (TV), and (TIII').

[0050] In some embodiments, the lipid has at least one tail selected from the group consisting of formulas (TII), (TIII), and (TII'), and at least one tail selected from the group consisting of (TIV), (TV), and (TIII').

[0051] In some embodiments, the lipid has at least two tails selected from the group consisting of (TII), (TIII), and (TII').

[0052] In some embodiments, the lipid has at least two tails selected from the group consisting of (TIV), (TV), and (TIII').

[0053] In some embodiments, the lipid has at least two tails selected from the group consisting of formulas (TII), (TIII), and (TII'), and at least one tail selected from the group consisting of (TIV), (TV), and (TIII').

[0054] In some embodiments, the lipid has at least one tail selected from the group consisting of formulas (TII), (TIII), and (TII'), and at least two tails selected from the group consisting of (TIV), (TV), and (TIII').

[0055] In some embodiments, the lipid has at least two tails selected from the group consisting of formulas (TII), (TIII), and (TII'), and at least two tails selected from the group consisting of (TIV), (TV), and (TIII').

[0056] In some embodiments, the lipid has at least three tails selected from the group consisting of (TII), (TIII), and (TII').

[0057] In some embodiments, the lipid has at least three tails selected from the group consisting of (TIV), (TV), and (TIII').

[0058] In some embodiments, the lipid has at least three tails selected from the group consisting of formulas (TII), (TIII), and (TII'), and at least one tail selected from the group consisting of (TIV), (TV), and (TIII').

[0059] In some embodiments, the lipid has at least one tail selected from the group consisting of formulas (TII), (TIII), and (TII'), and at least three tails selected from the group consisting of (TIV), (TV), and (TIII').

[0060] 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).

[0061] 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').

[0062] 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').

[0063] 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 to 5, u2 is 0 to 2, u3 is 1 to 4, and u4 is 1 to 4.

[0064] In some embodiments, the lipid has at least one tail of formula (TII) and / or at least one tail of formula (TIII), and the lipid further comprises at least one tail that does not have 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 Gem-di functional group attached to the same carbon atom adjacent to E (e.g., -C(O)O-).

[0065] 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'), i.e., the lipid further comprises at least one tail that does not contain a Gem-di functional group attached to the same carbon adjacent to E.

[0066] In some embodiments, the lipid further comprises at least one tail of formula (TNG-I): [ka] (TNG-I), During the ceremony, Each E is independently a biodegradable group as described herein, e.g., —OC(O)—, -C(O)O-, -N(R 7 )C(O)-, -SS-, or -C(O)N(R 7 )- and u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl.

[0067] In some embodiments, at least one tail of formula (TNG-I) is [ka] (TNG-II) or [ka] (TNG-III) wherein: u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; R b is independently at each occurrence H or C1-C4 alkyl.

[0068] The above-described E, R for tail groups containing Gem-di functional groups attached to the same carbon adjacent to E having the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TII'), or (TIII'). b , R t All of the above embodiments regarding the definitions of u1, u2, u3 and u4 are also applicable to tail groups having formula (TNG-I), (TNG-II) or (TNG-III) that do not contain a Gem-di functional group attached to the same carbon next to E.

[0069] In some embodiments, the lipid further comprises at least two tails that do not have 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), each tail group may be the same or different;

[0070] In some embodiments, the lipid further comprises at least three tails that do not have 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), each tail group may be the same or different;

[0071] Head group The head group of the lipid may be any amine-containing head group of a typical ionizable lipid.

[0072] In some embodiments, the head group of the lipid has the structure of formula (HA-I): [ka] (HA-I), During the ceremony, R 20 and R 30 are each independently H, a C1-C5 branched or unbranched alkyl, or a C2-C5 branched or unbranched alkenyl, optionally interrupted with one or more heteroatoms or substituted with OH, SH, halogen, or a cycloalkyl group; or 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 and optionally substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl groups; R1 and R2 are each 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; R 10 and R 11are each independently H, C1-C3 branched or unbranched alkyl, 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 or O, S, or NR 12 where R 12 is H or C1-C7 branched or unbranched alkyl, provided that when Z is absent, adjacent R1 and R2 are OH, NR 10 R 11 , SH should not be.

[0073] 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 and optionally substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl groups.

[0074] In some embodiments, the head group of the lipid has the structure of formula (HA-IA): [ka] (HA-IA), During the ceremony, R1 and R2 are each 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, R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or R 10 and R 11 together form a heterocycle, m is 1, 2, 3, 4, 5, 6, 7 or 8; n is 0, 1, 2, 3, or 4; Z is absent or O, S, or NR 12 where R 12 is H or C1-C7 branched or unbranched alkyl, provided that when Z is absent, adjacent R1 and R2 are OH, NR 10 R 11 , SH should not be, [ka] represents the bond connecting the head group to the tail group.

[0075] In some embodiments, m is 1, 2, 3, or 4.

[0076] In some embodiments, the head group of the lipid has the structure of formula (HA-III): [ka] (HA-III), wherein Z is absent, O, S, or NR 12 and R 12 is C1-C7 alkyl. The definitions of the other variables in (HA-III) are the same as those defined above in (HA-I).

[0077] 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.

[0078] In some embodiments, in any of the above formulas, such as (HA-I), (HA-IA), or (HA-III), R 1 and R 2 are each H.

[0079] In some embodiments, n is 0, 1, or 2 in any of the above formulas, such as (HA-I), (HA-IA), or (HA-III).

[0080] In some embodiments, the head group is [ka] or [ka] wherein: Rc is H or alkyl optionally substituted with OH; m1 is 1, 2, or 3;

[0081] In some embodiments, the head group of the lipid has the structure of formula (HA-V): [ka] (HA-V), During the ceremony, R1 is H, C1-C3 alkyl, OH, halogen, SH, or NR 10 R 11 and R2 is OH, halogen, SH, or NR 10 R 11 or R1 and R2 together can form a ring; R 10 and R 11 are each independently H or C1-C3 alkyl, or R 10 and R 11 may together form a heterocycle, 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 may be taken together to form a ring, v and y are each independently 1, 2, 3, or 4.

[0082] In some embodiments, the head group of the lipid has the structure of formula (HA-VI): [ka] (HA-VI). The definitions of all variables in (HA-VI) are the same as those defined above in (HA-V).

[0083] In some embodiments, in any of the above formulas, such as (HA-V) or (HA-VI), R 20 and R 30 are each independently C1-C3 alkyl. In one embodiment, R 20 and R 30 are each independently methyl.

[0084] In some embodiments, the head group of the lipid has the structure of formula (HA-VII): [ka]

[0085] In some embodiments, the head group of the lipid has the structure of formula (HB-I): [ka] (HB-I), In the formula, W is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] During the ceremony, R5 is OH, SH, (CH2) S OH, or NR 10 R 11 and R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; R7 and R8 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, (CH2) v OH, (CH2) v SH, (CH2) s N(CH3)2, or NR 10 R 11 where R 10 and R 11 are each independently H or C1-C3 alkyl, or R 10 and R 11 together form a heterocycle, or R7 and R8 together form a ring; R 20 are each independently H or C1-C3 branched or unbranched alkyl, R 14 is heterocyclic, NR 10 R 11 , C(O)NR 10 R 11 , N.R. 10 C(O)NR 10 R 11 , or NR 10 C(S)NR 10 R 11 and 、 In the formula, R 10 and R 11are each independently H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, optionally substituted with 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; s, u, and t are each 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, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl; Q is O, S, CH2, or NR 13 where R 13 are each H or C1-C5 alkyl, V is branched or unbranched C2 to C 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Alkynylene, or C2-C 10 heteroalkylene, optionally substituted with one or more OH, SH, and / or halogen groups; T is -NHC(O)O-, -OC(O)NH-, or a divalent heterocycle.

[0086] In some embodiments, R5 is OH or (CH2) s OH, and s is 1 or 2.

[0087] In some embodiments, R, R 7 and R8 are each independently H or C1-C3 alkyl.

[0088] In some embodiments, u and t are each independently 1, 2, or 3.

[0089] In some embodiments, each v is independently 0, 1, 2, or 3.

[0090] In some embodiments, R 16 is H or =O.

[0091] In some embodiments, each Z is independently absent, O, or NR 12 where R 12 is H or C1-C3 alkyl.

[0092] In some embodiments, T is a divalent heterocycle.

[0093] In some embodiments, Q is O or CH2.

[0094] In some embodiments, V is C2-C6 alkylene or C2-C6 alkenylene.

[0095] In some embodiments, the heterocyclic (or divalent heterocyclic) is piperazine, piperazinedione, piperazine-2,5-dione, piperidine, pyrrolidine, piperidinol, dioxopiperazine, bis-piperazine, aromatic, or heteroaromatic.

[0096] In some embodiments, in Formula (HB-I), W is [ka] where: R6, R7, and R8 are each independently H or methyl; u and t are each independently 1, 2, or 3.

[0097] In some embodiments, in Formula (HB-I), W is [ka] where: R 16 is H or =O, R 14 is a nitrogen-containing 5- or 6-membered heterocyclic ring, NR 10 R 11 , C(O)NR 10 R 11 , N.R. 10 C(O)NR 10 R 11 , or NR 10 C(S)NR 10 R 11 and 、 In the formula, R 10 and R 11 are each independently H or C1-C3 alkyl, u and v are each independently 1, 2, or 3.

[0098] In some embodiments, in Formula (HB-I), W is [ka] where: R6 are each independently H or methyl; each u is independently 1, 2, or 3; V is C2-C6 alkylene or C2-C6 alkenylene.

[0099] In some embodiments, in Formula (HB-I), W is [ka] where: R6 are each independently H or methyl; R7 is independently H; R8 is methyl at each occurrence; each u is independently 1, 2, or 3; V is C2-C6 alkylene or C2-C6 alkenylene.

[0100] In some embodiments, in Formula (HB-I), W is [ka] where: each u is independently 1, 2, or 3; T is a divalent nitrogen-containing 5- or 6-membered heterocycle.

[0101] In some embodiments, in Formula (HB-I), W is [ka] and During the ceremony, each u is independently 1, 2, or 3; Q is O, Z is independent of each other, NR 12 and R 12 is H or C1-C3 alkyl, In some embodiments, the head group is [ka] [ka] [ka] [ka] wherein u and t are each independently 1 or 2.

[0102] In some embodiments, the head group of the lipid has the structure of formula (HC-I): [ka] Y is alkyl, hydroxy, hydroxyalkyl, [ka] [ka] and A is absent or -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, -N(R7)C(O)N(R7)-, -S-, -SS-; X and Z are each independently absent, —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-, R 7 are each 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, W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl.

[0103] In some embodiments, W is hydroxyl, substituted or unsubstituted hydroxyalkyl, or [ka] During the ceremony, Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, or -(CH2) q -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and R 6are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, thiol, thiolalkyl, or N+(R 7 ) 3-alkylene-Q-, and R 8 are each independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, or thiolalkyl, heterocyclyl, heteroaryl, or together with the nitrogen atom, two R 8 may form a ring optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, aminoalkyl; q is 0, 1, 2, 3, 4, or 5; p is 0, 1, 2, 3, 4, or 5.

[0104] In some embodiments, the head group of the lipid has the structure of formula (HC-IA): [ka] (HC-IA) or [ka] (HC-IB). The definitions of all variables in (HC-IA) or (HC-IB) are the same as those defined above in (HC-I).

[0105] JPEG2025530781000061.jpg47161

[0106] JPEG2025530781000062.jpg53161 [ka] During the ceremony, G1, G2, G3, G4, G5, G6, and G7 are each independently C(R')(R''), O, or N, provided that no more than two of G1 through G7 are O or N; R' and R'' are each independently absent, H, alkyl, or two R's from two adjacent G's together form a second 5- to 7-membered ring or heterocycle; n1 and n2 each independently represent 0 or 1.

[0107] In some embodiments, in any of the above formulas, such as (HC-I), (HC-IA), or (HC-IB), pyrrolidine, piperidine, piperazine, cyclohexane, cyclopentane, tetrahydrofuran, tetrahydropyran, morpholine, and dioxane [ka] is selected from.

[0108] In some embodiments, in any of the above formulas, such as (HC-I), (HC-IA), or (HC-IB), [ka] teeth, [ka] be selected.

[0109] In some embodiments, in any of the above formulas, such as (HC-I), (HC-IA), or (HC-IB), [ka] teeth, [ka] is selected from the group consisting of:

[0110] In some embodiments, the head group of the lipid has the structure of formula (HC-IIA): [ka] (HC-IIA). R 7 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl. The definitions of all other variables in (HC-IIA) are the same as defined above in (HC-I).

[0111] In some embodiments, the head group of the lipid has the structure of formula (HC-IIA'): [ka] (HC-IIA'). R 7 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl. The definitions of all other variables in (HC-IIA') are the same as defined above in (HC-I).

[0112] In some embodiments, the head group of the lipid has the structure of formula (HC-IIC): [ka] (HC-IIC). R 7 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl. The definitions of all other variables in (HC-IIC) are the same as defined above in (HC-I).

[0113] In some embodiments, the head group of the lipid has the structure of formula (HC-IIC'): [ka] (HC-IIC'). R 7 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl. The definitions of all other variables in (HC-IIC') are the same as defined above in (HC-I).

[0114] 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, -O-, or -C(O)-.

[0115] 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)-.

[0116] In some embodiments, the head group of the lipid is a structure of one of the following formulas: [ka] (HC-IIIA) or [ka] (HC-IIIC). The definitions of all variables are the same as those defined above in (HC-I).

[0117] In some embodiments, the head group of the lipid is a structure of one of the following formulas: [ka] (HC-IIIA') or [ka] (HC-IIIC'). The definitions of all variables are the same as those defined above in (HC-I).

[0118] 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, -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 )-, wherein 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-.

[0119] In some embodiments, the head group of the lipid is [ka] (HC-IIID) or [ka] (HC-IIIE) where t1 is 0, 1, 2, or 3. The definitions of the other variables in these formulas are the same as those defined above for (HC-I).

[0120] In some embodiments, the head group of the lipid is [ka] (HC-IIID'), [ka] (HC-IIIE') wherein the definitions of the variables in these formulas are the same as defined above for (HC-I).

[0121] 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.

[0122] 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.

[0123] 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-IIIE), and (HC-IIIE'), W is OH, [ka]

[0124] In some embodiments, W is [ka] wherein Q is absent or -(CH2) q -C(R 7 )2-, or -N(R 7 ) where q is 0 or 1, and R 7 is H or methyl, and R 8 are each independently H or C1-C3 alkyl. In one embodiment, W is [ka] is.

[0125] In some embodiments, W is [ka] wherein Q is absent or -(CH2) q -C(R 7 )2-, or -N(R 7 ) where q is 0 or 1, and R 7 is H or methyl, and R 8 are each independently H or C1-C3 alkyl. In one embodiment, W is [ka] is.

[0126] In some embodiments, W is [ka] wherein Q is absent or -(CH2) q -C(R 7 )2-, or -N(R 7 ) where q is 0 or 1, and R 7 is H or methyl, and R 8 are each independently H or C1-C3 alkyl. In one embodiment, W is [ka] is.

[0127] In some embodiments, W is [ka] where Q is -(CH2) q -C(R 7 )2-, q is 0 or 1, and R 7 is H or methyl, and R 8 are each independently H or C1-C3 alkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] W is [ka] is. In some embodiments, W is [ka] where q is 0 and R 8 are each independently H, C1-C3 alkyl, hydroxyalkyl, heterocyclyl, or heteroaryl, optionally substituted with one or more alkyl. [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0128] In some embodiments, W is [ka] or [ka] where R 6 are each independently H, C1-C3 alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, or -O-alkylene-N(R 7 )2 and R 7 are each independently H or C1-C3 alkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0129] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0130] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0131] In some embodiments, W is [ka] [ka] [ka] where R 6 are each independently H, C1-C3 alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, or -O-alkylene-N(R7)2, and Q is -O-, -C(R7)2-, or -N(R 7 ) and R7 is H, C1-C3 alkyl, or hydroxyalkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka]

[0132] In some embodiments, W is [ka] where q is 0 and R 8 are each independently H, C1-C3 alkyl, or hydroxyalkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0133] In some embodiments, W is [ka] where R6 are each independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, or -O-alkylene-N(R 7 )2 and R 7 are each independently H or C1-C3 alkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0134] In some embodiments, W is [ka] where R 8 are each independently H, C1-C3 alkyl, or hydroxyalkyl, and each Q is independently absent, —O—, —CO—, —C(R 7 )2-, or -N(R 7 )- and R 7are each independently H, C1-C3 alkyl, alkylamino, alkylaminoalkyl, or aminoalkyl. [ka] In one embodiment, W is [ka] is.

[0135] In some embodiments, W is [ka] where R 8 are each independently H, C1-C3 alkyl, or hydroxyalkyl, and each Q is independently absent, —O—, —CO—, —C(R 7 )2-, or -N(R 7 )- and R 7 are each independently H, C1-C3 alkyl, alkylamino, alkylaminoalkyl, or aminoalkyl. [ka] In one embodiment, W is [ka] is.

[0136] In some embodiments, provided herein are lipids comprising at least one head group and at least one tail group, wherein: The tail group has the formula (TI) or (TI') [ka] (TI) or [ka] (TI'), a pharmaceutically acceptable salt thereof, or a compound having the structure of any of the aforementioned stereoisomers; During the ceremony, each E is independently a biodegradable group; R a are each independently C1 to C5 alkyl, C2 to C5 alkenyl, or C2 to C5 alkynyl; u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; R t are independently H, C1 to C 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted by OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; [ka] represents the bond connecting the tail group to the head group, The head group has a structure according to one of the following formulas: i) [ka] (HA-I), During the ceremony, R 20 and R 30 are each independently H, a C1-C5 branched or unbranched alkyl, or a C2-C5 branched or unbranched alkenyl, optionally interrupted with one or more heteroatoms or substituted with OH, SH, halogen, or a cycloalkyl group; or 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 and optionally substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl groups R1 and R2 are each 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; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, 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 or O, S, or NR 12 where R 12 is H or C1-C7 branched or unbranched alkyl, provided that when Z is absent, adjacent R1 and R2 are OH, NR 10 R 11 , SH should not be, ii) [ka] (HA-V) or [ka] (HA-VI). During the ceremony, R1 is H, C1-C3 alkyl, OH, halogen, SH, or NR 10 R 11 and R2 is OH, halogen, SH, or NR 10 R 11 or R1 and R2 together can form a ring; R 10 and R 11 are each independently H or C1-C3 alkyl, or R 10 and R 11 may together form a heterocycle, R 20 and R 30are each independently H, C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl, or R 20 and R 30 may be taken together to form a ring, v and y are each independently 1, 2, 3, or 4; iii) [ka] (HB-I), In the formula, W is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] During the ceremony, R5 is OH, SH, (CH2) s OH, or NR 10 R 11 and R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; R7 and R8 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, (CH2) v OH, (CH2) v SH, (CH2) s N(CH3)2, or NR 10 R 11 where R 10 and R 11 are each independently H or C1-C3 alkyl, or R 10 and R 11 together form a heterocycle, or R7 and R8 together form a ring; R 20 are each independently H or C1-C3 branched or unbranched alkyl, R 14 is heterocyclic, NR 10 R 11 , C(O)NR 10 R 11 , N.R. 10 C(O)NR 10 R 11 , or NR 10 C(S)NR 10 R 11 and 、 In the formula, R 10 and R 11 are each independently H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, optionally substituted with 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; s, u, and t are each 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 12is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl; Q is O, S, CH2, or NR 13 where R 13 are each H or C1-C5 alkyl, V is branched or unbranched C2 to C 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Alkynylene, or C2-C 10 heteroalkylene, optionally substituted with one or more OH, SH, and / or halogen groups; T is -NHC(O)O-, -OC(O)NH-, or a divalent heterocycle; iv) [ka] (HC-I), During the ceremony, [ka] is a cyclic or heterocyclic moiety, Y is alkyl, hydroxy, hydroxyalkyl, [ka] [ka] and A is absent or -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 -SS-; X and Z are each independently absent, —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-, R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl, or aminoalkyl; t1 is an integer from 0 to 10, W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl; Lipids have a pKa of about 4 to about 8.

[0137] In some embodiments, provided herein are lipids comprising at least one head group and at least one tail group, wherein: At least one tail group has a structure of at least one of the following formulas: [ka] (TII), [ka] (TIII), [ka] (TIV), [ka] (TV), [ka] (TII'), and [ka] (TIII'), During the ceremony, R 7 are each independently H or methyl; R b is independently at each occurrence H or C1-C4 alkyl; R a are each independently C1 to C5 alkyl, C2 to C5 alkenyl, or C2 to C5 alkynyl, u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6, or 7;

[0138] The head group has a structure of one of the following formulas: i) [ka] (HA-IA), wherein m is 1, 2, 3, 4, 5, 6, 7, or 8; ii) [ka] (HA-VI). iii) [ka] (HB-I), and iv) [ka] (HC-IB).

[0139] In some embodiments, in the lipids above, at least one tail group has a structure of formula (TII), (TIII), (TIV), (TV), (TII′), or (TIII′), where R a are each methyl, u1 is 3 to 5, u2 is 0 to 3, and u3 and u4 are each independently 1 to 7.

[0140] In some embodiments, in the lipids above, the head group has a structure of one of the following formulas: i) [ka] (HA-III), ii) [ka] (HA-VI), In the formula, R 20 and R 30 are each independently C1 to C3 alkyl. iii) [ka] During the ceremony, R6, R 7 and R8 are each independently H or methyl; u and t are each independently 1, 2, or 3; or W is, [ka] where: R 16 is H or =O, R 14 is a nitrogen-containing 5- or 6-membered heterocyclic ring, NR 10 R 11 , C(O)NR 10 R 11 , N.R. 10 C(O)NR 10 R 11 , or NR 10 C(S)NR 10 R 11 and 、 In the formula, R 10 and R 11 are each independently H or C1-C3 alkyl, u and v are each independently 1, 2, or 3; W is, [ka] R6 is independently H or methyl; R 7 are each independently H, Each R8 is methyl; each u is independently 1, 2, or 3; V is C2-C6 alkylene or C2-C6 alkenylene, or W is, [ka] or [ka] where: each u is independently 1, 2, or 3; Z is independent of each other, NR 12 and T is a divalent nitrogen-containing 5- or 6-membered heterocycle; iv) [ka] (HC-IIA) or [ka] (HC-IIC) where: W is hydroxyl, substituted or unsubstituted hydroxyalkyl; [ka] Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, or -(CH2) q -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and R6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, thiol, thiolalkyl, or N+(R 7 ) 3-alkylene-Q-; R 8 are each independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, heterocyclyl, heteroaryl, or together with the nitrogen atom, two R 8 form a ring optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, or aminoalkyl; q is 0, 1, 2, 3, 4, or 5; p is 0, 1, 2, 3, 4, or 5.

[0141] Another aspect of the invention is a lipid comprising at least two lipophilic tail groups and a head group of formula (G-HC-IIID): [ka] With respect to a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, During the ceremony, R a are each independently C1-C5 alkyl, C2-C5 alkenyl, or C2-C5 alkynyl, t2 is an integer between 0 and 5, W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl; [ka] represents the bond connecting the head group to the tail group.

[0142] In some embodiments, R a are methyl, and t2 is 0 to 3.

[0143] In some embodiments, W is hydroxyl, substituted or unsubstituted hydroxyalkyl, or [ka] Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, or -(CH2) q -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, thiol, thiolalkyl, or N+(R 7 ) 3-alkylene-Q-; R 8 are each independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, heterocyclyl, heteroaryl, or together with the nitrogen atom, two R 8 form a ring optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, or aminoalkyl; q is 0, 1, 2, 3, 4, or 5; p is 0, 1, 2, 3, 4, or 5.

[0144] In some embodiments, W is [ka] is.

[0145] In one embodiment, the lipid has the structure: [ka]

[0146] Also, nucleic acids, one or more lipid compounds comprising at least one head group (e.g., HA-I through HA-VII, HB-I, or HC-I through HC-IIIE, or any subgenus or species of these formulas disclosed herein) and at least one tail group of formula (T through TIII, or TI' through TIII', or any subgenus or species of these formulas disclosed herein); Helper lipids and Sterols and Disclosed herein are nucleic acid-lipid particles comprising:

[0147] Also disclosed herein are pharmaceutical compositions comprising lipid particles and a pharmaceutically acceptable diluent, wherein the lipid particles are Nucleic acids, 35-65 mol% of one or more lipid compounds comprising at least one head group (e.g., HA-I through HA-VII, HB-I, or HC-I through HC-IIIE, or any subgenus or species of these formulas disclosed herein) and at least one tail group of formula (T through TIII, or TI' through TIII', or any subgenus or species of these formulas disclosed herein); 3-12 mol% of helper lipids, 15-45 mol% sterol, and 0.5-10 mol % of a PEG-modified lipid.

[0148] Also disclosed herein are pharmaceutical compositions comprising one or more lipid compounds comprising at least one head group (e.g., HA-I through HA-VII, HB-I, or HC-I through HC-IIIE, or any subgenus or species of these formulas disclosed herein) and at least one tail group of the formula (T through TIII, or TI' through 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 may be useful for forming lipid nanoparticles for delivery of therapeutic agents.

[0149] In some embodiments, the present disclosure provides methods for delivering a therapeutic agent to a patient in need thereof, comprising one or more lipid compounds comprising at least one head group (e.g., HA-I through HA-VII, HB-I, or HC-I through HC-IIIE, or any subgenus or species of these formulas disclosed herein) and at least one tail group of the formula (T through TIII, or TI' through TIII', or any subgenus or species of these formulas disclosed herein), pharmaceutically acceptable salts thereof, and / or stereoisomers of any of the foregoing, and a therapeutic agent. In some embodiments, the method further includes preparing a lipid nanoparticle composition comprising one or more lipid compounds comprising at least one head group (e.g., HA-I through HA-VII, HB-I, or HC-I through HC-IIIE, or any subgenus or species of these formulas disclosed herein) and at least one tail group of the formula (T through TIII, or TI' through 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.

[0150] In some embodiments, the total therapeutic cargo administered to a subject has a spleen-to-liver ratio of 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 total therapeutic cargo administered to a subject has a spleen-to-liver ratio of at least 1. In some embodiments, the total therapeutic cargo administered to a subject has a spleen-to-liver ratio of at least 5.

[0151] These and other aspects of the present disclosure will become evident upon reference to the following detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0152] definition As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0153] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0154] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0155] Unless the context requires otherwise, throughout this specification and claims, the term "comprise" and variations thereof, such as "comprises" and "comprising," are to be interpreted in their open and inclusive sense, i.e., "including but not limited to."

[0156] 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 determine the level of protein expression, a test sample (e.g., a sample of cells in culture that express the desired protein) or a test mammal (e.g., a mammal such as a human or animal) model, such as a rodent (e.g., a mouse) or 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 sample of cells in culture that express the desired protein) or a control mammal (e.g., a mammal such as a human or animal) model, such as a rodent (e.g., a mouse) or non-human primate (e.g., a monkey) model that is not contacted with or administered with a nucleic acid. If the desired protein is present in the control sample or control mammal, the expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In some embodiments, induction of expression of a desired protein is achieved when the ratio of the expression of the desired protein in a test sample or test mammal to the level of expression of the desired protein in a control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. If the desired protein is not present in a control sample or control mammal, detection of any measurable level of the desired protein in the test sample or test mammal is achieved. Those skilled in the art will understand suitable assays for determining the level of protein expression in a sample, such as assays based on reporter proteins that can generate fluorescence or luminescence under appropriate conditions, for example, dot blots, Northern blots, insitu hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays.

[0157] The phrase "inhibit 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 determine the degree of gene silencing, a test sample (e.g., a sample of cells in culture that express the target gene) or a test mammal (e.g., a mammal such as a human or 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 sample of cells in culture that express the target gene) or a control mammal (e.g., a mammal such as a human or 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 administered with nucleic acid.The expression of the target gene in the control sample or control mammal may be assigned a value of 100%. In some embodiments, silencing, inhibition, or reduction of expression of a target gene is achieved when the level of target gene expression in a test sample or test mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to the level of target gene expression in a control sample or control mammal. In other words, compared with the level of target gene expression in a control sample or a control mammal that is not contacted with or administered with nucleic acid, the nucleic acid can silence, reduce or inhibit the expression of target gene in a test sample or a test mammal 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 target gene expression include, but are not limited to, testing protein or mRNA levels using techniques known to those skilled in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

[0158] 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 expression of a target sequence compared to the normal expression level detected in the absence of the nucleic acid. Increased expression of a target sequence is achieved when any measurable level of expression product not present in the absence of the nucleic acid is detected. When the expression product is present at a certain level before contact with the nucleic acid, increased expression is achieved when the fold increase in the value obtained with the nucleic acid, such as mRNA, compared to the control is 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, 5000, 10000, or more. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid, such as an antisense oligonucleotide, is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to a control. Suitable assays for measuring expression of a target gene or target sequence include, for example, examination of protein or RNA levels using techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of appropriate reporter proteins, and phenotypic assays known to those of skill in the art.

[0159] 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, including DNA, RNA, and hybrids thereof. DNA may be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, polyvalent 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, natural, or unnatural, and have similar binding properties to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-0-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the 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 set forth 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-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. CHem., 260:2605-2608 (1985); 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 linked via a phosphate group.

[0160] "Bases" include 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, including, but not limited to, modifications that place new reactive groups such as amines, alcohols, thiols, carboxylic acids, and alkyl halides.

[0161] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises partial or full-length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

[0162] "Gene product," as used herein, refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0163] The term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, generally characterized by poor solubility in water but solubility in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derivatives," such as steroids.

[0164] A "steroid" is a compound containing the following carbon skeleton: [ka] As used herein, the term "compound" is meant to include all isomers and isotopes of the depicted structure, all pharmaceutically acceptable salts, solvates, or hydrates thereof, and all crystalline forms (e.g., crystalline polymorphs), mixtures of crystalline forms, or anhydrates or hydrates thereof.

[0165] "Isotopes" refer to atoms having the same atomic number but different mass numbers, resulting from different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium (3H) and deuterium (2H). "Isomers." The compounds described herein, or pharmaceutically acceptable salts thereof, can include all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, and the like. For example, the compounds can contain one or more stereocenters and thus give rise to geometric isomers (e.g., double bonds giving rise to geometric E / Z isomers), enantiomers, diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers), and other stereoisomeric configurations that can be defined in terms of absolute stereochemistry, such as (R)- or (S)-, such as sugar isomers, or (D)- or (L)-, such as amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-) isomers, (R) and (S) isomers, or (D) and (L) isomers may be prepared using chiral synthesis or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of a racemate (or a racemate of a salt or derivative), using, for example, chiral high-pressure liquid chromatography (HPLC). Enantiomeric and stereoisomeric mixtures of compounds, as well as means for resolving them into their component enantiomers or stereoisomers, are well known. When compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers. Likewise, all tautomeric forms are intended to be included.

[0166] The terms "crystalline polymorph," "polymorph," or "crystalline form" refer to crystalline structures in which a compound (or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, density hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors may govern a single crystalline form. Crystalline polymorphs of a compound can be prepared by crystallization under different conditions. Crystallization of the compounds disclosed herein may produce solvates.

[0167] As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of an ionizable lipid of the present disclosure with one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, etc. Alternatively, the solvent may be an organic solvent.

[0168] As used herein, ionizable lipid refers to a lipid that can be charged. In some embodiments, the ionizable lipid comprises one or more positively charged amine groups. In some embodiments, the ionizable lipid is ionizable so that it can exist in a positively charged form or a neutral form depending on the pH. The ionization of the ionizable lipid affects the surface charge of lipid nanoparticles that contain the ionizable lipid under different pH conditions. The surface charge of lipid nanoparticles can affect their plasma protein absorption, blood clearance, and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)), as well as their ability to form endosomal non-bilayer structures, which can affect the intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)).

[0169] The term "polymer-conjugated lipid" refers to a molecule that contains both a lipid portion and a polymer portion. A non-limiting example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include, for example, l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG). As used herein, the terms "PEG lipid" and "PEGylated lipid" are interchangeable and refer to a lipid that contains a polyethylene glycol moiety.

[0170] The term "neutral lipid" refers to any lipid that exists either uncharged or neutral zwitterionic at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines, 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), phosphophytidylethanolamines, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramides, and steroids, such as sterols and their derivatives. Neutral lipids may be synthetic or naturally occurring.

[0171] As used herein, a "phospholipid" is a lipid containing a phosphate moiety and one or more carbon chains, such as an unsaturated fatty acid chain. A phospholipid may contain one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). Certain phospholipids may facilitate fusion with membranes. For example, cationic phospholipids may interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or intracellular membrane). The fusion of a phospholipid with a membrane may allow one or more components of a lipid-containing composition to pass through the membrane, for example, allowing one or more components to be delivered to a cell.

[0172] As used herein, the term "liposome" refers to a composition comprising an outer lipid membrane (e.g., a single lipid bilayer, known as a unilamellar liposome, or multiple lipid bilayers, known as a multilamellar liposome) enclosing an internal aqueous space that may contain cargo. See, e.g., Cullis et al., Biochim. Biophys Acta, 559: 399-420 (1987), which is incorporated herein by reference in its entirety. Unilamellar liposomes generally have diameters ranging from about 20 to about 400 nanometers (nm), about 50 to about 300 nm, about 100 to about 200 nm, or about 300 to about 400 nm. Multilamellar liposomes typically have diameters ranging from about 1 to about 10 μm and may contain two to several hundred concentric lipid bilayers alternating with layers of aqueous phase.

[0173] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) and comprising 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 any stereoisomers thereof, are included in compositions that can be used to deliver therapeutic agents, such as nucleic acids (e.g., mRNA), to target sites in a subject (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, lipid nanoparticles comprise one or more compounds of Formula (I), pharmaceutically acceptable salts thereof, and / or any stereoisomers thereof, and nucleic acids. In some embodiments, lipid nanoparticles comprise one or more compounds of Formula (I), pharmaceutically acceptable salts thereof, and / or any stereoisomers thereof, and nucleic acids, as well as 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, may be encapsulated in the lipid portion of the lipid nanoparticle, or in the aqueous space surrounded by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the host organism's or cellular machinery, e.g., a harmful immune response.

[0174] In some embodiments, the lipid nanoparticles are about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or have an average diameter of 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 are substantially non-toxic. In some embodiments, the nucleic acid, when present in the lipid nanoparticle, is resistant in aqueous solution to degradation by nucleases.Lipid nanoparticles containing nucleic acids and methods for preparing them are described, for example, in U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031, and PCT International Publication Nos. 2013 / 016058 and 2013 / 086373, U.S. Patent Nos. 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, No. 2014 / 0200257, No. 2013 / 086373, No. 2013 / 0338210, No. 2013 / 0323269, No. 2013 / 0245107, No. 2013 / 019592 No. 0, No. 2013 / 0123338, No. 2013 / 0022649, No. 2013 / 0017223, No. 2012 / 0295832, No. 2012 / 0183581, No. 2012 / 01 No. 72411, No. 2012 / 0027803, No. 2012 / 0058188, No. 2011 / 0311583, No. 2011 / 0311582, No. 2011 / 0262527, No. 201 No. 1 / 0216622, No. 2011 / 0117125, No. 2011 / 0091525, No. 2011 / 0076335, No. 2011 / 0060032, No. 2010 / 0130588, Nos. 2007 / 0042031, 2006 / 0240093, 2006 / 0083780, 2006 / 0008910, 2005 / 0175682, 2005 / 017054, 2005 / 0118253, 2005 / 0064595, 2004 / 0142025, 2007 / 0042031, 1999 / 009076 and PCT International Publication Nos. Nos. 99 / 39741, 2017 / 117528, 2017 / 004143, 2017 / 075531, 2015 / 199952, 2014 / 008334, 2013 / 086373, 2013 / 086322, 2013 / 016058, 2013 / 086373, 2011 / 141705, and 2001 / 07548, the entire disclosures of which are incorporated herein by reference for all purposes.

[0175] As used herein, the term "size" refers to the hydrodynamic diameter of a lipid nanoparticle population. Measurement of the size of a lipid nanoformulation can be used to indicate the size and population distribution (polydispersity index, PDI) of the composition.

[0176] As used herein, "polydispersity index" is the ratio between the weight average molar mass and the number average molar mass, Mn, which describes the homogeneity of the particle size distribution of a system. For example, a small value less than 0.3 indicates a narrow particle size distribution.

[0177] The polydispersity index can be used to indicate the homogeneity of a lipid composition (e.g., liposomes or LNPs), e.g., the particle size distribution of the liposomes or LNPs. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The lipid composition can have a polydispersity index of about 0 to about 0.25, e.g., 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 about 0.10 to about 0.20.

[0178] As used herein, the term "apparent pKa" refers to the pH at which a lipid nanoformulation (e.g., LNP) is 50% protonated. This can be used as an indicator of the pH range at which a lipid nanoformulation (e.g., LNP) is protonated and can therefore initiate the endosomal escape process in nucleotide delivery.

[0179] As used herein, the term "zeta potential" refers to the electrokinetic potential of lipids, for example, in lipid nanoformulations (e.g., LNP compositions). Zeta potential can describe the surface charge of LNP compositions. Zeta potential is useful for predicting organ tropism and potential interactions with serum proteins.

[0180] The zeta 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 zeta potential can describe the surface charge of the liposome or LNP. Lipid compositions (e.g., liposome or LNP) with a relatively low charge, positive or negative, are generally desirable, as more highly charged species can undesirably interact with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the liposome or LNP can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0181] As used herein, "encapsulated" by a lipid refers to a therapeutic agent, such as a nucleic acid (e.g., mRNA), that is fully or partially encapsulated by a lipid nanoparticle. In some embodiments, the nucleic acid (e.g., mRNA) is fully encapsulated within the lipid nanoparticle.

[0182] As used herein, "encapsulation efficiency" or "encapsulation efficiency" refers to the percentage of encapsulated cargo (e.g., a therapeutic agent and / or prophylactic agent) successfully incorporated into (e.g., encapsulated in, or otherwise associated with) a lipid composition (e.g., an LNP or liposome) relative to the initial total amount of therapeutic agent and / or prophylactic agent provided. For example, if 97 mg of therapeutic agent and / or prophylactic agent is encapsulated in a lipid composition out of a total of 100 mg of therapeutic agent and / or prophylactic agent initially provided, the encapsulation efficiency can be given as 97%. Encapsulation efficiency can be used to indicate the efficiency of encapsulated cargo (e.g., a nucleic acid molecule) loaded into a lipid composition using a particular formulation method and recipe.

[0183] The encapsulation efficiency of cargo, such as proteins and / or nucleic acids, describes the amount of protein and / or nucleic acid encapsulated or otherwise associated with a lipid composition (e.g., liposomes or LNPs) after preparation relative to the initial amount provided. A high encapsulation efficiency (e.g., at least 70%, 80%, 90%, approaching 95%, or 100%) is desirable. 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 dissolving the liposomes or LNPs with one or more organic solvents or detergents. Anion exchange resins can also be used to measure the amount of free protein or nucleic acid (e.g., RNA) in solution. Fluorescence can also 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 proteins and / or nucleic acids may be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0184] "Serum stability" in reference to nucleic acid-lipid nanoparticles means that the nucleic acid is not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0185] Some administration techniques may lead to systemic delivery of certain drugs, but not others. "Systemic delivery" means that a useful amount of a drug, such as a therapeutic agent, is delivered to most of the body. Systemic delivery of lipid nanoparticles can be achieved by any means 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.

[0186] As used herein, "local delivery" refers to the direct delivery of a drug to a target site within an organism.For example, a drug can be delivered locally by direct injection into a disease site such as a tumor, other target sites such as an inflammation site, or target organs such as the liver, heart, pancreas, or kidney.Local delivery can also include topical application or local injection techniques such as intramuscular, subcutaneous, or intradermal injection.Local delivery does not interfere with systemic pharmacological effects.

[0187] As used herein, "administration method" can include both systemic delivery and local delivery. "Systemic delivery" means that a useful amount of a drug, such as a therapeutic agent, is delivered to most of the body. Systemic delivery of liposomes or LNPs can be performed by any means known in the art, including, for example, intravenous, intraarterial, 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 the direct delivery of a drug to a target site within an organism. For example, a drug can be delivered locally by direct injection into a disease site such as a tumor, another target site such as a site of inflammation, or a target organ such as the liver, heart, pancreas, or kidney. Local delivery can also include topical administration or local injection techniques such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not interfere with systemic pharmacological effects.

[0188] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues typically joined by peptide bonds, which may be naturally (e.g., isolated or purified) or synthetically produced.

[0189] "Nucleic acid" is meant to define an oligonucleotide or polynucleotide sequence. Non-limiting examples of oligonucleotides or polynucleotides are DNA, plasmid DNA, self-amplifying RNA, mRNA, siRNA, and tRNA. The term also encompasses RNA / DNA hybrids. Nucleotides are typically linked in nucleic acids by phosphodiester bonds, but the term "nucleic acid" also encompasses nucleic acid analogs with other types of bonds or backbones (e.g., phosphoramide, phosphorothioate, phosphorodithioate, O-methylphosphoramidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threos nucleic acid (TNA), and peptide nucleic acid (PNA) bonds or backbones, among others). Nucleic acids may be single-stranded, double-stranded, or contain portions of both single-stranded and double-stranded sequences. Nucleic acids can contain any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including, for example, adenine, thymine, cytosine, guanine, uracil, and modified or non-standard bases, including, for example, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

[0190] As used herein, "RNA" refers to ribonucleic acid, which may be natural or non-natural. For example, RNA may contain modifications and / or non-natural components, such as one or more nucleic acid bases, nucleosides, nucleotides, or linkers. RNA may contain a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, for example, in vivo translation of an mRNA within a mammalian cell, can produce the encoded polypeptide. The 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.

[0191] "Alkyl" refers to and has a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, e.g., 1-24 carbon atoms (C1-C 24 alkyl), 4 to 20 carbon atoms (C4 to C 20 alkyl), 6 to 16 carbon atoms (C6 to C 16 alkyl), 6 to 9 carbon atoms (C6 to C9 alkyl), 1 to 15 carbon atoms (C1 to C 15 alkyl), 1 to 12 carbon atoms (C1 to C 12 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), or 1 to 6 carbon atoms (C1-C6 alkyl) and 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 (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless stated otherwise in the specification, alkyl groups are optionally substituted.

[0192] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain, consisting of and having only carbon and hydrogen, connecting the remainder of the molecule to a radical, e.g., 1 to 24 carbon atoms (C1 to C6). 24 Alkylene, 1 to 15 carbon atoms (C1 to C 15 Alkylene, 1 to 12 carbon atoms (C1 to C 12 alkylene), 1 to 8 carbon atoms (C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), 1 to 2 carbon atoms (C1-C2 alkylene), for example, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the free radical through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the free radical may be through one carbon or any two carbons within the chain.

[0193] The term "alkenyl" refers to a straight or branched 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). Typical examples of alkenyl include, but are not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl, and 3-octenyl groups. The term "alkynyl" refers to a straight or branched hydrocarbon chain containing 2 to 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, and 3-methylbutynyl, as well as propargyl. 2 and sp 3 The carbons can optionally serve as the point of attachment for the alkenyl and alkynyl groups, respectively.

[0194] As used herein, the term "cycloalkyl" or "cyclyl" includes saturated and partially unsaturated, but not aromatic, cyclic hydrocarbon groups having 3 to 12 carbons, e.g., 3 to 8 carbons, and e.g., 3 to 6 carbons, where the cycloalkyl groups may additionally be optionally substituted. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0195] The terms "heteroaryl" or "heteroar-" refer to an aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms in the monocyclic ring, 1 to 6 heteroatoms in the bicyclic ring, or 1 to 9 heteroatoms in the tricyclic ring, selected from O, N, or S (e.g., carbon atoms and 1 to 3, 1 to 6, or 1 to 9 heteroatoms of N, O, or S in the monocyclic, bicyclic, or tricyclic ring), and 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 group or point of attachment is on the heteroaromatic ring. Examples of heteroaryl groups include pyrrolyl, pyridyl, pyridazinyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, pyrazinyl, indolizinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, isothiazolyl, thiadiazolyl, purinyl, naphthyridinyl, pteridinyl, isoindolyl, benzothiazole, and the like. enyl, benzofuranyl, dibenzofuranyl, indazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one.

[0196] The term "heterocyclyl" refers to a 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, selected from O, N, or S (e.g., represented by carbon atoms and 1-3 groups, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), where 0, 1, 2, or 3 atoms in each ring may be substituted by substituents. As used herein, it generally can include both non-aromatic rings (e.g., those generally covered by heteroaryl) or aromatic rings. The term also includes groups in which a heterocycle is fused to one or more aryl, cycloalkyl, or heterocyclyl rings. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in n-substituted pyrrolidinyl).

[0197] Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, pyrrolidinyl, pyrrolidinyl, pyrrolinyl, pyrrolinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, quinuclidinyl, and the like.

[0198] Examples of heterocyclyl groups include pyrrolyl, pyridyl, pyridazinyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, pyrazinyl, indolizinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, isothiazolyl, thiadiazolyl, purinyl, naphthyridinyl, pteridinyl, isoindolyl, benzothienyl, Typical heteroaryl groups include benzofuranyl, dibenzofuranyl, indazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one.

[0199] Divalent alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl radicals are formed by removing a hydrogen atom from an alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl group, respectively (or by removing two hydrogen atoms from an alkane, alkene, arene, heteroarene, cycloalkane, or heterocycle, respectively).

[0200] The term "alkoxy" refers to an --O-alkyl group.

[0201] The term "aminoalkyl" refers to an alkyl substituted with an amino. The term "alkylamino" refers to an amino substituted with an alkyl.

[0202] The term "aminocarbonyl" refers to the group --C(O)-amino.

[0203] As used herein, the term substituted refers to any of the above groups (e.g., alkyl, hydroxyalkyl, alkylene, cycloalkyl, cycloalkylene, amino, aminocarbonyl, heterocyclyl, or heteroaryl) in which one or more hydrogen atoms have been replaced with, but are not limited to, a halogen atom such as F, CI, Br, or I, an oxo group (=O), a hydroxyl group (-OH), an alkoxy, alkoxyalkyl, aralkoxy, C1-C 12 Alkyl such as alkyl, cycloalkyl group, alkenyl, alkynyl, aryl, aralkylheterocyclyl, heterocyclyl, 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, aralkoxycarbonyl, sulfonyl, alkylaminolactam, alkylaminoheteroaryl, alkylaminoheterosil, and aminosulfonamido. Exemplary substituents also include -(C=O)OR, -O(C=O)R, -C(=O)R, -OR, -S(O)xR, -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)XR'R, -R'S(O) X R, and -S(O) x R, R', and R" are independently selected at each occurrence from H, C1 to C 15 alkyl or cycloalkyl, heterocyclyl, or optionally substituted heteroaryl, where x is 0, 1, or 2. In some embodiments, the substituent is C1-C 12In some embodiments, the substituent is an alkyl group. In some embodiments, the substituent is a cycloalkyl group. In some embodiments, the substituent is a halo group, such as fluoro. 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, =0, =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 selected from hydrogen, substituted or unsubstituted C1-6 alkyl, or an unsubstituted 5-6 membered saturated or partially unsaturated ring, or an aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0204] "Halo" or "halogen" refers to any radical of fluorine, chlorine, bromine, or iodine.

[0205] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described circumstance event may or may not occur, and the description includes instances where the event or circumstance occurs as well as instances where it does not. For example, "optionally substituted alkyl" means that the alkyl group may be substituted or unsubstituted, and that the specification includes both substituted and unsubstituted alkyl groups.

[0206] The present disclosure is also intended to encompass all pharmaceutically acceptable compounds of any formula specified herein that are isotopically labeled by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, for example, 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 Isotopically labeled compounds may be useful for determining or measuring the efficacy of compounds, for example, by characterizing the site or mode of action, or binding affinity to pharmacologically important sites of action. Certain isotopically labeled compounds are useful for drug and / or substrate tissue distribution studies. The radioactive isotope tritium, i.e. 、3 H, and carbon-14, i.e., 14 C may be useful for this purpose in view of its ease of incorporation and rapid means of detection.

[0207] Deuterium, i.e., 2 Substitution with heavier isotopes, such as H, may offer certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and may therefore be useful in some circumstances.

[0208] 11 C. 18 F, 15 O, and 13Substitution with positron-emitting isotopes, such as N, can be useful in positron emission topography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds can generally be prepared by conventional techniques known to those skilled in the art, or by processes analogous to those described in the preparations and examples described below, using appropriate isotopically labeled reagents in place of previously employed non-labeled reagents.

[0209] The present disclosure is also intended to encompass in vivo metabolic products of the disclosed compounds. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily due to enzymatic processes. Accordingly, embodiments of the present disclosure include compounds produced by a process comprising administering an ionizable lipid of the present disclosure to a mammal for a period of time sufficient to yield its metabolic products. Such products are typically identified by administering a detectable dose of a radiolabeled compound of the present disclosure to an animal, such as a rat, mouse, guinea pig, monkey, or human, allowing sufficient time for metabolism to occur, and isolating the conversion products from urine, blood, or other biological sample.

[0210] A "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for human or veterinary use.

[0211] "Pharmaceutically acceptable salts" include both acid and base addition salts.

[0212] "Pharmaceutically acceptable acid addition salts" refers to salts that retain the biological effectiveness and properties of the free bases, and which are not biologically or otherwise undesirable, such as, for example, inorganic acids including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, 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, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptan-1,2-one, hydroxybenzoic acid ... and the like.

[0213] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acid and are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Non-limiting examples of inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, n-ethylpiperidine, polyamine resins, etc. Non-limiting examples of organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0214] Crystallization of the ionizable lipid(s) disclosed herein may produce a solvate. As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of the ionizable lipid of the present disclosure with one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present disclosure may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., as well as corresponding solvate forms. The solvates of the compounds of the present disclosure may be true solvates, while in other cases, the compounds of the present disclosure may simply retain extraneous water or may be a mixture of water and some extraneous solvent.

[0215] A "pharmaceutical composition" refers to a composition that may include an ionizable lipid of the present disclosure and a vehicle generally accepted in the art for delivery of a biologically active compound to a mammal, e.g., a human. Such a vehicle includes a pharmaceutically acceptable carrier, diluent, or excipient therefor.

[0216] "Effective amount" or "therapeutically effective amount" refers to the amount of ionizable lipid of the present disclosure that is sufficient to cause treatment in a mammal, such as a human, when administered to the mammal, such as a human. The amount of lipid nanoparticles of the present disclosure that constitutes a therapeutically effective amount varies depending on the compound, condition and its severity, administration method, and age of the mammal being treated, but can be routinely determined by those skilled in the art based on their own knowledge and the present disclosure.

[0217] As used herein, treatment covers the treatment of a disease or condition of interest in a mammal, such as a human, having the disease or condition of interest; (i) preventing a disease or condition from occurring in a mammal, particularly when such mammal is predisposed to, but has not yet been diagnosed as having, said condition; (ii) inhibiting said disease or condition, i.e., arresting its development; (iii) alleviating said disease or condition, i.e., causing regression of said disease or condition; or (iv) Relieving symptoms resulting from a disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably or may differ in that a particular disease or condition may not have a known causative agent (and thus, the etiology has not yet been determined) and therefore is not yet recognized as a disease, but only as an undesirable state or syndrome with a more or less specific set of symptoms identified by clinicians.

[0218] The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may contain one or more stereocenters and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined in terms of absolute stereochemistry as (R)- or (S)-, or (D)- or (L)- for amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-) isomers, (R) and (S) isomers, or (D) and (L) isomers may be prepared using chiral synthesis or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). When the compounds described herein contain an olefinic double bond or other center of geometric asymmetry, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers, as well as all tautomers.

[0219] "Stereoisomers" refer to compounds composed of the same atoms joined by the same bonds but with different three-dimensional structures, which are not 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 one another.

[0220] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the present disclosure. However, it will be understood by those skilled in the art that the present disclosure may be practiced without these details.

[0221] Exemplary Lipid Compounds In some embodiments, an ionizable lipid of formula (LA-I): [ka] (LA-I), Pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing are disclosed; During the ceremony, R1 and R2 are each 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; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or R 10 and R 11 together form a heterocycle, m is 1, 2, 3, 4, 5, 6, 7 or 8; n is 0, 1, 2, 3, or 4; Z is absent or O, S, or NR 12 where R 12 is H or C1-C7 branched or unbranched alkyl, provided that when Z is absent, adjacent R1 and R2 are OH, NR 10 R11 , SH should not be, Each A is independently a C1-C optionally substituted with a heteroatom or substituted with OH, SH, or halogen. 16 is a branched or unbranched alkyl; Each B is independently a C1-C optionally substituted with a heteroatom or substituted with OH, SH, or halogen. 16 is a branched or unbranched alkyl; X', [ka] [ka] [ka] or [ka] and X' is a biodegradable moiety.

[0222] In some embodiments, each X is [ka] or [ka] is.

[0223] In some embodiments, X' is -OCO-, -COO-, -NR 7 CO-, -CONR 7 -, -C(OR 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 R13 is C3~C 10 It is alkyl.

[0224] In some embodiments, at least one X in the formula [ka] [ka] [ka] [ka] where R 7 is H or methyl. In one embodiment, each X is [ka] or [ka] In one embodiment, each X is [ka] or [ka] where R 7 is H or methyl.

[0225] In some embodiments, the present disclosure provides an ionizable lipid of formula (LA-II): [ka] (LA-II), With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, R1 is independently H, C1-C3 alkyl, OH, halogen, SH, or NR 10 R11 and R1 and R2 can together form a ring, and R 10 and R 11 are each independently H or C1-C3 alkyl, and R 10 and R 11 can together form a heterocycle, R2 is independently H, C1-C3 alkyl, OH, halogen, SH, or NR 10 R 11 and R1 and R2 can together form a ring, and R 10 and R 11 are each independently H or C1-C3 alkyl, and R 10 and R 11 can together form a heterocycle, m is 1, 2, 3, 4, 5, 6, 7 or 8; n is 0, 1, 2, 3, or 4; each r is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; R3 are each independently H or C3 to C 10 is alkyl, R4 is independently H or C3 to C 10 alkyl, with the proviso that at least one of R3 and R4 is not H; Z is absent, O, S, or NR 12 where R 12 is a C1-C7 alkyl, X', [ka] [ka] where at least one X is [ka] or [ka] and X' is a biodegradable moiety.

[0226] In some embodiments, each X is [ka] or [ka] is.

[0227] In some embodiments, X' is -OCO-, -COO-, -NR 7 CO-, -CONR 7 -, -C(OR 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 It is alkyl.

[0228] In some embodiments, at least one X in the formula is [ka] [ka] [ka] [ka] where R 7 is H or methyl. In one embodiment, each X is [ka] or [ka] In one embodiment, each X is [ka] or [ka] where R 7 is H or methyl.

[0229] In some embodiments, the present disclosure provides an ionizable lipid of formula (LA-III): [ka] (LA-III) With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, R1 and R2 are each 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; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or R 10 and R 11 together form a heterocycle, R2 is independently H, C1-C3 alkyl, OH, halogen, SH, or NR 10 R 11 and R1 and R2 can together form a ring, and R 10 and R 11 are each independently H or C1-C3 alkyl, and R 10 and R 11 can together form a heterocycle, n is 0, 1, 2, 3, or 4; each r is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; R3 are each independently H or C3 to C 10 is alkyl, R4 is independently H or C3 to C 10 alkyl, with the proviso that at least one of R3 and R4 is not H; Z is absent, O, S, or NR 12 where R 12 is a C1-C7 alkyl, X', [ka] [ka] where at least one X is [ka] or [ka] and X' is a biodegradable moiety.

[0230] In some embodiments, each X is [ka] or [ka] is.

[0231] In some embodiments, X' is -OCO-, -COO-, -NR 7 CO-, -CONR 7 -, -C(OR 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 It is alkyl.

[0232] In some embodiments, at least one X in the formula is [ka] [ka] [ka] [ka] [ka] where R 7 is H or methyl. In one embodiment, each X is [ka] Also [ka] In one embodiment, each X is [ka] or [ka] where R 7 is H or methyl.

[0233] In some embodiments, the present disclosure provides an ionizable lipid of formula (LA-IV): [ka] (LA-IV), With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, each r is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; q is independently C1 to C 10 is alkyl, Z is absent, O, S, or NR 12 where R 12 is a C1-C7 alkyl.

[0234] In some embodiments, Z is absent. In some embodiments, Z is S. In some embodiments, Z is O. In some embodiments, Z is NH. In some embodiments, r is 3. In some embodiments, r is 4. In some embodiments, q is 3. In some embodiments, q is 4. In some embodiments, Z is absent, r is 4, and q is 4.

[0235] In some embodiments, the present disclosure provides an ionizable lipid of formula (LA-V): [ka] (LA-V), With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, R1 is H, C1-C3 alkyl, OH, halogen, SH, or NR 10 R 11 and R2 is OH, halogen, SH, or NR 10 R 11 or R1 and R2 together can form a ring; R 10 and R 11are each independently H or C1-C3 alkyl, or R 10 and R 11 may together form a heterocycle, 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 be joined together to form a ring, v and y are each independently 1, 2, 3, or 4; A and B are each independently C1 to C 16 Branched or unbranched alkyl, or C2-C 16 branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted with OH, SH, or halogen; X', [ka] [ka] where at least one X in the formula is [ka] or [ka] and X' is a biodegradable moiety.

[0236] In some embodiments, each X is [ka] is.

[0237] In some embodiments, X' is -OCO-, -COO-, -NRCO-, -CONR-, -C(O-R)-O-(acetal), -COO(CH)-, -CONH(CH)-, -C(O-R)-O-(CH)-, where R 7 is H or C1-C3 alkyl, and R 13 is C3~C 10 It is alkyl.

[0238] In some embodiments, at least one X in the formula is [ka] [ka] [ka] [ka] [ka] where R 7 is H or methyl. In one embodiment, each X is [ka] or [ka] In one embodiment, each X is [ka] or [ka] where R 7 is H or methyl.

[0239] In some embodiments, the present disclosure provides an ionizable lipid of formula (LA-VI): [ka] (LA-VI), With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, R 20 and R 30 are each independently H or C1-C5 alkyl, and R 20 and R 30 can be joined together to form a ring, v is 1, 2, 3, or 4; y is 1, 2, 3, or 4; R3 are each independently H or C3 to C 10 is alkyl, R4 is independently H or C3 to C 10 alkyl, with the proviso that at least one of R3 and R4 is not H; each r is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; X', [ka] [ka] where at least one X is [ka] or [ka] and X' is -OCO-, -COO-, -NR7CO-, -CONR7-, -C(O-R13)-O- (acetal), -COO(CH2)s-, -CONH(CH2) s -, or -C(OR 13 )-O-(CH2) s -, wherein R 7is H or C1-C3 alkyl, and R 13 is C3~C 10 It is alkyl.

[0240] In some embodiments, each X is [ka] or [ka] is.

[0241] In some embodiments, at least one X in the formula is [ka] [ka] [ka] [ka] where R 7 is H or methyl. In one embodiment, each X is [ka] or [ka] In one embodiment, each X is [ka] or [ka] where R 7 is H or methyl.

[0242] In some embodiments, the present disclosure provides an ionizable lipid of formula (LA-VII): [ka] (LA-VII), With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, R 20 and R 30 are each independently H or C1-C5 alkyl, and R 20 and R 30 can be joined together to form a ring, v is 1, 2, 3, or 4; y is 1, 2, 3, or 4; each r is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; q is independently C1 to C 10 It is alkyl.

[0243] In some embodiments, r is 3. In some embodiments, r is 4. In some embodiments, q is 3. In some embodiments, q is 4. In some embodiments, r is 4 and q is 4. In some embodiments, B or [ka] is selected from the following: [ka] [ka] [ka] wherein t is 0, 1, 2, 3, 4, or 5.

[0244] In some embodiments, a lipid of formula (LB-I): [ka] (LB-I), pharmaceutically acceptable salts thereof, or stereoisomers of any of the foregoing are disclosed; During the ceremony, A is independently C1 to C 16 Branched or unbranched alkylene, or C1-C 16 branched or unbranched alkenylene, optionally substituted with heteroatoms or substituted with OH, SH, or halogen; B is independent of C1 to C 20 Branched or unbranched alkyl, or C1-C 20 branched or unbranched alkenyl, optionally substituted with heteroatoms or substituted with OH, SH, or halogen; X', [ka] [ka] where at least one X is [ka] or [ka] and X' is a biodegradable moiety; W is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] During the ceremony, R5 is (CH2) s OH, OH, SH, NR 10 R 11 and R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; R 7 and R8 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, (CH2) v OH, (CH2) v SH, (CH2) S N(CH3)2, or NR 10 R 11 where R 10 and R 11 are each independently H or C1-C3 alkyl, or R 10 and R 11 together form a heterocycle, R 20 are each independently H or C1-C3 branched or unbranched alkyl; R 14 is heterocyclic, NR 10 R 11 , C(O)NR 10 R 11 , N.R. 10 C(O)NR10 R 11 , or NR 10 C(S)NR 10 R 11であり、 In the formula, R 10 and R 11 are each independently H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, optionally substituted with 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; s, u, and t are each independently 1, 2, 3, 4, or 5; each v is independently 0, 1, 2, 3, 4, or 5; Each Z is independently absent, O, S, or NR 12 where R 12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl; each Y is a divalent heterocycle; Q is O, S, CH2, or NR 13 where R 13 are each H or C1-C5 alkyl, V is branched or unbranched C2 to C 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Alkynylene, or C2-C 10 It is a heteroalkylene, optionally substituted with one or more OH, SH, and / or halogen groups.

[0245] In some embodiments, each X is [ka] or [ka] is.

[0246] In some embodiments, X' is -OCO-, -COO-, -NR 7 CO-, -CONR 7 -, -C(OR 13 )-O-, -COO(CH2) r -, -CONH(CH2) r -, or -C(OR 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.

[0247] In some embodiments, at least one X in the formula is [ka] [ka] [ka] [ka] [ka] where R 7 is H or methyl. In one embodiment, each X is [ka] or [ka] In one embodiment, each X is [ka] or [ka] where R 7 is H or methyl.

[0248] In some embodiments, the heterocycle is piperazine, piperazinedione, piperazine-2,5-dione, piperidine, pyrrolidine, piperidinol, dioxopiperazine, bis-piperazine, aromatic or heteroaromatic.

[0249] In some embodiments, the present disclosure provides an ionizable lipid of formula (LB-II): [ka] (LB-II), With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, R1 and each R2 are each independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or R1 and R2 each independently form a ring together with the carbon atom(s) to which they are attached; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle, m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; X', [ka] [ka] where at least one X is [ka] or [ka] and X' is independently a biodegradable moiety; R3 and R4 are each independently H, C3 to 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; W is [ka] [ka] or [ka] is. During the ceremony, R5 is OH, SH, NR 10 R 11 and Each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; R 7 and R8 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, NR 10 R 11 where R 10 and R 11 are each independently H, C1-C3 alkyl, or R 10 and R 11 each form a heterocyclic ring together with the carbon atom(s) to which they are attached; each s is independently 1, 2, 3, 4, or 5; each u is independently 1, 2, 3, 4, or 5; t is 1, 2, 3, 4, or 5; Each Z is independently absent, O, S, or NR 12 where R12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, provided that when Z is absent, adjacent R1 and R2 are OH, NR 10 R 11 , or SH, Q is O, S, CH2, or NR 13 where R 13 are H and C1-C5 alkyl, respectively.

[0250] In some embodiments, each X is [ka] or [ka] is.

[0251] In some embodiments, X' is -OC(O)-, -C(O)O-, -NR 7 C(O)-, -C(O)NR 7 -, -C(OR 13 )-O-, -C(O)O(CH2)r-, -C(O)NH(CH2) r -, -CON(R 13 )-, or -C(OR 13 )—O—(CH)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.

[0252] In some embodiments, at least one X in the formula is [ka] [ka] [ka] [ka] [ka] where R 7 is H or methyl. In one embodiment, each X is [ka] or [ka] In one embodiment, each X is [ka] or [ka] where R 7 is H or methyl.

[0253] In some embodiments, W is [ka] is. During the ceremony, V is C2-C6 alkylene, C2-C6 alkenylene, C2-C 10 Alkynylene, or C2-C 10 is heteroalkylene, Each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; Each u is independently 2, 3, 4, or 5.

[0254] In some embodiments, in Formula (HB-I), W is [ka] where: R6 is independently H or methyl; R 7 are each independently H, Each R8 is methyl; each u is independently 1, 2, or 3; V is C2-C6 alkenylene.

[0255] In some embodiments, the present disclosure provides an ionizable lipid of formula (LB-III): [ka] (LB-III), With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, R1 and R2 are each independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or R1 and R2 each independently form a ring together with the carbon atom(s) to which they are attached; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle, R3 and R4 are each independently H, C3 to 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; X', [ka] [ka] where at least one X is [ka] Also [ka] is, X' is independently a biodegradable moiety; each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each s is independently 1, 2, 3, 4, or 5.

[0256] In some embodiments, each X is [ka] or [ka] is.

[0257] In some embodiments, X' is -OC(O)-, -C(O)O-, -NR 7 C(O)-, -C(O)NR 7 -, -C(OR 13 )-O-, -C(O)O(CH2) r -, -C(O)NH(CH2) r -, -CON(R 13 )-, or -C(OR 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.

[0258] In some embodiments, at least one X in the formula is [ka] [ka] [ka] [ka] [ka] where R 7 is H or methyl. In one embodiment, each X is [ka] or [ka] In one embodiment, each X is [ka] or [ka] where R 7 is H or methyl.

[0259] In some embodiments, the present disclosure provides an ionizable lipid of formula (LB-IV): [ka] (LB-IV), With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, R1 and R2 are each independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or R1 and R2 each independently form a ring together with the carbon atom(s) to which they are attached; R 10 and R 11are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle, R3 and R4 are each independently H, C3 to 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; X', [ka] [ka] where at least one X is [ka] or [ka] and X' is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0260] In some embodiments, each X is [ka] or [ka] is.

[0261] In some embodiments, X' is -OC(O)-, -C(O)O-, -NR 7 C(O)-, -C(O)R 7 H-, -C(OR 13 )-O-, -C(O)O(CH2)r -, -C(O)NH(CH2) r -, -CON(R 13 )-, or -C(OR 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.

[0262] In some embodiments, at least one X in the formula is [ka] [ka] [ka] [ka] [ka] where R 7 is H or methyl. In one embodiment, each X is [ka] or [ka] In one embodiment, each X is [ka] or [ka] where R 7 is H or methyl.

[0263] In some embodiments, the present disclosure provides an ionizable lipid of formula (LB-V): [ka] (LB-V), With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, R1 and R2 are each independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or R1 and R2 each independently form a ring together with the carbon atom(s) to which they are attached; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle, R3 and R4 are each independently H, C3 to 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; X', [ka] [ka] where at least one X in the formula is [ka] [ka] and X' is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0264] In some embodiments, each X is [ka] or [ka] is.

[0265] In some embodiments, X' is -OC(O)-, -C(O)O-, -NR 7 C(O)-, -C(O)NR 7 -, -C(OR 13 )-O-, -C(O)O(CH2) r -, -C(O)NH(CH2) r -, -CON(R 13 )-, or -C(OR 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.

[0266] In some embodiments, at least one X in the formula is [ka] [ka] [ka] [ka] [ka] where R 7is H or methyl. In one embodiment, each X is [ka] or [ka] In one embodiment, each X is [ka] or [ka] where R 7 is H or methyl.

[0267] In some embodiments, the present disclosure provides an ionizable lipid of formula (LB-VI): [ka] (LB-VI), With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, R1 and R2 are each independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or R1 and R2 each independently form a ring together with the carbon atom(s) to which they are attached; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle, R3 and R4 are each independently H, C3 to 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; X', [ka] [ka] [ka] [ka] X' is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0268] In some embodiments, each X is [ka] or [ka] is.

[0269] In some embodiments, X' is -OC(O)-, -C(O)O-, -NR 7 C(O)-, -C(O)NR 7 -, -C(OR 13 )-O-, -C(O)O(CH2) r -, -C(O)NH(CH2) r -, -CON(R 13 )-, or -C(OR 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.

[0270] In some embodiments, at least one X in the formula is [ka] [ka] [ka] [ka] [ka] where R 7 is H or methyl. In one embodiment, each X is [ka] or [ka] In one embodiment, each X is [ka] or [ka] where R 7 is H or methyl.

[0271] In some embodiments, the present disclosure provides an ionizable lipid of formula (LB-VII): [ka] (LB-VII), With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, R1 and R2 are each independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11or R1 and R2 each independently form a ring together with the carbon atom(s) to which they are attached; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle, R3 and R4 are each independently H, C3 to 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; X', [ka] [ka] where at least one X is [ka] or [ka] and X' is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0272] In some embodiments, each X is [ka] or [ka] is.

[0273] In some embodiments, X' is -OC(O)-, -C(O)O-, -NR 7 C(O)-, -C(O)NR 7 -, -C(OR 13 )-O-, -C(O)O(CH2) r -, -C(O)NH(CH2) r -, -CON(R 13 )-, or -C(OR 13 )—O—(CH)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.

[0274] In some embodiments, at least one X in the formula is [ka] [ka] [ka] [ka] [ka] where R 7 is H or methyl. In one embodiment, each X is [ka] or [ka] In one embodiment, each X is [ka] or [ka] where R 7 is H or methyl.

[0275] In some embodiments, B or [ka] is selected from the following: [ka] wherein t is 0, 1, 2, 3, 4, or 5.

[0276] In some embodiments, an ionizable lipid of formula (LC-I): [ka] (LC-I), a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing is disclosed; During the ceremony, [ka] is a cyclic or heterocyclic moiety, Y is alkyl, hydroxy, hydroxyalkyl, [ka] [ka] and A is absent or -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-, -SS-, or a divalent heterocycle; X and Z each independently represent either nothing, -O-, or -N(R 7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, or -S-, R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl, or aminoalkyl; M', [ka] [ka] where at least one M is [ka] or [ka] and each M' is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 70 , R 80 , R 90 , R 100 , R 110 , and R 120 are each independently H, C1 to C 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted with OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; each of l and m is an integer from 1 to 10; t is 0, 1, 2, or 3; t1 is an integer from 0 to 10, W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl; or [ka] During the ceremony, Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, or -(CH2) q -C(R 7 )2, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, N + (R 7 ) 3-alkylene-Q-, thiol, or thiolalkyl; R 8 are each independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, heterocyclyl, heteroaryl, thiol, or thiolalkyl, or together with the nitrogen atom, two R 8 may form a ring optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, or aminoalkyl; q is 0, 1, 2, 3, 4, or 5; p is 0, 1, 2, 3, 4, or 5.

[0277] In some embodiments, Y is hydroxy, [ka] [ka] is.

[0278] In some embodiments, R 70 and R 80 Each of is H and R 90 is C1~C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl, cycloalkyl, or substituted cycloalkyl. 90 is C1~C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 90 is C1~C 15 In some embodiments, R is a branched or unbranched alkyl. 90 is C1~C 12 It may be branched or unbranched alkyl.

[0279] In some embodiments, R 70 is H and R 80 and R 90 are independently C1 to C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl, or a cycloalkyl or substituted cycloalkyl. 80 and R 90 are independently C1 to C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 80 and R 90 are independently C1 to C 15 In some embodiments, R is a branched or unbranched alkyl. 80 and R 90 are independently C1 to C 12 In some embodiments, R is a branched or unbranched alkyl. 80 and R 90 are each independently a C1 to C8 branched or unbranched alkyl.

[0280] In some embodiments, R 100 is H and R 110 and R 120 are independently C1 to C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl, or a cycloalkyl or substituted cycloalkyl. 110 and R 120 are independently C1 to C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 110 and R 120 are independently C1 to C 15 In some embodiments, R is a branched or unbranched alkyl. 110 and R 120 are independently C1 to C 12 In some embodiments, R is a branched or unbranched alkyl. 110 and R 120 are each independently a C1 to C8 branched or unbranched alkyl.

[0281] In some embodiments, an ionizable lipid of formula (LC-IA) or (LC-IA-2): [ka] (LC-IA) or [ka] (LC-IA-2), a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, During the ceremony, [ka] is a cyclic or heterocyclic moiety, A is absent or -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(R7)-, -S-, -SS-, or a divalent heterocycle; X is absent, -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-, Z is absent or -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-, R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl, or aminoalkyl; M', [ka] [ka] where at least one M in the formula is [ka] or [ka] and each M' is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 100 , R 110 , and R120 are each independently H, C1 to C 16 Branched or unbranched alkyl, or C1-C 16 branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted with OH, SH, or halogen; R 90 However, C1~C 15 Branched or unbranched alkyl, C1-C 15 branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl; t is 0, 1, 2, or 3; t1 is an integer from 0 to 10, l is an integer from 1 to 10, m is an integer from 1 to 10, W is hydroxyl, or a divalent heterocyclic hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl: [ka] During the ceremony, Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, or -(CH2) q -C(R 7 )2, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, N + (R 7 ) 3-alkylene-Q-, thiol, or thiolalkyl; R 8are each independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, heterocyclyl, heteroaryl, thiol, or thiolalkyl, or together with the nitrogen atom, two R 8 may form a ring optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, or aminoalkyl; q is 0, 1, 2, 3, 4, or 5; p is 0, 1, 2, 3, 4, or 5.

[0282] In some embodiments, A is absent, —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 )-, wherein 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-.

[0283] Embodiments regarding the various variables in formulas (LC-I) and (LC-IA) are discussed further below.

[0284] In some embodiments, X is absent, —O—, or —C(O)—.

[0285] In some embodiments, Z is —O—, —C(O)O—, or —OC(O)—.

[0286] In some embodiments, R 30 , R 40 , R 50 , and R 60 are each H or C1-C4 branched or unbranched alkyl.

[0287] In some embodiments, R 30 , R 40、 R 50 , and R 60 are H, respectively.

[0288] In some embodiments, R 90 is C1~C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 90 is C1~C 15 In some embodiments, R is a branched or unbranched alkyl. 90 is C1~C 12 In some embodiments, R is a branched or unbranched alkyl. 90 is a C1-C8 branched or unbranched alkyl.

[0289] In some embodiments, R 100 is H and R 110 and R 120 are independently C1 to C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl, or a cycloalkyl or substituted cycloalkyl. 110 and R 120 are independently C1 to C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 110 and R 120 are independently C1 to C 15 In some embodiments, R is a branched or unbranched alkyl. 110 and R 120 are independently C1 to C 12 In some embodiments, R is a branched or unbranched alkyl. 110 and R 120 are each independently a C1 to C8 branched or unbranched alkyl.

[0290] In some embodiments, 1 is 3-10, 3-7, or 4-7.

[0291] In some embodiments, m is 4-10, 5-8, 1-7, 3-7, or 1-5.

[0292] 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.

[0293] 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.

[0294] In some embodiments, M is [ka] or [ka] is.

[0295] In some embodiments, M' is -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(OR 13 )—O—, —C(O)O(CH)—, —C(O)N(R)(CH)—, or —C(O—R)—O—(CH)—, wherein R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R 13 Branched or unbranched C3-C 10 alkyl, and r is 1, 2, 3, 4, or 5.

[0296] In some embodiments, at least one M in the formula [ka] [ka] [ka] [ka] where R 7 is H or methyl. In one embodiment, M is [ka] or [ka] In one embodiment, M is [ka] or [ka] where R 7 is H or methyl.

[0297] JPEG2025530781000438.jpg53161

[0298] In some embodiments, [ka] is a bicyclic or tricyclic ring, ie, contains two or more rings, including fused rings.

[0299] In some embodiments, [ka] is the expression [ka] During the ceremony, G1, G2, G3, G4, G5, and G6 are each independently C(R')(R''), O, or N, provided that no more than two of G1 through G6 are O or N; R' and R'' are each independently absent, H, alkyl, or two R's from two adjacent G's together form a second 5-7 membered ring or heterocycle; n1 and n2 each independently represent 0 or 1.

[0300] In some embodiments, [ka] is selected from pyrrolidine, piperidine, piperazine, cyclohexane, cyclopentane, tetrahydrofuran, tetrahydropyran, morpholine, and dioxane.

[0301] In some embodiments, [ka] teeth, [ka] [ka] It has the following structure. In some embodiments, [ka] teeth, [ka] [ka] [ka] [ka] It has the following structure.

[0302] JPEG2025530781000451.jpg18161 [ka] [ka] It has the following structure.

[0303] JPEG2025530781000454.jpg18161 [ka] [ka] It has the following structure.

[0304] JPEG2025530781000457.jpg18161 [ka] [ka] It has the following structure.

[0305] JPEG2025530781000460.jpg18161 [ka] [ka] It has the following structure.

[0306] [ka] [ka] [ka] It has the following structure.

[0307] In some embodiments, the present disclosure provides an ionizable lipid of formula (LC-IIA) or (LC-IIA-2): [ka] (LC-IIA) or [ka] (LC-IIA-2), With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, A is absent or -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-, -SS-, or a divalent heterocycle; X is absent, -O-, -CO-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)N(R 7 )-, or -S-, Z is absent or -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or -S-; R 7 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; M', [ka] [ka] where at least one M in the formula is [ka] [ka] and each M' is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 100 , R 110 , and R 120 are each independently H, C1 to C 16 Branched or unbranched alkyl, or C1-C 16 branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted with OH, SH, or halogen; R 90 However, C1~C 15 Branched or unbranched alkyl, C1-C 15 branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl; t is 0, 1, 2, or 3; l is an integer from 1 to 10, m is an integer from 1 to 10, W is hydroxyl, or a divalent heterocyclic hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl, or [ka] During the ceremony, Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, or -(CH2)qC(R 7 )2, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and R 6are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, N + (R 7 ) 3-alkylene-Q-, thiol, or thiolalkyl; R 8 are each independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, heterocyclyl, heteroaryl, thiol, or thiolalkyl, or together with the nitrogen atom, two R 8 may form a ring optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, or aminoalkyl; q is 0, 1, 2, 3, 4, or 5; p is 0, 1, 2, 3, 4, or 5.

[0308] In some embodiments, the present disclosure provides an ionizable lipid of formula (LC-IIIA): [ka] With respect to (LC-IIIA), its pharmaceutically acceptable salts, and any stereoisomers of the foregoing, the definitions of the variables in (LC-IIIA) are the same as those in (LC-IIA).

[0309] In some embodiments, the present disclosure provides an ionizable lipid of formula (LC-IIB): [ka] (LC-IIB), its pharmaceutically acceptable salts, and any stereoisomers of the foregoing: During the ceremony, A is absent or -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)N(R7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -SS-, X is absent, -O-, -CO-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)N(R 7 )-, or -S-, Z is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or -S-; R 7 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; M', [ka] [ka] where at least one M in the formula is [ka] or [ka] and each M' is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 70 , R 80 , R 90 , R 100 , R 110 , and R 120 are each independently H, C1 to C 16 Branched or unbranched alkyl, or C1-C 16branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted with OH, SH, or halogen; t is 0, 1, 2, or 3; l is an integer from 1 to 10, m is an integer from 1 to 10, W is hydroxyl, or a divalent heterocyclic hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl, or [ka] During the ceremony, Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, or -(CH2)qC(R 7 )2, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, N + (R 7 ) 3-alkylene-Q-, thiol, or thiolalkyl; R 8 are each independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, heterocyclyl, heteroaryl, thiol, or thiolalkyl, or together with the nitrogen atom, two R 8 may form a ring optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, or aminoalkyl; q is 0, 1, 2, 3, 4, or 5; p is 0, 1, 2, 3, 4, or 5.

[0310] In some embodiments, the present disclosure provides an ionizable lipid of formula (LC-IIIB): [ka] With respect to (LC-IIIB), its pharmaceutically acceptable salts, and any stereoisomers of the foregoing, the definitions of the variables in (LC-IIIB) are the same as those in (LC-IIB).

[0311] In some embodiments, the present disclosure provides an ionizable lipid of formula (LC-IIC): [ka] (LC-IIC), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing. During the ceremony, A is absent, -O-, -N(R7)-, -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-, -SS-, R 30 , R 40 , R 50 , R 60 , R 100 , R 110 , and R 120 are each independently H, C1 to C 16 Branched or unbranched alkyl, or C1-C 16 branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted with OH, SH, or halogen; R 90 However, C1~C 15 Branched or unbranched alkyl, C1-C 15 or unbranched alkenyl, cycloalkyl, or substituted cycloalkyl; R 7are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; M', [ka] [ka] where at least one M in the formula is [ka] or [ka] and each M' is independently a biodegradable moiety; t is 0, 1, 2, or 3; l is an integer from 1 to 10, m is an integer from 1 to 10, W is hydroxyl, or a divalent heterocyclic hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl, or [ka] During the ceremony, Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, or -(CH2) q -C(R 7 )2, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, N +(R 7 ) 3-alkylene-Q-, thiol, or thiolalkyl; R 8 are each independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, heterocyclyl, heteroaryl, thiol, or thiolalkyl, or together with the nitrogen atom, two R 8 may form a ring optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, or aminoalkyl; q is 0, 1, 2, 3, 4, or 5; p is 0, 1, 2, 3, 4, or 5.

[0312] In some embodiments, the present disclosure provides an ionizable lipid of formula (LC-IIIC): [ka] With respect to (LC-IIIC), its pharmaceutically acceptable salts, and any stereoisomers of the foregoing, the definitions of the variables in (LC-IIIA) are the same as those in (LC-IIC).

[0313] In some embodiments, the present disclosure provides an ionizable lipid of formula (LC-IIID): [ka] With respect to (LC-IIID), its pharmaceutically acceptable salts, and any stereoisomers of the foregoing, the definitions of the variables in (LC-IID) are the same as defined above.

[0314] In some embodiments, the present disclosure provides an ionizable lipid of formula (LC-IIIE): [ka] With respect to (LC-IIIE), its pharmaceutically acceptable salts, and any stereoisomers of the foregoing, the definitions of the variables in (IID) are the same as defined above.

[0315] Embodiments regarding the various variables in formula (LC-IIA), (LC-IIB), (LC-IIC), (LC-IIIA), (LC-IIIB), (LC-IIIC), (LC-IIID), or (LC-IIIE) are further discussed below.

[0316] In some embodiments, X is absent, -O-, or -C(O)-. In one embodiment, X is absent. In one embodiment, X is -O-. In one embodiment, X is -C(O)-.

[0317] 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)-.

[0318] In some embodiments, R 30 , R 40 , R 50 , and R 60 are each H or C1-C4 branched or unbranched alkyl.

[0319] In some embodiments, R 30 , R 40、 R 50 , and R 60 Each of is H.

[0320] In some embodiments, R 70 and R 80 are H and R, respectively. 90 is C1~C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 90 is C1~C 15 In some embodiments, R is a branched or unbranched alkyl.90 is C1~C 12 It may be branched or unbranched alkyl.

[0321] In some embodiments, R 70 is H and R 80 and R 90 are independently C1 to C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 80 and R 90 are independently C1 to C 15 In some embodiments, R is a branched or unbranched alkyl. 80 and R 90 are independently C1 to C 12 In some embodiments, R is a branched or unbranched alkyl. 80 and R 90 are each independently a C1 to C8 branched or unbranched alkyl.

[0322] In some embodiments, R 100 is H and R 110 and R 120 are independently C1 to C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 110 and R 120 are independently C1 to C 15 In some embodiments, R is a branched or unbranched alkyl. 110 and R 120 are independently C1 to C 12 In some embodiments, R is a branched or unbranched alkyl. 110 and R 120 are each independently a C1 to C8 branched or unbranched alkyl.

[0323] In some embodiments, 1 is 3-10, 3-7, or 4-7.

[0324] In some embodiments, m is 4-10, 5-8, 1-7, 3-7, or 1-5.

[0325] 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.

[0326] 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.

[0327] In some embodiments, M is [ka] or [ka] is.

[0328] In some embodiments, M' is -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(OR 13 )-O-, -C(O)O(CH2)r-, -C(O)N(R7)(CH2)r-, or -C(OR 13 )-O-(CH2) r -, wherein R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R 13 is branched or unbranched C3~C 10 alkyl, and r is 1, 2, 3, 4, or 5.

[0329] In some embodiments, at least one M in the formula is [ka] [ka] [ka] [ka] where R 7 is H or methyl. In one embodiment, M is [ka] or [ka] In one embodiment, M is [ka] or [ka] where R 7 is H or methyl.

[0330] For all the ionizable lipid formulas mentioned above, [ka] and [ka] Embodiments relating to are discussed further below.

[0331] In some embodiments, A is absent, —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 )-, wherein R 7is 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-.

[0332] In some embodiments, t is 0, 1, or 2.

[0333] In some embodiments, W is OH.

[0334] In some embodiments, W is [ka] wherein Q is absent or -(CH2) q -C(R 7 )2-, or -N(R 7 ) where q is 0 or 1, and R 7 is H or methyl, and R 8 is independently H or C1-C3 alkyl. In one embodiment, W is [ka] is.

[0335] In some embodiments, W is [ka] wherein Q is absent or -(CH2) q -C(R 7 )2-, or -N(R 7 ) where q is 0 or 1, and R 7 is H or methyl, and R 8 is independently H or C1-C3 alkyl. In one embodiment, W is [ka] is.

[0336] In some embodiments, W is [ka] wherein Q is absent or -(CH2) q -C(R 7 )2-, or -N(R 7 ) where q is 0 or 1, and R 7 is H or methyl, and R 8 are each independently H or C1-C3 alkyl. In one embodiment, W is [ka] is.

[0337] In some embodiments, W is [ka] where Q is -(CH2) q -C(R 7 )2-, q is 0 or 1, and R 7 is H or methyl, and R 8 are each independently H or C1-C3 alkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] W is [ka] is.

[0338] In some embodiments, W is [ka] where q is 0 and R 8 are each independently H, C1-C3 alkyl, hydroxyalkyl, heterocyclyl, or heteroaryl, optionally substituted with one or more alkyl. [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0339] In some embodiments, W is [ka] or [ka] where R 6 are each independently H, C1-C3 alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, or -O-alkylene-N(R 7 )2 and R 7 are each independently H or C1-C3 alkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0340] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0341] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0342] In some embodiments, W is [ka] [ka] [ka] where R 6 are each independently H, C1-C3 alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, or -O-alkylene-N(R7)2, and Q is -O-, -C(R7)2-, or -N(R 7 ) and R 7 is H, C1-C3 alkyl, or hydroxyalkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0343] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, [ka]

[0344] In some embodiments, W is [ka] where q is 0 and R 8 are each independently H, C1-C3 alkyl, or hydroxyalkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0345] In some embodiments, W is [ka] where R 6 are each independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, or -O-alkylene-N(R 7 )2 and R 7 are each independently H or C1-C3 alkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0346] In some embodiments, W is [ka] where R 8 are each independently H, C1-C3 alkyl, or hydroxyalkyl, and each Q is independently absent, —O—, —CO—, —C(R 7 )2-, or -N(R 7 )- and R 7 are each independently H, C1-C3 alkyl, alkylamino, alkylaminoalkyl, or aminoalkyl. [ka] In one embodiment, W is [ka] is.

[0347] In some embodiments, W is [ka] where R 8 are each independently H, C1-C3 alkyl, or hydroxyalkyl, and each Q is independently absent, —O—, —CO—, —C(R 7 )2-, or -N(R 7 )- and R 7 are each independently H, C1-C3 alkyl, alkylamino, alkylaminoalkyl, or aminoalkyl. In one embodiment, W is [ka] [ka] is.

[0348] For all the ionizable lipid formulas mentioned above, the variable R 70 , R 80、 R 90 , R 100 , R 110 , and R 120 Further embodiments relating to are discussed below.

[0349] In some embodiments, R 70 is H. In some embodiments, R 100 is H.

[0350] In these embodiments, [ka] or [ka] are independently selected from the following: [ka] wherein t is 0, 1, 2, 3, 4, or 5.

[0351] In some embodiments, the pKa of the protonated form of an ionizable lipid compound described herein is about 4 to about 8, e.g., 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 about 5.5 to about 6.0. In some embodiments, the pKa of the protonated form of the compound is about 6.1 to about 6.3. In some embodiments, the pKa of the protonated form of the compound is about 4.7 to about 5.1. In some embodiments, the pKa of the protonated form of the compound is about 5.4 to about 7.1.

[0352] Non-limiting examples of ionizable lipid compounds disclosed herein are listed in Table 1 below.

[0353] Examples of ionizable lipid compounds. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] [Table 1-16] [Table 1-17] Additional non-limiting examples of ionizable lipid compounds disclosed herein are listed in Table 2 below.

[0354] Examples of ionizable lipid compounds. [Table 2-1]

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

Table 2-30

Table 2-31

Table 2-32

Table 2-33

Table 2-34

Table 2-35

Table 2-36

Table 2-37

Table 2-38

Table 2-39

Table 2-40

Table 2-41

Table 2-42

Table 2-43

[0355] Process for Preparing Exemplary Lipid Compounds Also disclosed herein are various processes for making exemplary lipid compounds.

[0356] In some embodiments, provided herein are processes for making lipids comprising at least one head group and at least one tail group of formula (TI) or (TI'). [ka] (TI) or [ka] (TI'), During the ceremony, each E is independently a biodegradable group; R a are each independently C1 to C5 alkyl, C2 to C5 alkenyl, or C2 to C5 alkynyl; u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; R t are independently H, C1 to C 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted with OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; [ka] represents the bond connecting the tail group to the head group, Lipids have a pKa of about 4 to about 8.

[0357] The process is reacting a first precursor compound of a tail group of formula (TI) or (TI'); [ka] or [ka] forming a lipid using a head group precursor compound, wherein the head group precursor compound contains one or more attachment points for the tail group(s), each attachment point containing a halogen-reactive functional group, thereby attaching at least one tail group of formula (TI) or (TI') to the head group at one or more attachment points.

[0358] In some embodiments, one or more points of attachment of the tail group in the precursor compound of the head group contain one or more N's.

[0359] In some embodiments, one or more attachment points of the tail group in the head group precursor compound further comprise a non-N functional group, and one or more N contained in the one or more attachment points of the head group precursor compound are protected so that the attachment points containing the non-N functional group react with the tail group precursor compound. The process then comprises: deprotecting one or more N's contained at one or more attachment points of the lipid head group; a second precursor compound of the tail group of formula (TI) or (TI'), or a lipid containing one or more deprotected N's at one or more attachment points of the head group; [ka] thereby reacting with a compound of formula (TI) [ka] or (TI') to form a lipid by attaching a second tail group to the head group at 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 the same 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 different tail groups.

[0360] In some embodiments, at least one tail group has one of the following formulas: [ka] (TII), [ka] (TIII), [ka] (TIV), [ka] (TV), [ka] (TII'), and [ka] (TIII'), R 7 are each independently H or methyl; R b is independently at each occurrence H or C1-C4 alkyl; u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6, or 7.

[0361] In some embodiments, R in the above formula a are each methyl.

[0362] In some embodiments, provided herein is a process for producing a lipid comprising at least one head group and at least one tail group having the formula: [ka] The tail group is [ka] or [ka] and u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; u3 and u4 are each independently 0, 1, 2, 3, or 4. W is hydroxyl, hydroxyalkyl, or [ka] During the ceremony, Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, or -(CH2) q -C(R 7 )2, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, thiol, thiolalkyl, or N+(R 7 ) 3-alkylene-Q-; R 8 are independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, heterocyclyl, heteroaryl, or together with the nitrogen atom, two R 8 form a ring optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, or aminoalkyl; q is 0, 1, 2, 3, 4, or 5; p is 0, 1, 2, 3, 4, or 5.

[0363] The process is compound [ka] [ka] [ka] [ka] and Compound 33 [ka] [ka] The N-protecting group of compound 35 is removed to give compound [ka] and Compound 36 may be the same as or different from Compound 33. [ka] to form the compound [ka] and Compound 38 [ka] React with [ka]

[0364] The above process can be depicted in the following general reaction scheme: [ka] In some embodiments, provided herein is a process for producing a lipid comprising at least one head group and at least one tail group having the formula: [ka] During the ceremony, The tail group is [ka] or [ka] and u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; u3 and u4 are each independently 0, 1, 2, 3, or 4. R1 and R2 are each 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; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or R 10 and R 11 together form a heterocycle, m is 1, 2, 3, 4, 5, 6, 7 or 8; n is 0, 1, 2, 3, or 4; Z is absent or O, S, or NR 12 where R 12 is H or C1-C7 branched or unbranched alkyl, provided that when Z is absent, adjacent R1 and R2 are OH, NR 10 R 11 , SH should not be.

[0365] [ka] [ka] [ka] In some embodiments, provided herein is a process for producing a lipid comprising at least one head group and at least one tail group having the formula: During the ceremony, The tail group is [ka] or [ka] and u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; u3 and u4 are each independently 0, 1, 2, 3, or 4. R 7 and R8 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2) s N(CH3)2, or NR 10 R 11 where R 10 and R 11 are each independently H or C1-C3 alkyl, or R 10 and R 11 together form a heterocycle, and R 7 and R8 together form a ring, s, u, and t are each independently 1, 2, 3, 4, or 5.

[0366] [ka] [ka] and obtaining a lipid having the formula:

[0367] The above process can be depicted in the following general reaction scheme: [ka] [ka] Additional methods for preparing the lipid compounds described herein are exemplified in Examples 1-6 and 9.

[0368] lipid composition The ionizable lipids disclosed herein may 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 or are associated with one or more therapeutic agents.

[0369] In some embodiments, the present disclosure includes (i) one or more lipid compounds described herein, pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, comprising at least one head group (e.g., HA-I through HA-VII, HB-I, or HC-I through HC-IIIE, or any subgenus or species of these formulas disclosed herein) and at least one tail group of the formula (TI' through TIII', or any subgenus or species of these formulas disclosed herein), and (ii) stereoisomers of one or more lipid components different from 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 one or more lipid compounds.

[0370] In some embodiments, the present disclosure relates to compositions comprising (i) one or more lipid nanoparticles and (ii) one or more lipid components different from the lipid compounds described herein.

[0371] In some embodiments, the one or more lipid components different from the lipid compounds described herein include one or more helper lipids and one or more PEG lipids. In some embodiments, the lipid component(s) different from the lipid compounds described herein include one or more helper lipids, one or more PEG lipids, and one or more neutral lipid(s).

[0372] In some embodiments, the lipid composition may further comprise a sterol and a PEG-lipid. In some embodiments, the lipid composition may further comprise a sterol, a PEGylated lipid, a phospholipid, and / or a neutral lipid.

[0373] In some embodiments, one or more natural and / or synthetic lipid compounds may be used in preparing the lipid composition. The lipid composition may contain negatively charged lipids, positively charged lipids, or a combination thereof.

[0374] Nonionic lipid components Charged and neutral lipids Examples of suitable negatively charged (anionic) lipids include, but are not limited to, dimyristoyl-, dipalmitoyl-, and distearoyl-phosphatidylglycerol, dimyristoyl-, dipalmitoyl-, and dipalmitoyl-phosphatidic acid, dimyristoyl-, dipalmitoyl-, and dipalmitoyl-phosphatidylethanolamine, and their unsaturated diacyl and mixed acyl chain counterparts, and cardiolipin.

[0375] Examples of positively charged (cationic) lipids include, but are not limited to, N,N'-dimethyl-N,N'-dioctasylammonium bromide (DDAB) and chloride DDAC, n-(l-(2,3-dioleyloxy)propyl)-N,N,n-trimethylammonium chloride (DOTMA), 3β-[n-(N',N'-dimethylaminoethyl)carbamoyl)cholesterol (DC-cholesterol), 1,2-dioleoyloxy-3-[trimethylammonio]-propane (DOTAP), 1,2-dioctadecyloxy-3-[trimethylammonio]-propane (DSTAP), and 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium chloride (DORI), and cationic lipids described, for example, in Martin et al., Current Pharmaceutical Design, pages 1-394, which are incorporated herein by reference in their entirety.

[0376] Additional exemplary cationic lipids include N,n-dioleyl-N,n-dimethylammonium chloride (DODAC), N,n-distearyl-N,n-dimethylammonium bromide (DDAB), n-(1-(2,3-dioleoyloxy)propyl)-N,N,n-trimethylammonium chloride (DOTAP), n-(1-(2,3-dioleoyloxy)propyl)-N,N,n-trimethylammonium chloride (DOTMA), N,n-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), 1,2-dioleoyl-3-dimethylammonium- propane (DODAP), 1,2-dioleoylcarbamyl-3-dimethylammonium-propane (DOCDAP), 1,2-dilineoyl-3-dimethylammonium-propane (DLINDAP), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLin DMA), N,n-dimethyl-3,4-dioleyloxybenzylamine (DMOBA) and / or mixtures thereof. The neutral lipids may include dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), and / or mixtures thereof.

[0377] In some embodiments, the lipid component contains one or more neutral lipids. The neutral lipids may be one or more phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids may assemble to form one or more lipid bilayers. Generally, a phospholipid may comprise a phospholipid moiety and one or more fatty acid moieties. For example, the phospholipid may be a lipid according to the formula: [ka] R prepresents the phospholipid moiety, and R A and R B represents a fatty acid moiety, with or without unsaturation, which may be the same or different. The phospholipid moiety may be phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, or sphingomyelin. The fatty acid moiety may be lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, or docosahexaenoic acid. Non-natural species, including naturally occurring species with modifications and substitutions including branching, oxidation, cyclization, and alkynes, are also contemplated. For example, the phospholipid may be functionalized with one or more alkynes (e.g., an alkenyl group in which one or more double bonds are replaced with a triple bond) or crosslinked. Under appropriate reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition upon exposure to azides. Such reactions can be useful for functionalizing the lipid bilayer of lipid nanoparticles to facilitate membrane penetration or cell recognition, or for conjugating lipid nanoparticles to useful components such as targeting or imaging moieties (e.g., dyes).

[0378] In some embodiments, the neutral lipid may 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-diundecanoyl-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-cholesterylhemisuccinoyl-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-didocosahexanoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16).0 PE), 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-didocosahexanoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl The preferred phosphatidylcholine is sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a mixture thereof.

[0379] Additional non-limiting examples of neutral lipids also include phospholipids such as lecithin, phosphatidylethanolamine, lysolecytin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl phosphatidylethanolamine (POPE), palmitoyl oleyl phosphatidylglycerol (POPG), dioleoyl phosphatidylethanolamine 4-(n-maleimidomethyl)-cyclohexane-1-carboxylic acid (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), monomethyl phosphatidylethanolamine, dimethyl phosphatidylethanolamine, diazidoyl phosphatidylethanolamine (DEPE), stearoyloleoyl phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are C 10 -C 24 The acyl group may be derived from a fatty acid having a carbon chain, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0380] Steroids and other non-ionic lipid components In some embodiments, the lipid component in the lipid composition comprises one or more steroids or analogs thereof.

[0381] In some embodiments, the lipid components in the lipid composition include sterols such as cholesterol, sterol, and their derivatives.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, and cholesteryl decanoate, and mixtures thereof.In some embodiments, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[0382] In some embodiments, the non-ionic lipid component comprises or consists of a mixture of one or more phospholipids and cholesterol or its derivatives.In some embodiments, the non-ionic lipid component comprises or consists of one or more phospholipids, for example, cholesterol-free lipid particle formulations.In some embodiments, the non-ionic lipid component comprises or consists of cholesterol or its derivatives, for example, phospholipid-free lipid particle formulations.

[0383] In some embodiments, the lipid component in the lipid composition (e.g., LNP composition) comprises a phytosterol or a combination of a phytosterol and cholesterol. In some embodiments, the phytosterol is selected from the group consisting of β-sitosterol, stigmasterol, β-sitostanol, campesterol, brassicasterol, and combinations thereof. In some embodiments, the phytosterol is 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 phytosterol is 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 phytosterol is a combination of Compound S-141, Compound S-140, Compound S-143, and Compound S-148. In some embodiments, the phytosterol comprises sitosterol or a salt or ester thereof. In some embodiments, the phytosterol comprises stigmasterol or a salt or ester thereof. In some embodiments, the phytosterol comprises beta-sitosterol, [ka]

[0384] In some embodiments, the LNP composition comprises a phytosterol, or a salt or ester thereof, and cholesterol, or a salt thereof.

[0385] In some embodiments, the target cell is a cell described herein (e.g., a liver cell or a spleen cell), and the phytosterol or salt or ester thereof is selected from the group consisting of β-sitosterol, β-sitostanol, campesterol, and 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.

[0386] In some embodiments, the target cell is a cell described herein (e.g., a liver cell or a spleen cell), and the phytosterol or salt or ester thereof is selected from the group consisting of β-sitosterol, stigmasterol, and combinations thereof. In some embodiments, the phytosterol is β-sitosterol. In some embodiments, the phytosterol is stigmasterol.

[0387] Other examples of nonionic lipids include non-phosphorus-containing lipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin.

[0388] In some embodiments, the nonionic lipid comprises 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 thereof) of the total lipid present in the particle.

[0389] In embodiments in which the lipid particle composition contains a mixture of phospholipids and cholesterol or a cholesterol derivative, the mixture may comprise up to 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipid present in the particle.

[0390] In some embodiments, the phospholipid component in the mixture may comprise 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 thereof) of the total lipid present in the particle. In some embodiments, the phospholipid component in the mixture comprises 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 thereof) of the total lipid present in the particle.

[0391] In some embodiments, the cholesterol component in the mixture may comprise 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 fraction or range thereof) of the total lipid present in the particle. In some embodiments, the cholesterol component in the mixture comprises 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 fraction or range thereof) of the total lipid present in the particle.

[0392] In embodiments in which the lipid particle composition does not contain phospholipids, cholesterol or a derivative thereof may comprise up to 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipid present in the particle.

[0393] In some embodiments, cholesterol or a derivative thereof in a phospholipid-free lipid particle formulation may comprise 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 fraction or range thereof) of the total lipid present in the particle.

[0394] In some embodiments, the nonionic lipid comprises 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 a derivative thereof) of the total lipid present in the particle (or any fraction or range thereof).

[0395] The percentage of non-ionic lipid present in the lipid particle is a target amount, and the actual amount of non-ionic lipid present in the particle can vary, for example, by ±5 mol %.

[0396] A composition containing an ionizable lipid compound may contain 30-70% ionizable lipid compound, 0-60% cholesterol, 0-30% phospholipid, and 1-10% polyethylene glycol (PEG). In some embodiments, the composition contains 30-40% ionizable lipid compound, 40-50% cholesterol, and 10-20% PEG. In some embodiments, the composition contains 50-75% ionizable lipid compound, 20-40% cholesterol, 5-10% phospholipid, and 1-10% PEG. A composition may contain 60-70% ionizable lipid compound, 25-35% cholesterol, and 5-10% PEG. A composition may contain up to 90% ionizable lipid compound and 2-15% helper lipid.

[0397] The composition may be, for example, a lipid particle composition containing 8-30% compound, 5-30% helper lipid, and 0-20% cholesterol, 4-25% ionizable lipid, 4-25% helper lipid, 2-25% cholesterol, 10-35% cholesterol-PEG, and 5% cholesterol-amine, or 2-30% ionizable lipid, 2-30% helper lipid, 1-15% cholesterol, 2-35% cholesterol-PEG, and 1-20% cholesterol-amine, or up to 90% ionizable lipid and 2-10% helper lipid, or even 100% ionizable lipid.

[0398] lipid conjugates In addition to one or more ionizable lipids, the lipid particles described herein may further comprise one or more lipid conjugates. The conjugated lipids can prevent particle aggregation. Non-limiting examples of conjugated lipids include PEG-lipid conjugates, cationic polymer-lipid conjugates, and mixtures thereof.

[0399] In some embodiments, lipid conjugate is PEG lipid or PEG-modified lipid (alternatively referred to as PEGylated lipid).PEG lipid is the lipid modified with polyethylene glycol.The example of PEG lipid includes but is not limited to the PEG that is bonded to dialkyloxypropyl (PEG-DAA), the PEG that is bonded to diacylglycerol (PEG-DAG), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DEG), the PEG that is bonded to phospholipids such as phosphatidylethanolamine (PEG-PE), the PEG that is bonded to ceramide (PEG-CER), the PEG that is bonded to cholesterol or its derivatives, and their mixtures.

[0400] For example, the PEG lipid may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0401] In some embodiments, the PEG lipid is 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.

[0402] In some embodiments, the PEG lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-l,2-dimyristoyloxylpropyl-3-amine (PEG-c-DMA).

[0403] PEG is a linear water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified according to their molecular weight and include monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM), and terminal methoxy groups (e.g., OH-PEG-S, OH-PEG-S-NHS, HO-PEG-NH2).

[0404] The PEG moiety of the PEG-lipid conjugates described herein can comprise an average molecular weight ranging from 550 daltons to 10,000 daltons. In particular examples, the PEG moiety has an average molecular weight of 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 PEG moiety has an average molecular weight of 2,000 daltons or 750 daltons.

[0405] In certain examples, PEG can be optionally substituted with alkyl, alkoxy, acyl or aryl group.PEG can be directly conjugated to lipid, or can be linked to lipid via linker moiety.For example, any linker moiety suitable for connecting PEG to lipid can be used, including non-ester-containing linker moiety and ester-containing linker moiety.In some embodiments, linker moiety is non-ester-containing linker moiety. Suitable non-ester containing linker moieties include, but are not limited to, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCHCHC(O)-), succinamidyl (-NHC(O)CHCHC(O)NH-), ether, disulfide, and combinations thereof (such as linkers containing both carbamate and amide linker moieties). In some embodiments, a carbamate linker is used to attach PEG to a lipid.

[0406] In some embodiments, PEG is attached to the lipid using an ester-containing linker moiety. Suitable ester-containing linker moieties include, for example, carbonate (-OC(O)O-), succinoyl, phosphate (-O-(O)POH-O-), sulfonate ester, and combinations thereof.

[0407] Phosphatidylethanolamines with various acyl chain groups of various chain lengths and degrees of saturation can be conjugated to PEG to form lipid conjugates. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art.

[0408] In some embodiments, the phosphatidylethanolamine contains saturated or unsaturated fatty acids with carbon chain lengths ranging from C10 to C20. Phosphatidylethanolamines with mono- or di-unsaturated fatty acids, as well as mixtures of saturated and unsaturated fatty acids, can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0409] The term "diacylglycerol" or "DAG" includes compounds having two fatty acyl chains, R1 and R2, both of which have 2 to 30 carbons and are independently attached to the 1- and 2-positions of glycerol by ester bonds. The acyl groups may be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C12), myristoyl (CM), palmitoyl (C16), stearoyl (C18), and icosyl (C20). In some embodiments, R1 and R2 are the same, i.e., both R1 and R2 are myristoyl (i.e., dimyristoyl) and both R1 and R2 are stearoyl (i.e., distearoyl).

[0410] The term "dialkyloxypropyl" or "DAA" includes compounds having two alkyl chains, R and R', both of which independently contain 2 to 30 carbons. The alkyl groups may be saturated or have varying degrees of unsaturation.

[0411] In some embodiments, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C10) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG-dimyristoyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16) conjugate, or a PEG-distearyloxypropyl (C18) conjugate. In some embodiments, the PEG has an average molecular weight of 750 or 2,000 daltons. In some embodiments, the terminal hydroxyl group of the PEG is replaced with a methyl group.

[0412] In addition to the above, other hydrophilic polymers can be used instead of PEG. Examples of suitable polymers that can be used instead of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0413] In some embodiments, the PEG lipid has the formula [ka] or a salt thereof, wherein R 3PL1 -OR OPL1 and R OPL1 is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r PL1 is an integer between 1 and 100, L 1 is an optionally substituted C 1-10 alkylene and optionally substituted C 1-10At least one methylene of the alkylene is independently selected from 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 ), D is a residue obtained by click chemistry or cleavable under physiological conditions, m PL1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, A is of the formula: [ka] L 2 Each instance of is independently a bond or an optionally substituted C 1-6 alkylene and optionally substituted C 1-6 One methylene unit of alkylene is O, N(R NPL1 ), S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), C(O)O, OC(O)O, -OC(O)N(R NPL1 ), NR NPL1 C(O)O, or NR NPL1 C(O)N(R NPL1 ), optionally replaced by R 2SL Each instance of 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl, optionally wherein R 2SLwherein one or more methylene units 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)O, R NPL1 is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p SL is either 1 or 2.

[0414] In some embodiments, the PEG lipid has the formula [ka] or a salt thereof, PL1 , L 1 ,D,m PL1 , and A is as defined above.

[0415] In some embodiments, the PEG lipid is a compound of formula: [ka] or an isomer, wherein R 3PEG HA-OR O and R O is hydrogen, C 1~6 alkyl, or oxygen protecting group, r PEG is an integer between 1 and 100 (e.g., 40 and 50, e.g., 45), R 5PEG is C 10~40 Alkyl (e.g., C 17 alkyl), C 10~40 Alkenyl, or C 10~40 Alkynyl, R 5PEG Optionally, one or more methylene groups are independently selected from C 3-10 Carbocyclylene-substituted 4- to 10-membered heterocyclylene, 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-, R NPEG Each example is independently hydrogen, C 1-6 alkyl, or nitrogen protecting groups.

[0416] In some embodiments, the PEG lipid is a compound of the formula [ka] r PEG is an integer between 1 and 100 (e.g., 40-50, e.g., 45).

[0417] In some embodiments, the PEG lipid is a compound of the formula or [ka] salt or isomer, s PL1 is an integer from 1 to 100 (for example, 40-50, for example, 45).

[0418] In some embodiments, the PEG lipid has the formula [ka] R 8 and R 9 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally being interrupted by one or more ester bonds (e.g., R 8 and R 9 are each independently a straight, saturated alkyl chain containing 12-16 carbon atoms, and w has an average value in the range of 30-60 (eg, the average w is about 49).

[0419] In some embodiments, incorporating any 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 of the PEG-lipids discussed above into the lipid composition can reduce the accelerated blood clearance (ABC) effect.

[0420] Other ionizable lipids In some embodiments, the lipid composition may include one or more additional ionizable lipids different from the ionizable lipids described herein. Examples of ionizable lipids include, but are not limited to: [ka] ), SM-86( [ka] SM-102( [ka] ALC-0315( [ka] Lipid 10( [ka] MC3( [ka] and Acuitas Lipid 10 (see WO 2017 / 004143, incorporated herein by reference in its entirety).

[0421] In one embodiment, the additional ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid (SM-102), e.g., as described in Example 1 of U.S. Pat. No. 9,867,888, which is incorporated herein by reference in its entirety.

[0422] 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), synthesized, for example, in Example 13 of WO 2015 / 095340, which is incorporated herein by reference in its entirety.

[0423] In one embodiment, the additional ionizable lipid is di((Z)-non-2-en-1-yl)9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., as synthesized in Example 7, 8, or 9 of U.S. Application Publication No. 2012 / 0027803, which is incorporated herein by reference in its entirety.

[0424] 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.

[0425] In one embodiment, the additional ionizable lipid is the imidazole cholesterol ester (ICE) lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthron-3-yl 3-(1H-imidazol-4-yl)propanoate, such as that described in WO 2020 / 106946, which is incorporated herein by reference in its entirety.

[0426] In one embodiment, the additional ionizable lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), for example, as described in Example 9 of WO 2019 / 051289, which is incorporated herein by reference in its entirety.

[0427] In one embodiment, the additional ionizable lipid is lipid ATX-002, for example, as described in Example 10 of WO 2019 / 051289, which is incorporated herein by reference in its entirety.

[0428] In one embodiment, the additional ionizable lipid is (l3Z,l6Z)-A,A-dimethyl-3-nonyldocosa-13,l6-dien-l-amine (compound 32), as described, for example, in Example 11 of WO 2019 / 051289 A9 (herein incorporated by reference in its entirety).

[0429] In one embodiment, the additional ionizable lipid is, for example, compound 6 or compound 22, described in Example 12 of WO 2019 / 051289, which is incorporated herein by reference in its entirety.

[0430] Examples of additional ionizable lipids useful in the lipid composition include those listed in Table 1 of WO 2019 / 051289, which is incorporated herein by reference.

[0431] Additional lipid ingredients 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 lipid compositions include the following: [ka] (i) [ka] (ii) [ka] (iii) [ka] (iv), [ka] (v), [ka] (vi) [ka] (vii), [ka] (viii), or [ka] (ix)

[0432] In some embodiments, the lipid composition further comprises a lipid of formula (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), or (ix).

[0433] In some embodiments, the lipid composition further comprises a compound having the following structure: [ka] (x), During the ceremony, X 1 , O, NR 1 , or a direct bond, X 2 is C2-5 alkylene, and X 3 is C(=O) or a direct bond, R 1 is H or Me, and R 3 is C 1-3 alkyl, and R 2 is C 1-3 alkyl, or R 2 together with the nitrogen atom to which it is attached, X 2 1-3 carbon atoms of form a 4-, 5-, or 6-membered ring, or X 1 is NR 1 , R 1, and R 2 form a 5- or 6-membered ring together with the nitrogen atom to which they are attached, or R 2 But R 3 and together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y 1 is C 2-12 is alkylene, and Y 2 teeth, [ka] (either orientation), (either orientation), (either orientation), (either orientation), n is 0 to 3, R 4 But C 1~15 is alkyl, Z 1 is C 1-6 is an alkylene or a direct bond, [ka] (in either orientation) or absent, Z 1 If is a direct bond, Z 2 does not exist, R 5 But C 5-9 Alkyl or C 6-10 is alkoxy, and R 6 But C 5-9 Alkyl or C 6-10 is an alkoxy, W is methylene or a direct bond; R 7 is H or Me, or a salt thereof, However, R 3 and R 2 is a C2 alkyl, and X 1 is O and X 2 is a linear C3 alkylene, and X 3 is C(=O) and Y 1 is a linear C5 alkylene, and (Y 2 )nR 4 but [ka] and R 4 is a linear C5 alkyl, and Z 1 is C2 alkylene, and Z 2 is absent, W is methylene, and R 7 If is H, then R 5 and R 6 is not a C2 alkoxy.

[0434] In some embodiments, the lipid composition further comprises one or more compounds of formula (x).

[0435] Additional non-limiting examples of lipid compounds that may be further included in the lipid composition further include (e.g., in combination with the lipid compounds and other lipid components described herein): [ka] (xi), [ka] [ka] [ka] (xiii), [ka] (xiv), [ka] (xv), [ka] (xvi), [ka] (xvii), [ka] (xviii), [ka] [ka] (xviii)(b), [ka] (xix).

[0436] 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).

[0437] In some embodiments, the lipid composition further comprises a lipid formed by one of the following reactions: [ka] [ka] (xx)(a) [ka] [ka] (xx)(b).

[0438] In some embodiments, the lipid composition has the formula (xxi): [ka] (xxi) (e.g., in combination with lipid compounds and other lipid components described herein), wherein: each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently a straight or branched chain C9-C 20 Alkyl or C9-C 20 alkenyl, optionally substituted by one or more substituents selected from the group consisting of oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(a ... (alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; R2 is selected from the group consisting of: [ka]

[0439] In some embodiments, the lipid composition further comprises one or more compounds of formula (xxi). In some embodiments, the compound of formula (xxi) comprises a compound described in WO 2022 / 113777 (e.g., a lipid of formula (1), such as a lipid in Table 1 of WO 2022 / 113777), which is incorporated herein by reference in its entirety.

[0440] In some embodiments, the lipid composition has formula (xxii): [ka] (xxii) (e.g., in combination with lipid compounds and other lipid components described herein), wherein: n's are each independently an integer of 1 to 15, R1 and R2 are each independently selected from the group consisting of: [ka] R3 is selected from the group consisting of: [ka]

[0441] In some embodiments, the lipid composition further comprises one or more compounds of formula (xxii). In some embodiments, the compound of formula (xxii) comprises a compound described in WO 2022 / 113777 (e.g., a lipid of formula (2), such as the lipids in Table 2 of WO 2022 / 113777), which is incorporated herein by reference in its entirety.

[0442] In some embodiments, the lipid composition has formula (xxiii): [ka] (xxiii) (e.g., in combination with lipid compounds and other lipid components described herein), wherein: X is selected from -O-, -S-, or -OC(O)-*, where * indicates the point of attachment to R1; R1 is selected from the group consisting of: [ka] R2 is selected from the group consisting of: [ka]

[0443] In some embodiments, the lipid composition further comprises one or more compounds of formula (xxiii). In some embodiments, the compound of formula (xxiii) comprises a compound described in WO 2022 / 113777 (e.g., a lipid of formula (3), such as the lipids in Table 3 of WO 2022 / 113777), which is incorporated herein by reference in its entirety.

[0444] Examples of additional lipids that may be used in the lipid composition include, but are not limited to, one or more of the following formulas: X of U.S. Patent Application Publication No. 2016 / 0311759, I of U.S. Patent Application Publication No. 20150376115 or U.S. Patent Application Publication No. 2016 / 0376224, I, II, or III of U.S. Patent Application Publication No. 2016 / 0151284, I, IA, II, or IIA of U.S. Patent Application Publication No. 2017 / 0210967, Ic of U.S. Patent Application Publication No. 2015 / 0140070, A of U.S. Patent Application Publication No. 2013 / 0178541, I of U.S. Patent Application Publication No. 2013 / 0303587 or U.S. Patent Application Publication No. 2013 / 0123338, I of U.S. Patent Application Publication No. 2015 / 0141678 , II, III, IV, or V of U.S. Patent Application Publication No. 2015 / 0239926, I of U.S. Patent Application Publication No. 2017 / 0119904, I or II of WO 2017 / 117528, A of U.S. Patent Application Publication No. 2012 / 0149894, A of U.S. Patent Application Publication No. 2015 / 0057373, A of WO 2013 / 116126, A of U.S. Patent Application Publication No. 2013 / 0090372, A of U.S. Patent Application Publication No. 2013 / 0274523, A of U.S. Patent Application Publication No. 2013 / 02 74504 A, U.S. Patent Application Publication No. 2013 / 0053572 A, WO 2013 / 016058 A, WO 2012 / 162210 A, U.S. Patent Application Publication No. 2008 / 042973 I, U.S. Patent Application Publication No. 2012 / 01287670 I, II, III, or IV, U.S. Patent Application Publication No. 2014 / 0200257 I or II, U.S. Patent Application Publication No. 2015 / 0203446 I, II, or III, U.S. Patent Application Publication No. 2015 / 0005363 I or III, I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of U.S. Patent Application Publication No. 2014 / 0308304, U.S. Patent Application Publication No. 2013 / 0338210, I, II, III, or IV of WO 2009 / 132131, A of U.S. Patent Application Publication No. 2012 / 01011478, I or XXXV of U.S. Patent Application Publication No. 2012 / 0027796, XIV or XVII of U.S. Patent Application Publication No. 2012 / 0058144,U.S. Patent Application Publication No. 2013 / 0323269, U.S. Patent Application Publication No. 2011 / 0117125, I, II, or III of U.S. Patent Application Publication No. 2011 / 0256175, I, II, III, IV, V, VI, VII, VIII, IX, X, XI, and XII of U.S. Patent Application Publication No. 2012 / 0202871, and I, II, III, IV, V, VI, VII, VIII, X, XII, and X of U.S. Patent Application Publication No. 2011 / 0076335 III, XIV, XV, or XVI, I or II of U.S. Patent Application Publication No. 2006 / 008378, I of U.S. Patent Application Publication No. 2013 / 0123338, I or XAYZ of U.S. Patent Application Publication No. 2015 / 0064242, XVI, XVII, or XVIII of U.S. Patent Application Publication No. 2013 / 0022649, I, II, or III of U.S. Patent Application Publication No. 2013 / 0116307, U.S. Patent Application Publication No. 2013 / 0116307 No. 2010 / 0062967, U.S. Patent Application Publication No. 2013 / 0189351, U.S. Patent Application Publication No. 2014 / 0039032, U.S. Patent Application Publication No. 2018 / 0028664, U.S. Patent Application Publication No. 2016 / 0317458, U.S. Patent Application Publication No. 2013 / 0195920, U.S. Patent Application Publication No. 10,221,127, U.S. Patent ... III-3 of WO 2020 / 081480, I-5 or I-8 of WO 2020 / 081938, 18 or 25 of U.S. Patent Application Publication No. 9,867,888, A of U.S. Patent Application Publication No. 2019 / 0136231, II of WO 2020 / 219876, 1 of U.S. Patent Application Publication No. 2012 / 0027803, OF-02 of U.S. Patent Application Publication No. 2019 / 0240349, 23 of U.S. Patent Application Publication No. 10,086,013, Miao cKK-E12 / A6 from WO 2010 / 053572, C12-200 from Dahlman et al. (2017), 7C1 from Dahlman et al. (2017), 304-O13 or 503-O13 from Whitehead et al., TS-P4C2 from U.S. Patent Application Publication No. 9,708,628, I from WO 2020 / 106946, I from WO 2020 / 106946,(1), (2), (3), or (4) of WO 2021 / 113777, and any of Tables 1-16 of WO 2021 / 113777, all of which are incorporated herein by reference in their entirety.

[0445] 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 fraction or range thereof) 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 fraction or range thereof) of the total lipid present in the particle.

[0446] 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 fraction or range thereof) of the total lipid present in the particle.

[0447] 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, for example, by ±2 mol%. Those skilled in the art will understand that the concentration of lipid conjugate can vary depending on the lipid conjugate used and the rate at which the lipid particles become fusogenic.

[0448] By controlling the composition and concentration of lipid complex, the rate at which lipid complex is exchanged from lipid particle can be controlled, and then the rate at which lipid particle becomes fuming can 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 lipid particle becomes fuming.Other methods that can be used to control the rate at which lipid particle becomes fuming will be clear to those skilled in the art after reading this disclosure.In addition, by controlling the composition and concentration of lipid conjugate, the size of lipid particle can be controlled.

[0449] In some embodiments, the composition further comprises one or more nucleic acids, ionizable lipids, amphiphilic lipids, phospholipids, cholesterol, and / or PEG-linked cholesterol.

[0450] Other components of the LNP composition The lipid nanoparticle composition may include one or more ingredients in addition to those described above. 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.

[0451] The lipid nanoparticle composition may also include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents, or other components.

[0452] Suitable carbohydrates can include simple sugars (eg, glucose) and polysaccharides (eg, glycogen and its derivatives and analogs).

[0453] Polymers can be used to encapsulate or partially encapsulate nanoparticle compositions. The polymers can be biodegradable and / or biocompatible. Suitable polymers include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, The polymer may include poly(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 acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters, polyvinyl halides such as poly(vinyl acetate), poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, and cellulose esters.Nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polymers of acrylic acid, such as poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), Poly(octadecyl acrylate), poly(octadecyl acrylate) and copolymers and blends thereof, polydioxanone and its copolymers, polyhydroxyalkanoic acids, polypropylene fumarate, polyoxymethylene, poloxamers, polyoxamines, poly(ortho)esters, 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.

[0454] Suitable surface altering agents 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), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum bromhexine, carbocysteine, eprazinone, menthol, ambroxol, sobrerol, domiodol, letostein, stearonine, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexin, and erdosteine), and DNase (e.g., rhDNase). The surface altering agent may be disposed within and / or on the surface of the lipid nanoparticle (e.g., by coating, adsorption, covalent attachment, or other process).

[0455] The lipid nanoparticle composition may also comprise one or more functional lipids.For example, lipids may be functionalized with alkyne groups that can undergo cycloaddition when exposed to azide under suitable reaction conditions.In particular, lipid bilayers may be functionalized in this manner with one or more groups that are useful for promoting membrane permeation, cell recognition, or imaging.The surface of lipid nanoparticles may also be conjugated with one or more useful antibodies.Functional groups and conjugates that are useful for target cell delivery, imaging, and membrane permeation are well known in the art.

[0456] Lipid nanoparticle compositions can contain any substance useful in pharmaceutical compositions.For example, lipid nanoparticle compositions can contain one or more pharmaceutically acceptable excipients or auxiliary ingredients, 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, isotonicity agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and other species.Excipients such as waxes, butters, colorants, coating agents, flavors, and fragrances can also be included.

[0457] Suitable diluents 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 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 sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0458] Suitable surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia agar, alginic acid, sodium alginate, tragacanth, etc.). cartilage, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [ TWEEN® 20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]), polyoxyethylene esters (e.g., polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monooleate [SPAN® 80]), polyoxyethylene sorbitan monopalmitate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]), polyoxyethylene esters (e.g., polyoxyethylene sorbitan monooleate ..., polyoxyethylene sorbitan monooleate [SPAN® 80]), polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monoole polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate,Triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC® F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0459] Suitable binders may 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., acacia sodium alginate, Irish mosquito extract, breadwort gum, ghatti gum, isopole shell mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohols, and combinations thereof, or any other suitable binder.

[0460] Suitable preservatives include, but are not limited to, antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium sulfite, 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 antibacterial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, 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, hydroxybenzoates, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium sulfite, sodium metabisulfite, potassium sulfite, sodium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE™, KATHON™, and / or EUXYL®.

[0461] Suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0462] Suitable oils include almond, apricot kernel, avocado, babassu, bergamot, blackcurrant seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, primrose, fish, flaxseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cuba, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, and safflower. The following ingredients may be used: sandalwood, sasquama, savory, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, camellia, vetiver, walnut, wheat germ oil, and / or aqua (water), including but not limited to: 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.

[0463] 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-propanediol), 1,3-propanediol, glycerol, (+ / -)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), non-deterrent sulfobetaines (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), osmolytes (e.g., L-proline or trimethylamine n-oxide dihydrate), polymers (e.g., polyethylene glycol 200 (PEG200), PEG400, PEG600, PEG1000, PEG 2k-DMG, PEG3350, PEG4000, PEG8000, PEG10000, PEG 20000, polyethylene glycol monomethyl ether 550 (mPEG550), mPEG600, mPEG2000, mPEG3350, mPEG4000, mPEG5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K15), pentaerythritol propoxylate, or polypropylene glycol P 400), an organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), a sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, mesoerythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, or D-(+)-glucose monohydrate), or a salt (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 hydrate thereof), or any combination thereof.

[0464] 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.

[0465] In some embodiments, the composition further comprises one or more buffers. Suitable buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, aminosulfonic acid buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof.

[0466] In some embodiments, the buffer is an acetate buffer, a citrate buffer, a phosphate buffer, a Tris buffer, or a combination thereof.

[0467] Pharmaceutical Composition Another aspect of the present disclosure also provides a pharmaceutical composition comprising the lipid composition described herein, which comprises 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.

[0468] All of the above descriptions and all of the embodiments discussed in the above aspects relating to lipid compounds and exemplary variable and compound aspects are all applicable to these aspects of the invention relating to pharmaceutical compositions.

[0469] All statements and all embodiments discussed in the above aspects relating to lipid composition aspects, including various other lipid components, are applicable to these aspects of the invention relating to pharmaceutical compositions.

[0470] In lipid compositions containing a therapeutic agent, the ratio of total lipid components to cargo (e.g., an encapsulated therapeutic agent such as a nucleic acid) can be varied as desired. For example, the total lipid components to cargo ratio (mass or weight) can be from about 10:1 to about 30:1. In some embodiments, the total lipid components to cargo ratio (mass / mass ratio, w / w ratio) can be within 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 amount of total lipid components and cargo can be adjusted to provide a desired N / P ratio, for example, 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 greater. Generally, the overall lipid content of the lipid composition can range from about 5 mg / ml to about 30 mg / mL.

[0471] Treatment drugs nucleic acid molecule In some embodiments, the composition further comprises one or more nucleic acid components. The nucleic acid molecule may be a plasmid, an immunostimulatory oligonucleotide, an antisense oligonucleotide, an antagomir, an aptamer, a deoxyribozyme (DNAzyme), and a ribozyme.

[0472] In some embodiments, the composition further comprises one or more RNA and / or DNA components.

[0473] In some embodiments, the composition further comprises one or more DNA components, hi some embodiments, the DNA is linear, circular, single-stranded, or double-stranded.

[0474] 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.

[0475] In some embodiments, the one or more RNA components are selected from mRNA. In some embodiments, the mRNA is a modified mRNA.

[0476] 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 in the substrate binding region to a specific gene target and also has the enzymatic activity of specifically cleaving target RNA.That is, the enzymatic nucleic acid molecule can intermolecularly cleave RNA, thereby inactivating the target RNA molecule.The term enzymatic nucleic acid is used interchangeably with terms such as ribozyme, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulable ribozyme, catalytic oligonucleotide, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme.All of these terms describe nucleic acid molecules with enzymatic activity.

[0477] 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 (protein nucleic acid) interactions and alters the activity of the target RNA.

[0478] In some embodiments, the nucleic acid molecule may be a 2-5A antisense chimera. The term "2-5A antisense chimera" refers to an antisense oligonucleotide containing a 5'-phosphorylated 2'-5'-linked adenylic acid residue.

[0479] In some embodiments, the nucleic acid molecule may be a triplex-forming oligonucleotide. The term "triple-strand-forming oligonucleotide" refers to an oligonucleotide that can bind to double-stranded DNA in a sequence-specific manner to form a triple-stranded helix.

[0480] In some embodiments, the nucleic acid molecule may be a decoy RNA. The term "decoy RNA" refers to an RNA molecule or aptamer that is designed to preferentially bind to a specific ligand. Such binding can result in the inhibition or activation of the target molecule.

[0481] In some embodiments, the nucleic acid molecule (e.g., RNA or DNA) encodes a therapeutic peptide or polypeptide 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 and, e.g., a nuclease component of a CRISPR system, e.g., Cas9, dCas9, aCas9-nickase, Cpf / Cas12a), a CRISPR-binding enzyme, e.g., a base editor or prime editor, a mobile genetic element protein (e.g., a transportase, retrotranspotase, recombinase, integrase, or the like), or a polypeptide encoding a polypeptide encoding a polypeptide encoding a polypeptide (e.g., a polypeptide encoding a polypeptide), or ... enzymes), gene makers, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, ligases, deubiquitinases, integrases, recombinases, topoisomerases, gyrases, helicases, lysosomal acid hydrolases, antibodies, receptor ligands, receptors, clotting factors, membrane proteins, mitochondrial proteins, nuclear proteins, antibodies or other protein scaffold binders such as centrins, darpins, or adnectins.

[0482] In some embodiments, the one or more RNA components comprise a gRNA nucleic acid. In some embodiments, the gRNA nucleic acid is a gRNA.

[0483] In some embodiments, the one or more RNA components comprise a Class 2 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 in one or more of the first five nucleotides at the 5' end. In some embodiments, the modified gRNA comprises a modification in one or more of the last five nucleotides at the 3' end.

[0484] In some embodiments, one or more RNA components comprise an mRNA. In some embodiments, one or more RNA components comprise an RNA-guided DNA binding agent, such as a Cas nuclease mRNA (such as a class 2 Cas nuclease mRNA) or a Cas9 nuclease mRNA.

[0485] 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 contains one or more modifications selected from the group consisting of pseudouridine, 5-bromouracil, 5-methylcytosine, peptide nucleic acid, heterologous nucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threos nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugars), thiol-containing nucleotides, biotin-conjugated nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, quosine, and wyosine. In some embodiments, the antisense oligonucleotide can be a locked nucleic acid oligonucleotide (LNA). The term "locked nucleic acid (LNA)" refers to an oligonucleotide containing one or more nucleotide components in which an extra methylene bridge locks the ribose moiety in either a C3'-endo (beta-D-LNA) or C2'-endo (alpha-L-LNA) configuration (Grunweller A, Hartmann RK, BioDrugs, 21(4): 235-243 (2007)).

[0486] In some embodiments, the composition further comprises one or more template nucleic acids.

[0487] Additional examples of nucleic acid molecules (including tumor suppressor genes, antisense oligonucleotides, siRNA, miRNA, or shRNA) can be found in U.S. Published Patent Application No. 2007 / 0065499 and U.S. Patent No. 7,780,882, which are incorporated by reference in their entireties.

[0488] In some embodiments, a pharmaceutical composition may include multiple nucleic acid molecules that may be of the same or different types.

[0489] Nucleic acids for use in the embodiments of the present disclosure can be prepared according to any available technique. For mRNA, the primary method of preparation is enzymatic synthesis (also known as in vitro transcription), which represents, but is not limited to, the most efficient method for producing long, sequence-specific mRNA. In vitro transcription describes the process of template-specific synthesis of RNA molecules from an engineered DNA template composed of an upstream bacteriophage promoter sequence (e.g., including but not limited to, those from T7, T3, and SP6 coliphages) linked to a downstream sequence encoding a gene of interest. Template DNA can be prepared for in vitro transcription from many sources using suitable techniques known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (Linpinsel, JL, and Conn GL, General Protocol for Preparation of Plasmid DNA Templates and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD, RNA Purification by Denaturing PAGE in In Vitro RNA Transcription and Recombinant RNA Synthesis and In Vitro RNA Synthesis Methods v.941 Conn GL (ed), New York: NY Humana Press, 2012).

[0490] Transcription of RNA occurs in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) 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, including, but not limited to, the RiboMax Large Scale RNA Production System (Promega), the MegaScript Transcription Kit (Life Technologies), and commercially available reagents containing RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are well known in the art (see, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v. 41 409-46; Kamaka, RT and Kraus, WL 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 in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter 5 - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology. 530, 101-114, all of which are incorporated herein by reference).

[0491] The desired in vitro transcribed mRNA can be purified from undesired components of the transcription or related reaction, including unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc. Techniques for isolating mRNA transcripts are well known in the art. Well-known procedures include, for non-limiting examples, phenol / chloroform extraction or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride.

[0492] Additional, non-limiting examples of purification procedures that can be used include size exclusion chromatography (Lukavsky, PJ, and Puglisi JD 2004, Large-Scale Preparation and Purification of Polyacrylamide-Free RNA Oligonucleotides, RNA v. 10, 889-893), silica-based affinity chromatography, and polyacrylamide gel electrophoresis (Bowman, JC, Azizi B., Lentz TK, Ray P., and Williams LDin RNA in vitro transcription and recombinant RNA synthesis and RNA purification by denaturing PAGE in In vitro RNA synthesis methods v. 941 Conn GL (ed.), New York NY Humana Press, 2012). Purification can be performed using various commercially available kits, including, but not limited to, the SV Total Isolation System (Promega) and the In Vitro Transcription Wash Concentration Kit (Norgen Biotek).

[0493] Furthermore, while reverse transcription can produce large amounts of mRNA, the product may contain numerous aberrant RNA impurities associated with undesired polymerase activity that may need to be removed from full-length mRNA preparations. These include short RNAs resulting from abortive transcription initiation, as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and self-complementary 3' extensions. It has been demonstrated that these contaminants, which have dsRNA structures, can result in unwanted immunostimulatory activity through interactions with various innate immune sensors in eukaryotic cells that recognize specific nucleic acid structures and function to elicit strong immune responses. This, in turn, can dramatically reduce mRNA translation because protein synthesis is reduced during the innate cellular immune response. Therefore, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including, but not limited to, scalable HPLC purification (e.g., Kariko, K., Muramatsu, H., Ludwig J. Weissman D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 el42; 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 in Methods in Molecular Biology v.969 (Rabinovich, PHEd), 2013). HPLC-purified mRNA has been reported to be translated at much higher levels, especially in primary cells and in vivo.

[0494] A wide variety of modifications have been described in the art to alter specific properties of in vitro transcribed mRNA and improve its usefulness. These include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNAs typically contain a cap structure at the 5' end of the mature molecule, which plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which in turn plays a role in enhancing intracellular mRNA stability and the efficiency of mRNA translation. Therefore, the highest level of protein expression is achieved with capped mRNA transcripts. The 5'-cap contains a 5'-5'-triphosphate linkage between the 5'-proximal nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the terminal and penultimate 2'-hydroxyl groups of most 5'-nucleotides.

[0495] Multiple distinct 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 chemical cap analogs (i.e., capping during in vitro transcription). For example, the anti-reverse cap analog (ARC A) cap contains a 5'-5'-triphosphate guanine-guanine linkage, with one guanine containing an N7 methyl group and a 3'-0-methyl group. However, up to 20% of transcripts remain uncapped during this co-transcription process, and synthetic capping analogs are not identical to the 5'-cap structure of authentic cellular mRNAs, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules can be enzymatically capped after transcription. These may produce more authentic 5'-cap structures that structurally or functionally mimic endogenous 5'-caps with enhanced cap-binding protein binding, increased half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' decapping. Numerous synthetic 5' cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, synthesis RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PHEd), 2013).

[0496] At the 3'-end, a long chain of adenine nucleotides (poly-A tail) is usually added to mRNA molecules during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved to free a 3' hydroxyl at which poly-A polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. Poly(A) tails have been extensively shown to enhance both the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A)-binding proteins and the regulation of mRNA stability, Trends Bio Sci v. 14 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dryfus, M. and Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v. 11, 611-613).

[0497] Poly(A) tailing of in vitro transcribed mRNA can be achieved using various approaches, including, but not limited to, cloning a poly(T) tract into a DNA template or post-transcriptional addition using poly(A) polymerase. The first approach allows for in vitro transcription of mRNA with a poly(A) tail of defined length, depending on the size of the poly(T) tract, but requires additional manipulation of the template. The latter approach involves enzymatically adding a poly(A) tail to in vitro transcribed mRNA using poly(A) polymerase, which catalyzes the incorporation of adenine residues into the 3' end of the RNA. This approach does not require additional manipulation of the DNA template, but can result in mRNAs with poly(A) tails of heterogeneous lengths. 5'-capping and 3'-poly(A) tailing can be performed using a variety of commercially available kits, including, but not limited to, the Poly(A) Polymerase Tailing Kit (EpiCenter), the mMESSAGE mMACHINE T7 Ultra Kit and the Poly(A) Tailing Kit (Life Technologies), as well as commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0498] In addition to 5' caps 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 in eukaryotes and induce a strong innate immune response. Because most nucleic acids from natural sources contain modified nucleosides, the ability to distinguish pathogenic DNA from self-DNA and RNA has been shown to be based, at least in part, on structural and nucleoside modifications. In contrast, in vitro-synthesized RNA lacks these modifications and therefore provides immunostimulatory properties, which, as mentioned above, can inhibit effective mRNA translation.Introducing modified nucleosides into in vitro transcribed mRNA can prevent the recognition and activation of RNA sensors, alleviating this undesirable immunostimulatory activity and enhancing translational competence (see, for example, 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, v.10 pp. 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 in Methods in Molecular Biology v.969 (Rabinovich, PHEd), 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 v.16, 1833-1840). The modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared and utilized using general methods and procedures known in the art. A wide variety of nucleoside modifications are available that can be incorporated into in vitro transcribed mRNA alone or in some combination with other modified nucleosides (see, for example, U.S. Patent Application Publication No. 2012 / 0251618).In vitro synthesis of nucleoside-modified mRNA has been reported to have a reduced ability to activate immunosensors with a concomitant enhanced translational capacity.

[0499] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include 5' and 3' untranslated regions (UTRs).Optimizing UTRs (preferable 5' and 3' UTRs can be obtained from cellular or viral RNA) has been shown to increase mRNA stability and translation efficiency of in vitro transcribed mRNA, both or independently (see, for example, Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in synthetic melanoma RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PHEd), 2013).

[0500] In addition to mRNA, other nucleic acid payloads may be used in the present disclosure. For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis and enzymatic cleavage, chemical cleavage of longer precursors, in vitro transcription as described above, etc. Methods for synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, for example, Gait, MJ (ed.) Oligonucleotide Synthesis: A Practical Approach, Oxford [Oxfordshire], Ishington, DC: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, v. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005, both of which are incorporated herein by reference). For plasmid DNA, preparations for use in embodiments of the present disclosure generally utilize, but are not limited to, in vitro propagation and isolation of the plasmid DNA in liquid cultures of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows those bacteria containing the plasmid of interest to selectively grow in antibiotic-containing cultures. Methods for isolating plasmid DNA are widely used and well known in the art (e.g., 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 U.S. Patent No. 6,197,553). Plasmid isolation can be performed using a variety of commercially available kits, including, but not limited to, PlasmidPlus (Qiagen), GenJET Plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits, as well as commercially available reagents.

[0501] In some embodiments, the lipid nanoparticle compositions are useful for expressing proteins encoded by mRNA. In some embodiments, provided herein are methods for expressing proteins encoded by mRNA.

[0502] In some embodiments, the LNP composition has an N / P ratio of about 1:1 to about 30:1, e.g., about 3:1 to about 20:1, about 3:1 to about 15:1, about 3:1 to about 10:1, or about 3:1 to about 6:1. For example, the N / P ratio of the nucleic acid molecule-encapsulating lipid composition may be about 6±1, or the N / P ratio of the nucleic acid molecule-encapsulating lipid composition may be about 6±0.5. In some embodiments, the N / P ratio of the nucleic acid molecule-encapsulating lipid composition ranges from about 3:1 to about 15:1. In some embodiments, the N / P ratio of the nucleic acid molecule-encapsulating lipid composition is about 6. The N / P ratio refers to the molar ratio of amines (e.g., amines in ionizable lipids) present in the lipid composition or lipid nanoformulation to phosphate present in the nucleic acid molecule. This is a factor in efficient packaging and efficacy.

[0503] Other medications The therapeutic agent may be a peptide or protein, a small molecule drug, encapsulated in the lipid composition. The pharmaceutical composition may contain two or more different therapeutic agents from a nucleic acid molecule, a peptide or protein, and a small molecule drug.

[0504] In some embodiments, the protein may be a peptide or polypeptide, such as, for example, a transcription factor, a chromatin remodeling factor, an antigen, a hormone, an enzyme (such as a nuclease, e.g., an endonuclease and, e.g., a nuclease component of a CRISPR system, e.g., Cas9, dCas9, aCas9-nickase, Cpf / Cas12a), a CRISPR-binding enzyme, e.g., a base editor or prime editor, a mobile genetic element protein (e.g., a transportase, retrotranspotase, recombinase, integrase), a gene writer, a polymerase, a methylase, a demethylase, an acetylase, a deacetylase, a kinase, a phosphatase, a ligase, a deubiquitinase, an integrase, a topoisomerase, a gyrase, a helicase, a lysosomal acid hydrolase, an antibody, a receptor ligand, a receptor, a coagulation factor, a membrane protein, a mitochondrial protein, a nuclear protein, an antibody, or other protein scaffold binders such as centrins, darpins, or adnectins.

[0505] In some embodiments, a pharmaceutical composition may include multiple protein molecules, which may be of the same or different types.

[0506] In some embodiments, the therapeutic agent is a small molecule drug, eg, a small molecule drug approved for human use by an appropriate regulatory agency.

[0507] In some embodiments, a pharmaceutical composition may include multiple small molecule drugs, which may be of the same or different type.

[0508] 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).

[0509] Other ingredients Pharmaceutical compositions may contain one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are selected based on the mode and route of administration. Suitable pharmaceutical carriers or excipients for use in pharmaceutical formulations are described in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippencott Williams & Wilkins (2005); Handbook of Pharmaceutical Excipients, 6th Edition, Rowe et al., Eds., Pharmaceutical Press (2009), and USP / NF (United States Pharmacopeia and the National Formulary), which are incorporated herein by reference in their entirety.

[0510] In some embodiments, the pharmaceutically acceptable excipient comprises one or more of an antioxidant, binder, anti-adhesive, buffer, colorant, diluent (e.g., solid or liquid), disintegrant (e.g., coating disintegrates), dispersant, digestive agent, filler, emulsifier, flavoring agent, lubricant, pH adjuster, pigment, preservative, stabilizer, solubilizer, solvent, suspending agent, sweetener, or wetting agent, or a combination thereof.

[0511] Examples of suitable excipients include acacia alginate, calcium phosphate, calcium carbonate, calcium silicate, carbopol gel, carboxymethylcellulose, carnauba wax, cellulose, crospovidone, dextrose, diacetylated monoglyceride, ethylcellulose, gelatin, glyceryl monostearate 40-50, acacia gum, gum arabic, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hypromellose phthalate, hypromellose, lactose, lecithin, magnesium stearate, kaolin, methacrylic acid copolymer type C, mannitol, methylcellulose, methylhydroxybenzoate, microcrystalline cellulose, povidone, polyethylene glycol, polysorbate 80, polyvinylpyrrolidone, propylhydroxybenzoate, carboxymethylcellulose, sodium hydroxide, sodium stearyl fumarate, sodium starch glycolate, starch, sorbitan sorbitol monooleate, sorbic acid, sucrose, talc, and tragacanth. These include, but are not limited to, talc, triethyl citrate, titanium dioxide, yellow ferric oxide, talc, oil vehicles (e.g., peanut oil, liquid paraffin, mineral oil, olive oil, mondo oil, glycerin, propylene glycol), and water.

[0512] When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material (e.g., saline) that acts as a vehicle, carrier, or medium for the active ingredient. As is known in the art, the type of diluent can vary depending on the intended route of administration.

[0513] The pharmaceutical composition may contain pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, serum albumin, etc. The formulation may comprise a hydrophilic polymer such as gelatin, or an immunoglobulin, polyvinylpyrrolidone, an amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine, a monosaccharide, a disaccharide, or other carbohydrate including glucose, and may contain mannose, or dextran, a chelating agent such as EDTA, a sugar such as sucrose, mannitol, trehalose, or sorbitol, a salt-forming counterion such as sodium, a metal complex (e.g., a Zn-protein complex), and / or a non-ionic surfactant such as TWEEN™, a buffer such as PLURONICS™, or polyethylene glycol (PEG).

[0514] Suitable carriers or excipients for pharmaceutical compositions may also include substances that enhance the individual's body's ability to absorb LNPs or liposomes. Suitable carriers and / or excipients also include any substance that can be used to bulk formulations containing LNPs or liposomes to allow for convenient and accurate dosing. In addition, carriers and / or excipients may be used in the manufacturing process to facilitate handling of LNPs or liposomes. Different carriers and / or excipients may be used depending on the route of administration and the form of the drug.

[0515] Carriers and / or excipients may also include vehicles and / or diluents. "Vehicle" generally refers to any of a variety of media that act as a solvent or carrier, and "diluent" refers to a diluent used to dilute the active ingredients of the composition. Suitable diluents include any substance that can reduce the viscosity of a pharmaceutical product. The type and amount of carrier and / or excipient are selected as a function of the selected pharmaceutical form. Suitable pharmaceutical forms include liquid systems such as solutions, injections, and suspensions; semi-solid systems such as colloids, gels, pastes, or creams; and solid systems such as powders, granules, tablets, capsules, pellets, microparticles, minitablets, microcapsules, micropellets, and suppositories.

[0516] Each of the above systems may be suitably formulated for normal, delayed, or accelerated release using techniques well known in the art.

[0517] Formulation, Dosage, and Route of Administration The pharmaceutical compositions described herein can be prepared according to standard techniques and the techniques described herein.For example, pharmaceutical compositions can be prepared by conventional methods, such as conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, encapsulating or lyophilizing processes.The method for preparing formulations well known in the art is well known in the art.For example, it can be found in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippencott Williams & Wilkins (2005), and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York.

[0518] The therapeutic agent may be encapsulated within the lipid composition; for example, the therapeutic agent may be associated with the interior space of the LNP, within the lipid layer / membrane, or with the outer surface of the lipid layer / membrane. One purpose of incorporating a therapeutic agent into an LNP is to protect the therapeutic agent from the environment, which may contain enzymes, chemicals, or conditions that degrade the therapeutic agent and / or systems or receptors that cause rapid excretion of the therapeutic agent. Furthermore, incorporating a therapeutic agent into an LNP may facilitate uptake of the therapeutic agent and thus enhance therapeutic efficacy.

[0519] In some embodiments, in pharmaceutical compositions, the ratio of lipid component to therapeutic agent (mass / mass ratio, w / w ratio) can range from 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.

[0520] The lipid or pharmaceutical composition may contain about 5 to about 95% by weight of the therapeutic agent, based on the weight of the lipid or pharmaceutical composition. In some embodiments, the lipid or pharmaceutical composition contains about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% by weight of the therapeutic agent, based on the weight of the LNP or pharmaceutical composition. In some embodiments, the lipid composition or pharmaceutical composition contains a therapeutic agent in an amount of 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 10-80%, about 10-70%, about 10-60%, about 10-50%, about 10-40%, about 10-30%, about 10-20%, or Approximately 5-10%, approximately 10-95%, approximately 10-90%, approximately 10-80%, approximately 10-70%, approximately 10-60%, approximately 10-50%, approximately 10-40%, approximately 10-30%, approximately 10-20%, approximately 20-95%, approximately 20-90%, approximately 20-80%, approximately 20-70%, approximately 20-60%, approximately 20-50%, approximately 20-40%, The amount may be 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%.

[0521] The lipid composition or pharmaceutical composition may contain total lipids in an amount of about 5 to about 95% by weight based on the weight of the lipid composition or pharmaceutical composition. In some embodiments, the lipid composition or pharmaceutical composition may contain total lipids in an amount of about 5 to about 95%, about 5 to 90%, about 5 to 80%, about 5 to 70%, about 5 to 60%, about 5 to 50%, about 5 to 40%, about 5 to 30%, about 5 to 20%, about 5 to 10%, about 10 to 95%, about 10 to 90%, about 10 to 80%, about 10 to 70%, about 10 to 60%, about 10 to 50%, about 10 to 40%, about 10 to 30%, about 10 to 20%, about 20 to 95%, about 20 to 90%, about 20 to 80%, about 20 to 70%, about 20 to 60%, about 20 to 50%, or about 20 to 40% based on the weight of the lipid composition or pharmaceutical composition. The total lipid content may be 0%, 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%.

[0522] The composition of the present disclosure can be administered by various routes, for example, to achieve systemic delivery through intravenous, parenteral, intraperitoneal or local route.In some embodiments, siRNA can be delivered intracellularly in the cells of target tissues such as lung or liver, or in inflammatory tissue.In some embodiments, the present disclosure provides a method for delivering siRNA in vivo.Nucleic acid-lipid composition can be administered to subject intravenously, subcutaneously or intraperitoneally.

[0523] The compositions and methods of the present disclosure can be administered to a subject by various mucosal administration modes, including oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal or cutaneous delivery, or topical delivery to the eye, ear, skin, or other mucosal surfaces. In some aspects of the present disclosure, the mucosal tissue layer comprises an epithelial cell layer. The epithelial cells can be pulmonary, tracheal, bronchial, alveolar, nasal, oral, epidermal, or gastrointestinal. The compositions of the present disclosure can be administered using conventional actuators, such as mechanical spray devices, as well as pressurized, electrically actuated, or other types of actuators.

[0524] The compositions of the present disclosure may be administered in aqueous solution as nasal or pulmonary sprays, or may be dispensed in spray form by various methods known to those skilled in the art. Pulmonary delivery of the compositions of the present disclosure is achieved, for example, by administering the compositions in the form of droplets, particles, or sprays, which may be aerosolized, atomized, or nebulized. The particles of the composition, spray, or aerosol may be in either liquid or solid form. A non-limiting example of a system for dispensing liquids as nasal sprays is described in U.S. Patent No. 4,511,069. Such formulations can be conveniently prepared by dissolving a composition according to the present disclosure in water to produce an aqueous solution and sterilizing the solution. Formulations are described, for example, in U.S. Patent No. 4,511,069. Other suitable nasal spray delivery systems are described in TRANSDERMAL SYSTEMIC MEDICATION, Y.W. Chien, ed., Elsevier Publishers, New York, 1985, and U.S. Patent No. 4,778,810. Additional forms of aerosol delivery include compressed air jet nebulizers, ultrasonic nebulizers, and piezoelectric nebulizers, which deliver bioactive agents dissolved or suspended in a pharmaceutical solvent such as water, ethanol, or mixtures thereof.

[0525] The nasal and pulmonary spray solutions of the present disclosure typically contain the 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 may be between pH 6.8 and 7.2. The pharmaceutical solvent used may also be a slightly acidic aqueous buffer solution with a pH of between 4 and 6. Other ingredients, including preservatives, surfactants, dispersants, or gases, may be added to enhance or maintain chemical stability.

[0526] In some embodiments, the present disclosure is a pharmaceutical product comprising a solution containing a composition of the present disclosure and an actuator for a pulmonary, mucosal, or intranasal spray or aerosol.

[0527] The dosage form of the composition of the present disclosure may be in the form of a liquid, droplets or emulsion, or in the form of an aerosol.

[0528] The dosage form of the composition of the present disclosure may be solid and can be reconstituted in liquid before administration.The solid can be administered as a powder.The solid can be in the form of a capsule, tablet, or gel.

[0529] To prepare compositions for pulmonary delivery within the present disclosure, bioactive agents can be combined with various pharmaceutically acceptable additives, as well as bases or carriers for dispersion of the active agent(s).

[0530] 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), tonicity agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancers (e.g., cyclodextrin and its 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 measured with reference to the tonicity of 0.9% (w / v) saline taken as a single unit and is typically adjusted to a value that does not induce substantial irreversible tissue damage to the mucosa at the administration site. Generally, the tonicity of the solution is adjusted to a value of 1 / 3 to 3, more typically 1 / 2 to 2, and most often 3 / 4 to 1.7.

[0531] The bioactive agent may be dispersed in a base or vehicle, which may contain a hydrophilic compound capable of dispersing the active agent and any desired additives. The base may be selected from a wide range of suitable carriers, including, but not limited to, copolymers of polycarboxylic acids or their salts with carboxylic acid anhydrides (e.g., maleic anhydride) and other monomers (e.g., methyl (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 non-toxic metal salts thereof. Biodegradable polymers are often chosen as the base or carrier, such as polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer, and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters, such as polyglycerin fatty acid esters and sucrose fatty acid esters, can be used as carriers. Hydrophilic polymers and other carriers can be used alone or in combination, and improved structural integrity can be imparted to the carrier by partial crystallization, ionic bonding, cross-linking, etc. Carriers can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres, and films for direct application to the nasal mucosa. The use of selected carriers in this context can result in enhanced absorption of the bioactive agent.

[0532] Compositions for mucosal, nasal, or pulmonary delivery may contain hydrophilic low-molecular-weight compounds as bases or excipients. Such hydrophilic low-molecular-weight compounds can provide a pathway through which water-soluble active agents, such as physiologically active peptides or proteins, can diffuse to the body surface where the active agent is absorbed through the base. The hydrophilic low-molecular-weight compounds may optionally absorb moisture from the mucous membrane or the administration atmosphere and dissolve the water-soluble active peptide. In some embodiments, the molecular weight of the hydrophilic low-molecular-weight compounds is 10,000 or less, for example, 3,000 or less. Examples of hydrophilic low-molecular-weight compounds include polyol compounds, such as sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentiose, glycerin, polyethylene glycol, and mixtures thereof. Further examples of hydrophilic low molecular weight compounds include n-methylpyrrolidone, alcohols (eg, oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.), and mixtures thereof.

[0533] Alternatively, the compositions of the present disclosure may contain pharmaceutically acceptable carrier materials required to approximate physiological conditions, such as pH adjusting and buffering agents, isotonicity adjusting agents, and wetting agents, for example, 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, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc.

[0534] In certain embodiments of the present disclosure, the bioactive agent may be administered in a sustained-release formulation, such as a composition comprising a sustained-release polymer. The active agent may be formulated with a carrier that protects it from rapid release, e.g., a controlled-release vehicle such as a polymer, a microencapsulated delivery system, or a bioadhesive gel. Extended delivery of the active agent in various compositions of the present disclosure may be achieved by including it in an absorption-delaying composition, such as, for example, aluminum monosterate hydrogel and gelatin.

[0535] In some embodiments, the lipid composition, pharmaceutical composition, or dosage unit comprises about 0.01 to about 1000 mg of one or more lipid compounds described herein. In some embodiments, the lipid composition, pharmaceutical composition, or dosage unit comprises about 0.01, about 0.1, about 0.5, about 1, about 5, about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 mg of one or more lipid compounds described herein.In some embodiments, the lipid composition, pharmaceutical composition, or unit dose is 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, or from about 0. 1 to about 1000mg, about 0.1 to about 750mg, about 0.1 to about 500mg, about 0.1 to about 250mg, about 0.1 to about 100mg, about 0.1 to about 50mg, about 0. 1 to about 25, about 0.1 to about 10 mg, about 0.1 to about 5 mg, about 0.1 to about 1 mg, about 1 to about 1000 mg, about 1 to about 750 mg, about 1 to about 500 mg, about 1 to about 2 50mg, about 1 to about 100mg, about 1 to about 50mg, about 1 to about 25mg, about 1 to about 10mg, about 1 to about 5mg, about 5 to about 1000mg, about 5 to about 750mg, about 5 to about 500 mg, about 5 to about 250 mg, about 5 to about 100 mg, about 5 to about 50 mg, about 5 to about 25 mg, about 5 to about 10 mg, about 10 to about 1000 mg, about 10 to about 750mg, about 10 to about 500 pieces, about 10 to about 250mg, about 10 to about 100mg, about 10 to about 50mg, about 10 to about 25mg, about 25 to about 1000mg, about 2 5 to about 750 mg, about 25 to about 500 mg, about 25 to about 250 mg, about 25 to about 100 mg, about 25 to about 50 mg, about 50 to about 1000 pieces, mg about 50 to about 750 mg, about 50 to about 500 mg, about 50 to about 250 mg, about 50 to about 100 mg, about 100 to about 1000 mg, about 100 to about 750 mg, about 100 to about 500 mg, about 100 to about 250 mg, about 250 to about 1000 mg, about 250 to about 750 mg, about 250 to about 500 mg, about 500 to about 1000 mg, about 500 to about 750 mg, or about 750 to about 1000 mg of one or more lipid compounds described herein.

[0536] Methods of Use of Lipid Compositions 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.

[0537] In some embodiments, provided herein are methods for delivering a therapeutic cargo to at least one organ selected from the pancreas, one or both of the lungs, and the spleen of a subject in a minimal amount that would be delivered elsewhere in the body, such as the liver of the subject. In some embodiments, the methods deliver a therapeutic cargo to the pancreas and / or one or both of the lungs of a subject in need thereof in a minimal amount that would be delivered elsewhere in the body, such as the liver of the subject.

[0538] 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 a subject is delivered to the subject's liver, hi 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 a subject is delivered to the subject's liver.

[0539] 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 a subject is delivered to the subject's pancreas, spleen, and / or one or both lungs. 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 a subject is delivered to the subject's pancreas. 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 a subject is delivered to the subject's lungs. 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 a subject is delivered to the subject's spleen.

[0540] In some embodiments, the total therapeutic cargo administered to a subject has a spleen-to-liver ratio of 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 total therapeutic cargo administered to a subject has a spleen-to-liver ratio of at least 1. In some embodiments, the total therapeutic cargo administered to a subject has a spleen-to-liver ratio of at least 5.

[0541] As used herein, the percentage of total therapeutic cargo administered to a subject and delivered to a location within the subject is measured by the level of protein expression, or mRNA knockdown level.

[0542] In some embodiments, the method of delivering a therapeutic cargo disclosed above comprises administering to a 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 any stereoisomers thereof. 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 any stereoisomers thereof. 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 any stereoisomers thereof. 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 any stereoisomers thereof. 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 any stereoisomers thereof. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from an ionizable lipid of formula (V), a pharmaceutically acceptable salt thereof, and any stereoisomers of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from an ionizable lipid of formula (VI), a pharmaceutically acceptable salt thereof, and any stereoisomers of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from an ionizable lipid of formula (VII), a pharmaceutically acceptable salt thereof, and any stereoisomers of the foregoing.

[0543] In some embodiments, the lipid nanoparticles and lipid nanoparticle compositions disclosed herein can be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells, in vitro and / or in vivo. Accordingly, in some embodiments, methods are provided for treating or preventing a disease or disorder in a subject in need thereof, comprising administering lipid nanoparticles to the subject. In some embodiments, the lipid nanoparticles encapsulate or are associated with a suitable therapeutic agent, and the lipid nanoparticles comprise one or more of the novel ionizable lipids described herein, pharmaceutically acceptable salts thereof, and / or stereoisomers of any of the foregoing.

[0544] In some embodiments, the lipid nanoparticles of the present disclosure are useful for delivering therapeutic cargoes. 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 (antagomirs), messenger RNA interference complementary RNA (micRNA), DNA, polyvalent RNA, Dicer substrate RNA, complementary DNA (cDNA), etc. Thus, in some embodiments, disclosed herein are methods for inducing expression of a desired protein in vitro and / or in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel ionizable lipids described herein, wherein the lipid nanoparticles encapsulate or are associated with a nucleic acid that is expressed to produce the desired protein (e.g., a messenger RNA or plasmid encoding the desired protein) or inhibit a process that terminates mRNA expression (e.g., a miRNA inhibitor). In some embodiments, disclosed herein are methods for reducing target gene and protein expression in vitro and / or in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel ionizable lipids described herein, wherein the lipid nanoparticles encapsulate or are associated with a nucleic acid (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)) that reduces target gene expression. In some embodiments, disclosed herein are methods for the co-delivery of one or more nucleic acids (e.g., mRNA and plasmid DNA). For example, these methods may be useful for providing effects that require co-localization of different nucleic acids (e.g., mRNA and DNA segment(s) encoding a gene-modifying enzyme suitable for integration into the host genome).

[0545] In some embodiments, the lipid nanoparticle compositions are useful for upregulating endogenous protein expression by delivering miRNA inhibitors that target one specific miRNA or a group of miRNAs that regulate one target mRNA or several mRNAs. In some embodiments, provided herein are methods for upregulating endogenous protein expression, comprising delivering miRNA inhibitors that target one or more miRNAs that regulate one or more mRNAs. In some embodiments, the lipid nanoparticle compositions are useful for downregulating (e.g., silencing) the protein and / or mRNA levels of target genes. In some embodiments, provided herein are methods for downregulating (e.g., silencing) the protein and / or mRNA levels of target genes.

[0546] In some embodiments, lipid nanoparticles are useful for delivering mRNA and plasmids for transgene expression. In some embodiments, methods for delivering mRNA and plasmids for transgene expression are provided herein.

[0547] In some embodiments, lipid nanoparticle compositions are useful for inducing a pharmacological effect resulting from protein expression, such as increased red blood cell production through delivery of a suitable erythropoietin mRNA, or protection against infection through delivery of mRNA encoding a suitable antigen or antibody. In some embodiments, provided herein are methods for inducing a pharmacological effect resulting from protein expression, such as increased red blood cell production through delivery of a suitable erythropoietin mRNA, or protection against infection through delivery of mRNA encoding a suitable antigen or antibody. Non-limiting exemplary embodiments of the disclosed ionizable lipids, lipid nanoparticles, and compositions comprising them, and their uses for delivering pharmaceutical agents (e.g., therapeutic agents such as nucleic acids) and / or modulating gene and / or protein expression, are described in further detail below.

[0548] In some embodiments, the present disclosure relates to methods of gene editing comprising contacting a cell with an LNP, hi some embodiments, the present disclosure relates to any method of gene editing described herein comprising cleaving DNA.

[0549] In some embodiments, the present disclosure relates to methods of cleaving DNA comprising contacting a cell with an LNP composition.

[0550] In some embodiments, the present disclosure relates to any method of cleaving DNA described herein, wherein the cleaving step comprises introducing a single-stranded DNA nick. In some embodiments, the present disclosure relates to any method of cleaving DNA described herein, wherein the cleaving step comprises introducing a double-stranded DNA break. In some embodiments, the present disclosure relates to any method of cleaving 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 cleaving DNA described herein, wherein the LNP composition further comprises introducing at least one template nucleic acid into a cell. In some embodiments, the present disclosure relates to any method of cleaving DNA described herein, comprising contacting a cell with an LNP composition comprising a template nucleic acid.

[0551] In some embodiments, the present disclosure relates to any method of gene editing 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 method of gene editing described herein, wherein the method comprises administering an LNP composition to a cell, such as a eukaryotic cell.

[0552] In some embodiments, the present disclosure relates to any method of gene editing described herein, wherein the method comprises administering mRNA formulated in a first LNP composition and a second LNP composition, the first LNP composition comprising one or more of an mRNA, a gRNA, a gRNA nucleic acid, and a template nucleic acid. In some embodiments, the present disclosure relates to any method of gene editing described herein, wherein the first and second LNP compositions are administered simultaneously. In some embodiments, the present disclosure relates to any method of gene editing described herein, wherein the first and second LNP compositions are administered sequentially.

[0553] In some embodiments, the present disclosure relates to any of the methods of gene editing described herein, wherein the method comprises administering mRNA and guide RNA nucleic acids formulated in a single LNP composition.

[0554] In some embodiments, the present disclosure relates to any of the methods of gene editing described herein, wherein the gene editing results in a gene knockout.

[0555] In some embodiments, the present disclosure relates to any of the methods of gene editing described herein, wherein the gene editing results in gene correction.

[0556] In some embodiments, the present disclosure relates to methods for in vivo delivery of interfering RNA to the lungs of a mammalian subject.

[0557] In some embodiments, the present disclosure relates to methods of treating a disease or disorder in a mammalian subject, hi 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 expression or overexpression of a gene that can be reduced, decreased, downregulated, or silenced by the composition. [Example]

[0558] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way. [Example]

[0559] Synthesis of compound 2243 [ka]

[0560] Step A1 To a solution of methoxymethyl(triphenyl)phosphonium chloride (24.16 g, 70.47 mmol, 3 equiv.) in THF (360 mL), n-BμLi (2.5 M, 26.31 mL, 2.8 equiv.) was added dropwise at 0 °C, and the mixture was stirred at 25 °C for 2 h. A solution of undecan-6-one (B) (4 g, 23.49 mmol, 1 equiv.) in THF (120 mL) was added to the mixture at 0 °C, followed by stirring at 25 °C for 12 h. The mixture was poured into HO (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 NaSO, 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) (18 g, 90.8 mmol, 77% yield) as a colorless oil.

[0561] Step A2: A solution of 6-(methoxymethylene)undecane (C) (18 g, 90.75 mmol, 1 equiv.) in THF (72 mL) and aqueous HCl (3 M, 18.00 mL, 0.595 equiv.) was stirred at 70 °C for 12 h. The mixture was poured into HO (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 NaSO, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 100 / 1 to 20 / 1) to give 2-pentylheptanal (D) (15 g, 81.38 mmol, 90% yield) as a colorless oil.

[0562] Step A3: To a solution of NaH (3.95 g, 98.74 mmol, 7.05 mL, 60% purity, 1.3 equiv) in THF (280 mL) was added ethyl 2-diethoxyphosphorylacetate (22.14 g, 98.74 mmol, 19.59 mL, 1.3 equiv) dropwise at 0 °C, and the mixture was stirred at 25 °C for 0.5 h. A solution of 2-pentylheptanal (D) (14 g, 75.96 mmol, 1 equiv) in THF (70 mL) was added to the mixture at 0 °C, and the mixture was then warmed to 25 °C and stirred at 25 °C for 2 h. The mixture was poured into HO (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 NaSO, 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 obtain ethyl 4-pentylnon-2-enoate (E) (16 g, 62.89 mmol, yield 82.80%) as a colorless oil.

[0563] Step A4 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 under H (15 Psi) at 25° C. for 1 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give ethyl 4-pentylnonanoate (F) (15 g, crude) as a colorless oil.

[0564] Step A5 To a solution of LAH (1.48 g, 39.00 mmol, 7.05 mL, 2 equiv.) in THF (50 mL) was added a solution of ethyl 4-pentylnonanoate (F) (5 g, 19.50 mmol, 1 equiv.) in THF (10 mL) at 0 °C and stirred at 0 °C for 1 h. The mixture was poured into HO (30 mL) at 0 °C, then the mixture was filtered, and the filtrate was extracted with EtOAc (50 mL × 3). The combined organic layer was washed with brine (50 mL × 2), dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 100 / 1 to 20 / 1) to give 4-pentylnonan-1-ol (compound A) (10 g, 46.6 mmol, 80% yield) as a colorless oil.

[0565] Step 1 To 2-methylpropanoyl chloride (25.50 g, 239 mmol, 25 mL, 1 equiv) in DCM (400 mL) was added 2-methylpropan-2-ol (1) (18.63 g, 251 mmol, 24 mL, 1.05 equiv) in DCM (400 mL), followed by the addition of TEA (36.33 g, 359 mmol, 50 mL, 1.5 equiv) and DMAP (1.46 g, 11.97 mmol, 0.05 equiv) and the mixture was stirred at 25 °C for 8 h. The mixture was added to HO (1000 mL) and extracted with DCM (300 mL × 2). The organic layer was washed with brine (200 mL × 2), dried over NaSO, 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) (46 g, 319 mmol, 33% yield) as a colorless oil.

[0566] Step 2 To a solution of diisopropylamine (10.5 g, 104 mmol, 14.7 mL, 1.5 equiv) in THF (120 mL) was added n-BμLi (2.5 M, 41.6 mL, 1.5 equiv) under N at −40° C. The mixture was stirred at −40° C. for 0.5 h, then cooled to −70° C., and 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 N. Then, 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 at −70° C. and stirred at 25° C. for 8 h under N. The mixture was added to aqueous NH4Cl (200 mL) and extracted with EtOAc (200 mL × 3). The combined organic phase was washed with brine (100 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 = 1 / 0 to 50 / 1) to give tert-butyl 8-bromo-2,2-dimethyloctanoate (5) (10 g, 32.5 mmol, 47% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3), 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).

[0567] Step 3 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. The residue was then dissolved in EtOAc (200 mL), washed with NaHCO (200 mL × 3), brine (200 mL × 2), dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure to give 8-bromo-2,2-dimethyl-octanoic acid (6) (6.6 g, crude) as a colorless oil...

Claims

1. at least one head group and at least one tail group of formula (TI) or (TI') 【Chemistry 1】 (TI) or 【Chemistry 2】 (TI'), a pharmaceutically acceptable salt thereof, or a lipid comprising any of the aforementioned stereoisomers, During the ceremony, 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(CH 2 ) r -, -C(O)N(R 7 )(CH 2 ) r -, -S-S-, or -C(O-R 13 )-O-(CH 2 ) r -; in the formula, each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl, R 13 is branched or unbranched C 3 to C 10 alkyl, and r is 1, 2, 3, 4 or 5, R a Each of them operates independently, C 1 ~C 5 Alkyl, C 2 ~C 5 Alkenyl, or C 2 ~C 5 It is alkinyl, u1 and u2 are independently 0, 1, 2, 3, 4, 5, 6, or 7. R t However, H and C are independent of each other. 1 ~C 16 Branched or unbranched alkyl or C 1 ~C 16 Branched or unbranched alkenyls, optionally interrupted by heteroatoms, or substituted with OH, SH, or halogens, or cycloalkyl or substituted cycloalkyl groups. 【Transformation 3】 This represents a bond that connects the tail group to the head group. The lipid is the lipid having a pKa of approximately 4 to approximately 8.

2. The lipid comprises at least one tail group of the following formula: 【Chemistry 4】 (TII), 【Transformation 5】 (TIII) 【Transformation 6】 (TIV), 【Transformation 7】 (TV) 【Transformation 8】 (TII'), and 【Chemistry 9】 (TIII'), During the ceremony, R 7 However, each is independently either H or methyl, R b However, on each occasion, independently, H or C 1 ~C 4 It is alkyl, u3 and u4 are independently 0, 1, 2, 3, 4, 5, 6, or 7. The lipid is the lipid according to claim 1, having a pKa of about 4 to about 8.

3. The lipid according to claim 2, wherein the lipid comprises two, three, four or more tail groups of the formula (TII), (TIII), (TIV), (TV), (TII'), or (TIII'), and the tail groups may be the same or different.

4. R a Each of them is methyl, and / or The lipid according to claim 1, wherein u1 is 3 to 5, u2 is 0 to 3, and u3 and u4 are each independently 1 to 7.

5. The aforementioned lipids (i) comprising at least one tail of formula (TIII), where R a Each of them is methyl, and R b However, in each instance independently, the lipid is H, ethyl, or butyl, u1 is 3 to 5, u2 is 0 to 3, u3 is 1 to 7, and optionally the lipid contains at least two, three, or four tails of formula (TIII), and the two, three, or four tails of formula (TIII) may be the same or different. (ii) comprising at least one tail of formula (TII), wherein Ra is methyl, u1 is 3 to 5, u2 is 0 to 3, u3 is 1 to 4, u4 is 1 to 4, and optionally the lipid has at least two, three, or four tails of formula (TII), and the two, three, or four tails of formula (TII) may be the same or different. (iii) Having at least one tail of formula (TII) and at least one tail of formula (TIII), optionally the lipid having at least two tails of formula (TII) and at least two tails of formula (TIII), wherein the two tails of formula (TII) may be the same or different, and the two tails of formula (TIII) may be the same or different. (iv) Having at least one tail of formula (TIV), where Ra is methyl, u1 is 3 to 5, u2 is 0 to 3, u3 is 1 to 4, u4 is 1 to 4, and optionally the lipid has at least two, three, or four tails of formula (TIV), and the two, three, or four tails of formula (TIV) may be the same or different. (v) Having at least two, three, or four tails of formula (TV), wherein the two, three, or four tails of formula (TV) are either identical or different. (vi) Having at least two, three, or four tails of formula (TII'), wherein the two, three, or four tails of formula (TII') are either identical or different. (vii) Having at least two, three, or four tails of formula (TIII'), wherein the two, three, or four tails of formula (TIII') are either identical or different. (viiii) Having at least one tail selected from the group consisting of formulas (TII), (TIII), and (TII'), and at least one tail selected from the group consisting of (TIV), (TV), and (TIIII'), (ix) Having at least two tails selected from the group consisting of (TII), (TIII), and (TII'), (x) Having at least two tails selected from the group consisting of (TIV), (TV), and (TIII'), (xi) Having at least two tails selected from the group consisting of formulas (TII), (TIII), and (TII'), and at least one tail selected from the group consisting of (TIV), (TV), and (TIIII'), (xii) Having at least one tail selected from the group consisting of formulas (TII), (TIII), and (TII'), and at least two tails selected from the group consisting of (TIV), (TV), and (TIIII'), (xiiii) Having at least two tails selected from the group consisting of formulas (TII), (TIII), and (TII'), and at least two tails selected from the group consisting of formulas (TIV), (TV), and (TIIII'), (xiv) Having at least three tails selected from the group consisting of (TII), (TIII), and (TII'), (xv) Having at least three tails selected from the group consisting of (TIV), (TV), and (TIII'), (xvi) Having at least three tails selected from the group consisting of formulas (TII), (TIII), and (TII'), and at least one tail selected from the group consisting of (TIV), (TV), and (TIIII'), (xvii) Having at least one tail selected from the group consisting of formulas (TII), (TIII), and (TII'), and at least three tails selected from the group consisting of formulas (TIV), (TV), and (TIII'), (xviiii) having at least two tails of formula (TII) or (TIII), and at least two tails of formula (TIV) or (TV), (xix) having at least two tails of formula (TII) or (TIII), and at least two tails of formula (TII') or (TIII'), (xx) having at least two tails of formula (TIV) or (TV), and at least two tails of formula (TII') or (TIII'), The lipid according to claim 2.

6. The formula further includes at least one tail of the formula (TNG-I), 【Chemistry 10】 (TNG-I), in the formula, E is independent of each other: -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -SS-, or -C(O)N(R 7 ) - and u1 and u2 are independently 0, 1, 2, 3, 4, 5, 6, or 7. R 7 The lipid according to claim 1, wherein is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl.

7. The at least one tail of formula (TNG-I) is 【Chemistry 11】 (TNG-II) or 【Chemistry 12】 Represented by (TNG-III), u3 and u4 are independently 0, 1, 2, 3, 4, 5, 6, or 7. R b In each instance, independently, H or C 1 ~C 4 The lipid according to claim 6, which is alkyl.

8. The head group has one of the following structures: (i) 【Chemistry 13】 (HA-I), During the ceremony, R 20 and R 30 These are H and C, which are independent of each other. 1 ~C 5 Branched or unbranched alkyl, or C 2 ~C 5 It is a branched or unbranched alkenyl, optionally interrupted by one or more heteroatoms, or substituted with OH, SH, halogens, or cycloalkyl groups, or R 20 and R 30 Together with adjacent N atoms, it forms a 3- to 7-membered heterocycle or heteroaromatic ring containing one or more heteroatoms, and is optionally substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl groups. R 1 and R 2 These are H and C, which are independent of each other. 1 ~C 3 Branched or unbranched alkyl, C 2 ~C 3 Branched or unbranched alkenyl, OH, halogen, SH, or NR 10 R 11 is or R 1 and R 2 They form a ring together, R 10 and R 11 These are H and C, which are independent of each other. 1 ~C 3 Branched or unbranched alkyl, C 2 ~C 3 It is either a branched or unbranched alkenyl, or R 10 and R 11 Together they form a complex ring, n is 0, 1, 2, 3, or 4, Z does not exist, or O, S, or NR 12 And in the formula, R 12 is H or C 1 ~C 7 It is a branched or unbranched alkyl group, provided that Z is present, and adjacent R 1 and R 2 OH, NR 10 R 11 It must not be SH. (ii) 【Chemistry 14】 (HA-V), During the ceremony, R1 is H, C1-C3 alkyl, OH, halogen, SH, or NR10 R11. R2 is OH, halogen, SH, or NR10 R11, or R1 and R2 together can form a ring. R10 and R11 may each be independently H or C1-C3 alkyl, or R10 and R11 may together form a heterocycle. R20 and R30 are independently H, C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl, or R20 and R30 can form a ring together. v and y are independently 1, 2, 3, or 4. (iii) 【Chemistry 15】 (HB-I), In the formula, W is 【Chemistry 16】 【Chemistry 17】 [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 And, During the ceremony, R 5 is OH, SH, (CH 2) s OH, or NR 10 R 11. Each of R6 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, or a cycloalkyl. R7 and R8 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, (CH2)vOH, (CH2)vSH, (CH2)sN(CH3)2, or NR10R11, where R10 and R11 are each independently H or C1-C3 alkyl, or R10 and R11 together form a heterocycle, or R7 and R8 together form a ring. Each of R20 is independently H, or a C1-C3 branched or unbranched alkyl group. R14 is a heterocyclic compound, NR10R11, C(O)NR10R11, NR10C(O)NR10R11, or NR10C(S)NR10R11, where R10 and R11 are independently H, C1-C3 alkyl, C3-C7 cycloalkyl, or C3-C7 cycloalkenyl, and are optionally substituted with one or more NH and / or oxo groups, or R10 and R11 together form a heterocycle. R 16 is H, =O, =S, or CN. s, u, and t are independently 1, 2, 3, 4, or 5. v is independently 0, 1, 2, 3, 4, or 5. Each of Y is a divalent heterocyclic ring, Each Z is either absent, O, S, or NR12, where R12 is H, a C1-C7 branched or unbranched alkyl, or a C2-C7 branched or unbranched alkenyl. Q is O, S, CH2, or NR13, where R13 is H, C1-C5 alkyl, respectively. V is a branched or unbranched C2-C10 alkylene, C2-C10 alkenylene, C2-C10 alkynylene, or C2-C10 heteroalkylene, which is optionally substituted with one or more OH, SH, and / or halogen groups. T is -NHC(O)O-, -OC(O)NH-, or a divalent heterocycle, or (iv) 【Chemistry 26】 (HC-I), During the ceremony, 【Chemistry 27】 teeth, 【Chemistry 28】 【Chemistry 29】 【Transformation 30】 【Chemistry 31】 【Chemistry 32】 【Transformation 33】 【Transformation 34】 Selected from the group consisting of, Y is alkyl, hydroxy, hydroxyalkyl, 【Chemistry 35】 【Transformation 36】 And, A is either nonexistent, -O-, -N(R7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, -N(R7)C(O)N(R7)-, -S-, or -S-S-. X and Z are, independently, nonexistent, -O-, -C(O)-, -N(R7)-, alkylene, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, or -S-. Each of R7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl, or aminoalkyl. t is 0, 1, 2, or 3, t1 is an integer between 0 and 10. W is a hydroxyl, substituted or unsubstituted hydroxyalkyl, or 【Chemistry 37】 During the ceremony, Each Q is either nonexistent, -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2)q, -C(R7)2, -C(O)N(R7)-, -C(S)N(R7)-, or -N(R7). R 6 independently contains H, alkyl, hydroxyl, hydroxyalkyl, and alkoxy. -O-alkylene-O-alkyl, -O-alkylene-N(R7)2, amino, alkylamino, aminoalkyl, thiol, thiolalkyl, or N+(R7)3-alkylene-Q- Each R8 is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, thioalkyl, heterocyclyl, heteroaryl, or two R8s together with a nitrogen atom form a ring optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, or aminoalkyl groups. q is 0, 1, 2, 3, 4, or 5, p is 0, 1, 2, 3, 4, or 5. The lipid according to claim 1.

9. The head base has the structure of formula (i) (HA-I), wherein, R 20 and R 30 However, together with the adjacent N atoms, they form a 3- to 7-membered heterocycle or heteroaromatic ring containing one or more heteroatoms, which are optionally substituted with one or more OH, SH, halogens, alkyls, or cycloalkyl groups. Z is either nonexistent, or O, S, or NH. Each R1 and R2 is H, and / or The lipid according to claim 8, wherein n is 0, 1, or 2.

10. The head base is 【Transformation 38】 or 【Chemistry 39】 It has the structure, and in the formula, Rc is an alkyl group substituted with H, alkyl, or OH. The lipid according to claim 9, wherein m1 is 1, 2, or 3.

11. The head group has the structure of formula (ii) (HA-V), The lipid according to claim 8, having the structure of the following formula (HA-VI). 【Chemistry 40】 The lipid (HA-VI), wherein R20 and R30 are optionally each independently C1-C3 alkyl.

12. The head base has the structure of formula (HB-I), and R 5 However, OH or (CH 2 ) s It is OH, and s is 1 or 2. R 6 , R 7 , and R 8 Each of them independently is H or C 1 ~C 3 It is alkyl, u and t are independently 1, 2, or 3. v is independently 0, 1, 2, or 3. R 16 However, H or = O, Each Z is either non-existent, or O or NR. 12 And in the formula, R 12 However, H or C 1 ~C 3 It is alkyl, T is a divalent heterocycle, Q is O or CH 2 and V is C 2 ~C 6 Alkylene or C 2 ~C 6 The lipid according to claim 8, which is an alkenylene.

13. W (a) 【Chemistry 41】 And in the formula, R 6 , R 7 , and R 8 are each independently H or methyl, u and t are independently 1, 2, or 3. (b) 【Chemistry 42】 And in the formula, R 16 is H or = O, R 14 is a nitrogen-containing five- or six-membered heterocyclic 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 R 10 and R 11 are each independently H or C1-C3 alkyl. u and v are independently 1, 2, or 3. (c) 【Chemistry 43】 or 【Chemistry 44】 And in the formula, Each of R6 is independently either H or methyl. Each of R7 is independently H, R8 is methyl, Each of u is independently 1, 2, or 3. V is a C2-C6 alkylene or a C2-C6 alkenylene, or (d) 【Chemistry 45】 or 【Chemistry 46】 And, During the ceremony, Each of u is independently 1, 2, or 3. Q is O, Each Z is independent and is NR 12, R12 is H or C1-C3 alkyl, T is a five- or six-membered heterocycle containing divalent nitrogen. The lipid according to claim 12.

14. The head base is 【Chemistry 47】 【Chemistry 48】 【Chemistry 49】 [Transformation 50] The lipid according to claim 8, having the structure, wherein u and t are each independently 1 or 2.

15. The head group has the structure of formula (iv) (HC-I), The following formula: 【Chemistry 51】 (HC-IA) or 【Chemistry 52】 (HC-IB) During the ceremony, W is OH, 【Chemistry 53】 【Chemistry 54】 【Transformation 55】 【Transformation 56】 【Chemistry 57】 【Chemistry 58】 【Chemistry 59】 【Transformation 60】 【Chemistry 61】 【Transformation 62】 【Transformation 63】 【Chemistry 64】 【Transformation 65】 [Chem 66] 【Transformation 67】 【Transformation 68】 【Transformation 69】 【Transformation 70】 【Chemistry 71】 【Chemistry 72】 【Transformation 73】 【Chemistry 74】 【Chemistry 75】 【Transformation 76】 [Chem 77] 【Transformation 78】 【Transformation 79】 【Chemistry 80】 【Chemistry 81】 【Chemistry 82】 【Chemistry 83】 【Chemical 84】 【Chemical 85】 [Chem. 86] 【Transformation 87】 [Chemical 88] 【Chemistry 89】 【Chemistry 90】 【Chemistry 91】 【Chemistry 92】 【Chemistry 93】 【Chemistry 94】 【Chemical 95】 【Chemistry 96】 【Chemistry 97】 【Chem.98】 【Chem.99】 【Chemistry 100】 【Chemistry 101】 【Chemical Engineering 102】 【Chemistry 103】 【Chemical 104】 【Chemistry 105】 【Chemistry 106】 It is one of the following, and / or A does not exist, or is -O-, -N(R7)-, -OC(O)-, or -C(O)O-, and X does not exist, or is -O-, or -C(O)-, Z is -O-, -C(O)O-, or -OC(O)-. The lipid according to claim 8, having the structure.

16. The head base is defined by the following formula 【Chemistry 107】 (HC-IIA) or 【Chemistry 108】 The lipid according to claim 15, having the structure (HC-IIC).

17. The head base is defined by the following formula 【Chemistry 109】 (HC-IIID) or 【Chemical 110】 The lipid according to claim 16, having the structure (HC-IIIE), wherein t1 is 0, 1, 2, or 3.

18. A lipid comprising at least one head group and at least one tail group, At least one tail group has the structure of formula (TI) or (TI'), 【Chemistry 111】 (TI) or 【Chemistry 112】 (TI'), During the ceremony, Each of E is an independent biodegradable group. R a Each of them operates independently, C 1 ~C 5 Alkyl, C 2 ~C 5 Alkenyl, or C 2 ~C 5 It is alkinyl, u1 and u2 are independently 0, 1, 2, 3, 4, 5, 6, or 7. R t However, H and C are independent of each other. 1 ~C 16 Branched or unbranched alkyl or C 1 ~C 16 Branched or unbranched alkenyls, optionally interrupted by heteroatoms, or substituted with OH, SH, or halogens, or cycloalkyl or substituted cycloalkyl groups. 【Chemistry 113】 teeth, This represents a bond connecting the tail group to the head group. The head group has one of the following structures: 【Chemical 114】 During the ceremony, R 20 and R 30 These are H and C, which are independent of each other. 1 ~C 5 Branched or unbranched alkyl, or C 2 ~C 5 It is a branched or unbranched alkenyl, optionally interrupted by one or more heteroatoms, or substituted with OH, SH, halogens, or cycloalkyl groups, or R 20 and R 30 Together with adjacent N atoms, it forms a 3- to 7-membered heterocycle or heteroaromatic ring containing one or more heteroatoms, and is optionally substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl groups. R 1 and R 2 These are H and C, which are independent of each other. 1 ~C 3 Branched or unbranched alkyl, C 2 ~C 3 Branched or unbranched alkenyl, OH, halogen, SH, or NR 10 R 11 is or R 1 and R 2 They form a ring together, R 10 and R 11 These are H and C, which are independent of each other. 1 ~C 3 Branched or unbranched alkyl, C 2 ~C 3 It is either a branched or unbranched alkenyl, or R 10 and R 11 Together they form a complex ring, n is 0, 1, 2, 3, or 4, Z does not exist, or O, S, or NR 12 And in the formula, R 12 is H or C 1 ~C 7 It is a branched or unbranched alkyl group, provided that Z is present, and adjacent R 1 and R 2 OH, NR 10 R 11 It must not be SH, 【Chemical 115】 During the ceremony, R 1 H, C 1 ~C 3 Alkyl, OH, halogen, SH, or NR 10 R 11 And, R 2 OH, halogen, SH, or NR 10 R 11 is, or R 1 and R 2 They can form a ring together, R 10 and R 11 Each is independently H or C 1 ~C 3 Alkyl or R 10 and R 11 They may together form a complex ring, R 20 and R 30 These are H and C, which are independent of each other. 1 ~C 5 Branched or unbranched alkyl, C 2 ~C 5 It is either a branched or unbranched alkenyl, or R 20 and R 30 They can form a ring together, v and y are independently 1, 2, 3, or 4. 【Chemistry 116】 In the formula, W is 【Chemistry 117】 【Chemistry 118】 【Chemical 119】 【Chemical 120】 【Chemistry 121】 【Chemistry 122】 [Chem 123] 【Chemistry 124】 【Chemistry 125】 【Chemistry 126】 During the ceremony, R 5 is OH, SH, (CH 2 ) s OH, or NR 10 R 11 And, R 6 These are H and C, which are independent of each other. 1 ~C 3 Branched or unbranched alkyl, C 2 ~C 3 It is a branched or unbranched alkenyl, or a cycloalkyl, R 7 and R 8 These are H and C, which are independent of each other. 1 ~C 3 Branched or unbranched alkyl, C 2 ~C 3 Branched or unbranched alkenyls, halogens, (CH 2 ) v OH, (CH 2 ) v SH, (CH 2 ) s N(CH 3 ) 2 , or NR 10 R 11 And in the formula, R 10 and R 11 Each of them independently is H or C 1 ~C 3 Alkyl or R 10 and R 11 They together form a complex ring, or R 7 and R 8 They form a ring together, R 20 These are H or C, respectively, independently. 1 ~C 3 It is a branched or unbranched alkyl group. R 14 This is a heterocyclic algebra, 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 and 、 In the formula, R 10 and R 11 These are H and C, which are independent of each other. 1 ~C 3 Alkyl, C 3 ~C 7 Cycloalkyl, C 3 ~C 7 It is a cycloalkenyl, optionally substituted with one or more NH and / or oxo groups, or R 10 and R 11 Together they form a complex ring, R 16 However, H, =O, =S, or CN, s, u, and t are independently 1, 2, 3, 4, or 5. v is independently 0, 1, 2, 3, 4, or 5. Each of Y is a divalent heterocyclic ring, Each Z is independent, either non-existent, or O, S, or NR 12 And in the formula, R 12 H, C 1 ~C 7 Branched or unbranched alkyl, or C 2 ~C 7 It is a branched or unbranched alkenil. Q is O, S, CH 2 , or NR 13 And R 13 is H, C respectively. 1 ~C 5 It is alkyl, V is branched or unbranched C 2 ~C 10 Alkylene, C 2 ~C 10 Alkenylene, C 2 ~C 10 Alkynylene, or C 2 ~C 10 It is a heteroalkylene, optionally substituted with one or more OH, SH, and / or halogen groups. T is -NHC(O)O-, -OC(O)NH-, or a divalent heterocycle. iv) 【Chemistry 127】 (HC-I), During the ceremony, 【Chemistry 128】 This is a ring or complex ring part, Y is alkyl, hydroxy, hydroxyalkyl, 【Chemistry 129】 【Chemistry 130】 And, A does not exist, -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-, X and Z are either nonexistent, -O-, -C(O)-, or -N(R) respectively. 7 ) - -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 ) - or -S-, R 7 Each of these is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl, or aminoalkyl. t1 is an integer between 0 and 10. W is a hydroxyl, a substituted or unsubstituted hydroxyalkyl, a substituted or unsubstituted amino, a substituted or unsubstituted aminocarbonyl, or a substituted or unsubstituted heterocyclyl or heteroaryl. The lipids are lipids having a pKa of approximately 4 to approximately 8.

19. At least one tail group has at least one structure from the following formulas: 【Chemistry 131】 (TII), 【Chemistry 132】 (TIII) 【Chemistry 133】 (TIV), 【Chemistry 134】 (TV) 【Chemistry 135】 (TII'), and 【Transformation 136】 (TIII'), In the formula, Ra is methyl, u1 is 3 to 5, u2 is 0 to 3, u3 and u4 are independently 1 to 7, and The head group has one of the following structures: i) 【Chemistry 137】 (HA-III) ii) 【Chemistry 138】 (HA-VI), where R 20 and R 30 Each is independent of C 1 ~C 3 It is alkyl, iii) 【Chemistry 139】 (HB-I), in the formula, W is [Chemistry 140] And in the formula, R 6 , R 7 , and R 8 Each of them is independently either H or methyl, u and t are independently 1, 2, or 3, or W is 【Chemistry 141】 And in the formula, R 16 However, H or = O, R 14 This refers to a nitrogen-containing 5-membered or 6-membered heterocyclic molecule, 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 and 、 In the formula, R 10 and R 11 Each of these is independently H or C 1 ~C 3 It is alkyl, u and v are independently 1, 2, or 3, or 【Chemistry 142】 【Chemistry 143】 And in the formula, R 6 Each of them is independently either H or methyl, R 7 Each of them is independently H, R 8 Each of them is methyl, Each of u is independently 1, 2, or 3. V is C 2 ~C 6 Alkylene or C 2 ~C 6 It is an alkenylene, or W is 【Chemistry 144】 or 【Chemistry 145】 And in the formula, Each of u is independently 1, 2, or 3. Q is O, Each Z is independent, NR 12 and 、 T is a five- or six-membered heterocycle containing divalent nitrogen. iv) 【Chemistry 146】 (HC-IIA) or 【Chemistry 147】 (HC-IIC), where, W is a hydroxyl, substituted, or unsubstituted hydroxyalkyl compound. 【Chemistry 148】 Q is either independent and does not exist, or -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH 2 ) q -C(R 7 ) 2 , -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and R 6 These independently include H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 ) 2 , amino, alkylamino, aminoalkyl, thiol, thiolalkyl, or N+(R 7 ) 3 -Alkilen-Q-, R 8 Each of these is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, heterocyclyl, heteroaryl, or two R atoms together with a nitrogen atom. 8 However, it forms a ring optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, or aminoalkyl groups. q is 0, 1, 2, 3, 4, or 5, The lipid according to claim 18, wherein p is 0, 1, 2, 3, 4, or 5.

20. At least one tail group has the structure of formula (TII), where Ra is methyl, u1 is 3 to 5, u2 is 0 to 3, u3 is 1 to 4, u4 is 1 to 4, and optionally the lipid has at least two, three, or four tails of formula (TII), and the two, three, or four tails of formula (TII) are identical or different. The head base is defined by the following formula 【Chemistry 149】 (HC-IIID) or [Chemical 150] It has the structure (HC-IIIE), where t1 is 0, 1, 2, or 3. W is OH, 【Chemistry 151】 【Chemistry 152】 【Chemistry 153】 【Chemistry 154】 【Chemistry 155】 【Chemistry 156】 【Chemistry 157】 【Chemistry 158】 【Chemistry 159】 [Chemical 160] 【Chemistry 161】 【Chemistry 162】 【Chemistry 163】 【Chemistry 164】 【Chemistry 165】 【Chemistry 166】 【Chemistry 167】 [Chem 168] 【Chemistry 169】 【Chemistry 170】 【Chemistry 171】 【Chemistry 172】 【Chemistry 173】 【Chemistry 174】 【Chemistry 175】 【Chemistry 176】 【Chemistry 177】 【Chemistry 178】 【Chemistry 179】 【Chemistry 180】 【Chemistry 181】 【Chemistry 182】 【Chemistry 183】 【Chemistry 184】 【Chemistry 185】 【Chemistry 186】 【Chemistry 187】 [Chem. 188] [Chemical 189] 【Chemistry 190】 【Chemistry 191】 【Chemistry 192】 【Chemistry 193】 【Chemistry 194】 【Chemistry 195】 【Chemistry 196】 【Chemistry 197】 【Chemistry 198】 【Chemistry 199】 【Chemistry 200】 【Chemical Engineering 201】 【Chemical Engineering 202】 【Chemical 203】 【Chemical 204】 The lipid described in claim 19, which is one of the lipids described in claim 19.

21. The following formula, 【Chemical 205】 (TNG-II) or 【Chemical 206】 (TNG-III) one or more tail groups, In the formula, E is independently -OC(O)-, -C(O)O-, -N(R7)C(O)-, -S-S-, or -C(O)N(R7)-, u1 and u2 are independently 0, 1, 2, 3, 4, 5, 6, or 7. R7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, and u3 and u4 are independently 0, 1, 2, 3, 4, 5, 6, or 7, Rb is independently H or C1-C4 alkyl in each instance. The lipid according to claim 20, further comprising:

22. A lipid according to claim 1 or claim 18, having one of the following structures. Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 2-7 Table 2-8 Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 Table 2-23 Table 2-24 Table 2-25 Table 2-26 Table 2-27 Table 2-28 Table 2-29 Table 2-30 Table 2-31 Table 2-32 Table 2-33 Table 2-34 Table 2-35 Table 2-36 Table 2-37 Table 2-38 Table 2-39 Table 2-40 Table 2-41 Table 2-42 Table 2-43 Table 2-44 Table 2-45 Table 2-46 Table 2-47 Table 2-48 Table 2-49 Table 2-50 Table 2-51 Table 2-52 Table 2-53 Table 2-54 Table 2-55 Table 2-56

23. At least two lipophilic tail groups and a head group of formula (G-HC-IIID): 【Chemical 207】 There is, During the ceremony, R a However, each is independent of C 1 ~C 5 Alkyl, C 2 ~C 5 Alkenyl, or C 2 ~C 5 It is alkinyl, t2 is an integer between 0 and 5. W is a hydroxyl, substituted or unsubstituted hydroxyalkyl, or 【Chemical 208】 It was one of them, and during the ceremony, Q is either nonexistent, -O-, -C(O)-, -C(S)-, -C(O)O-, -C(R7)2-, -C(O)N(R7)-, -C(S)N(R7)-, or -N(R7), R 6 independently contains H, alkyl, hydroxyl, hydroxyalkyl, and alkoxy. -O-alkylene-O-alkyl, -O-alkylene-N(R7)2, amino, alkylamino, aminoalkyl, thiol, thiolalkyl, or N+(R7)3-alkylene-Q- Each R8 is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, heterocyclyl, heteroaryl, or two R8s together with a nitrogen atom form a ring optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, or aminoalkyl groups. q is 0, 1, 2, 3, 4, or 5, and p is 0, 1, 2, 3, 4, or 5, and 【Chemical Engineering 209】 This represents a bond that connects the head group to the tail group, and is a lipid.

24. A composition comprising a lipid according to any one of claims 1 to 21 and 23, and one or more lipid components different from the lipid, wherein the combination may optionally be an LNP composition.

25. The composition according to claim 24, wherein the lipid component comprises one or more of sterols, PEG lipids, phospholipids, and neutral lipids.

26. A pharmaceutical composition comprising the composition described in Claim 24, a therapeutic agent selected from the group consisting of nucleic acid molecules, proteins, and low molecular weight drugs, and a pharmaceutically acceptable excipient.

27. ​​The pharmaceutical composition according to claim 26, wherein the therapeutic agent is RNA or DNA, and optionally the therapeutic agent may be RNA containing mRNA.

Images