Glutamic acid-based lipids, lipid nanoparticles containing glutamate-based lipids, and their formulations.

Abstract

An ionizable lipid compound of formula (I) based on glutamic acid or glutaric acid, or a pharmaceutically acceptable salt thereof, is provided. The compound can be used to obtain lipid nanoparticles. In some embodiments, the lipid nanoparticles may comprise: (a) about 40 mol% to about 100 mol% of the compound of formula (I); (b) 0 to about 20 mol% of neutral lipids; (c) 0 to about 50 mol% of accessory lipids; (d) 0 to about 5 mol% of polymer-conjugated lipids; and (e) 0 to about 10 mol% of a hydrophobic component; wherein the mol% is based on the total lipids present in the nanoparticles. In some embodiments, the ionizable lipid compound is an ionizable lipid compound based on glutamic acid. (I)

Description

This disclosure of the field

[0001] This disclosure relates to glutamate or glutaric acid-based lipids and their use in systems for preparing encapsulated goods such as nucleic acid sequences, polypeptides, or peptides. More specifically, this disclosure relates to ionizable glutamate or glutaric acid-based lipid compounds that can be used to prepare lipid nanoparticles (LNPs). This disclosure also relates to LNPs comprising such glutamate or glutaric acid-based lipids.

[0002] This disclosure also relates to lipid nanoparticles (LNPs) for delivering goods such as nucleic acid sequences, polypeptides, or peptides, and methods for using these LNPs to treat diseases, conditions, and / or illnesses. background

[0003] In recent years, the development of systems for delivering therapeutic drugs (e.g., proteins, nucleic acid sequences, peptides, or polypeptides) to prevent or treat diseases (such as lipid nanoparticle delivery systems) has been booming. Lipid nanoparticles (LNPs) typically contain four components: ionizable lipids, phospholipids, cholesterol, and PEGylated lipids. The main component of LNPs is the ionizable lipid. Phospholipids support the formation of the lipid bilayer, while cholesterol stabilizes the lipid bilayer. Amphiphilic PEGylated lipids are retained on the surface of the LNP to provide colloidal stability through steric hindrance shielding. There is a need to design novel ionizable lipids with appropriate efficacy, stability, and / or biodegradability to allow for the preparation of LNPs.

[0004] Proteins have long been the benchmark for therapeutic drugs, but in recent years, the use of nucleic acids as a treatment approach for a variety of diseases and therapeutic indications has gained increasing attention. Several companies have demonstrated that nucleic acids (e.g., siRNA, mRNA, circular RNA, DNA, etc.) can be more effective than protein-based therapies. New delivery systems, such as novel LNPs, are needed for both nucleic acid and protein therapeutics. Overview

[0005] This disclosure provides novel lipid compounds, and more specifically, lipid compounds comprising a central glutamate or glutaric acid moiety, referred to herein as "glutamate- or glutaric acid-based lipids". In some embodiments, the lipid compound comprises a central glutamate moiety and is therefore referred to as "glutamate-based lipids". In some embodiments, the lipid compound comprises a central glutaric acid moiety and is therefore referred to as "glutaric acid-based lipids".

[0006] This disclosure also provides LNPs, and more specifically, LNPs formulated with lipid compounds based on glutamate or glutaric acid. It also provides particles (e.g., nanoparticles) comprising the compound; constructs comprising the nanoparticles and cargo, wherein the cargo may be a small molecule, antibody, polynucleotide, or peptide; methods for using the particles / constructs; and methods for preparing the compound, particles, and constructs.

[0007] Therefore, according to one aspect, compounds of formula (I) are provided: (I) Or its pharmaceutically acceptable salt. in A does not exist or is -CH2-; B is -CH2- or -CH2CH2-, where B is -CH2CH2- when A is not present, and B is -CH2- when A is -CH2-. X is either -NR1R2 or -O-(C=O)R 1’ ; R1 is H or an optional substituted straight-chain C1-C4 alkyl group; R2 is H, an optionally substituted straight-chain C1-C8 alkyl group, -C(=O)R7, -S(=O)2-R7, or... ; R 1’ It is a straight-chain C1-C8 alkyl group that can be optionally substituted; R 20 It is H or an optional substituted straight-chain C1-C8 alkyl group; R 21 It is an optional substituted straight-chain C1-C8 alkyl or -(C=O)-R 22 , where R 22 It is an optional substituted C1-C8 alkyl group; R3 and R4 are independently H, a straight-chain or branched C4-C60 alkyl, a straight-chain or branched C4-C60 alkenyl, or a straight-chain or branched C4-C60 alkynyl, wherein at least one of R3 or R4 is different from H; R5 and R6 are independently straight-chain or branched C4-C60 alkyl, straight-chain or branched C4-C60 alkenyl, or straight-chain or branched C4-C60 alkynyl. R7 is an optionally substituted straight-chain C1-C8 alkyl, optionally substituted straight-chain C2-C8 alkenyl, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group. When any one of R3, R4, R5, and R6 represents an alkenyl group, the alkenyl group independently contains 1 to 6 C=C bonds, each C=C bond independently having an E or Z configuration; When any one of R3, R4, R5, and R6 represents an alkynyl group, the alkynyl group independently contains 1 to 6 C≡C bonds; When R7 represents an alkenyl group, the alkenyl group contains one to two C=C bonds, each C=C bond having an E or Z configuration independently; When any alkyl and / or alkenyl group is substituted, the group is independently substituted by one or more of the following: -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 , -O-(C=O)-CH3, -O-(C=O)-(CH2) p CH3, -O-(C=O)-(CH2) p -R 19 ,-(C=O)-OCH3,-(C=O)-O-(CH2) p CH3、-(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -R 19 -(C=O)NH-(CH2) p -R 19 ,-(C=S)-CH3,-O-CH3,-O-(CH2) p CH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p -R 19 -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, optionally substituted 5- to 10-membered heterocyclic groups, C5-C12 aralkyl, C5-C10 aryl, or optionally substituted 5- to 10-membered heteroaryl groups, wherein p and p' are independently 1 to 5; and wherein R 19 It is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5 to 10 heterocyclic groups or optionally substituted 5 to 10 heteroaryl groups; When any 5- to 10-membered heteroaryl and / or 5- to 10-membered heterocyclic group is substituted, the group is independently selected from C1-C4 alkyl, -(CH2) ... p’’N(CH3)2 is substituted with one or more substituents of oxo (=O), wherein p'' is 1 to 5; The conditions are that A is absent, B is -CH2CH2-, X is -NR1R2, R1 is -CH3, and each of R3, R4, R5, and R6 has carbons at C9 and C6. 10 A C=C bond between carbon and carbon C 12 and C 13 When R2 is a straight-chain C18 alkyl group with one C=C bond, then R2 is different from -CH3, -CH2CH2OH and -CH2CH2NH2.

[0008] In one embodiment, the compound of formula (I) may have formula (I1). (I1) Or its pharmaceutically acceptable salt. in R1 is H or an optional substituted straight-chain C1-C4 alkyl group; R2 is H, an optionally substituted straight-chain C1-C8 alkyl group, -C(=O)R7, -S(=O)2-R7, or... ; R 20 It is H or an optional substituted straight-chain C1-C8 alkyl group; R 21 It is an optional substituted straight-chain C1-C8 alkyl or -(C=O)-R 22 , where R 22 It is an optional substituted C1-C8 alkyl group; R3 and R4 are independently H, a straight-chain or branched C4-C60 alkyl, a straight-chain or branched C4-C60 alkenyl, or a straight-chain or branched C4-C60 alkynyl, wherein at least one of R3 or R4 is different from H; R5 and R6 are independently straight-chain or branched C4-C60 alkyl, straight-chain or branched C4-C60 alkenyl, or straight-chain or branched C4-C60 alkynyl. R7 is an optionally substituted straight-chain C1-C8 alkyl, optionally substituted straight-chain C2-C8 alkenyl, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group. When any one of R3, R4, R5, and R6 represents an alkenyl group, the alkenyl group independently contains 1 to 6 C=C bonds, each C=C bond independently having an E or Z configuration; When any one of R3, R4, R5, and R6 represents an alkynyl group, the alkynyl group independently contains 1 to 6 C≡C bonds; When R7 represents an alkenyl group, the alkenyl group contains one to two C=C bonds, each C=C bond having an E or Z configuration independently; When any alkyl and / or alkenyl group is substituted, the group is independently substituted by one or more of the following: -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 , -O-(C=O)-CH3, -O-(C=O)-(CH2) p CH3, -O-(C=O)-(CH2) p -R 19 ,-(C=O)-OCH3,-(C=O)-O-(CH2) p CH3、-(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -R 19 -(C=O)NH-(CH2) p -R 19 ,-(C=S)-CH3,-O-CH3,-O-(CH2) p CH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p -R 19 -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, optionally substituted 5- to 10-membered heterocyclic groups, C5-C12 aralkyl, C5-C10 aryl, or optionally substituted 5- to 10-membered heteroaryl groups, wherein p and p' are independently 1 to 5; and wherein R 19 It is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5- to 10-membered heterocyclic groups or optionally substituted 5- to 10-membered heteroaryl groups. When any 5- to 10-membered heteroaryl and / or 5- to 10-membered heterocyclic group is substituted, the group is independently selected from C1-C4 alkyl, -(CH2) ... p’’ N(CH3)2 is substituted with one or more substituents of oxo (=O), where p'' is 1 to 5.

[0009] According to another aspect, a compound is provided, the compound being selected from compounds 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320a, 320b, 321a, 321b, 322, 323, 324, 3... in Table 1. 25, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 358, 359, 360 and 362 or their pharmaceutically acceptable salts. In one embodiment, the compound may be selected from compounds 301, 302, 303, 304, 305, 306, 307, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320a, 320b, 321a, 321b, 322, 323, 324, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 358, 359, 360, and 362 of Table 1, or pharmaceutically acceptable salts thereof. In one embodiment, the compound may be selected from compounds 301, 302, 303, 304, 305, 306, 307, 309, 311, 312, 313, 314, 315, 316, 318, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 347, 350, 351, 352, 353, 355, 358, 359, 360 and 362 of Table 1, or pharmaceutically acceptable salts thereof. In another embodiment, the compound may be selected from compounds 305, 313, 314, 315, 316, 317, 318, 320a, 320b, 321a, 321b, 323, 324, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 340, 350, 351 and 360 of Table 1, or pharmaceutically acceptable salts thereof. In yet another embodiment, the compound may be selected from compounds 301, 303, 304, 305, 306, 307, 311, 312, 313, 314, 315, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 347, 350, 351, 352, 353, 355, 358 and 359 of Table 1, or pharmaceutically acceptable salts thereof.In another embodiment, the compound may be selected from compounds 305, 306, 307, 313, 314, 315, 319, 320a, 320b, 321a, 321b, 325, 326, 350, 351, 352, 353, 355, 358 and 359 of Table 1 or pharmaceutically acceptable salts thereof.

[0010] In some embodiments, the compounds of this disclosure, namely the lipid compounds of this disclosure based on glutamic acid or glutaric acid or pharmaceutically acceptable salts thereof, may be in the form of any enantiomer, any diastereomer, any cis or trans geometric isomer or any mixture thereof.

[0011] According to another aspect, lipid nanoparticles are provided that comprise at least one compound of the present disclosure, namely, a lipid compound of the present disclosure based on glutamic acid or glutaric acid or a pharmaceutically acceptable salt thereof.

[0012] According to another aspect, lipid nanoparticles are provided, comprising: (a) Approximately 40 mol% to approximately 100 mol% of ionizable lipids; (b) 0 to approximately 20 mol% of neutral lipids; (c) 0 to 50 mol% of auxiliary lipids; (d) 0 to 5 mol% polymer-conjugated lipids; and (e) 0 to approximately 10 mol% of hydrophobic components; The mol% figure is based on the total lipids present in the nanoparticles; and The ionizable lipid is at least one compound of formula (I): (I) Or its pharmaceutically acceptable salt. in A does not exist or is -CH2-; B is -CH2- or -CH2CH2-; Where A does not exist, then B is -CH2CH2-, and when A is -CH2-, then B is -CH2-; X is either -NR1R2 or -O-(C=O)R 1’ ; R1 is H or an optional substituted straight-chain C1-C4 alkyl group; R2 is H, an optionally substituted straight-chain C1-C8 alkyl group, -C(=O)R7, -S(=O)2-R7, or... ; R 1’ It is a straight-chain C1-C8 alkyl group that can be optionally substituted; R20 It is H or an optional substituted straight-chain C1-C8 alkyl group; R 21 It is an optional substituted straight-chain C1-C8 alkyl or -(C=O)-R 22 , where R 22 It is an optional substituted C1-C8 alkyl group; R3 and R4 are independently H, a straight-chain or branched C4-C60 alkyl, a straight-chain or branched C4-C60 alkenyl, or a straight-chain or branched C4-C60 alkynyl, wherein at least one of R3 or R4 is different from H; R5 and R6 are independently straight-chain or branched C4-C60 alkyl, straight-chain or branched C4-C60 alkenyl, or straight-chain or branched C4-C60 alkynyl. R7 is an optionally substituted straight-chain C1-C8 alkyl, optionally substituted straight-chain C2-C8 alkenyl, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group. When any one of R3, R4, R5, and R6 represents an alkenyl group, the alkenyl group independently contains 1 to 6 C=C bonds, each C=C bond independently having an E or Z configuration; When any one of R3, R4, R5, and R6 represents an alkynyl group, the alkynyl group independently contains 1 to 6 C≡C bonds; When R7 represents an alkenyl group, the alkenyl group contains one to two C=C bonds, each C=C bond having an E or Z configuration independently; When any alkyl and / or alkenyl group is substituted, the group is independently substituted by one or more of the following: -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 , -O-(C=O)-CH3, -O-(C=O)-(CH2) p CH3, -O-(C=O)-(CH2) p -R 19 ,-(C=O)-OCH3,-(C=O)-O-(CH2) p CH3、-(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -R 19 -(C=O)NH-(CH2) p-R 19 ,-(C=S)-CH3,-O-CH3,-O-(CH2) p CH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p -R 19 -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, optionally substituted 5- to 10-membered heterocyclic groups, C5-C12 aralkyl, C5-C10 aryl, or optionally substituted 5- to 10-membered heteroaryl groups, wherein p and p' are independently 1 to 5; and wherein R 19 It is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5 to 10 heterocyclic groups or optionally substituted 5 to 10 heteroaryl groups; When any 5- to 10-membered heteroaryl and / or 5- to 10-membered heterocyclic group is substituted, the group is independently selected from C1-C4 alkyl, -(CH2) ... p’’ N(CH3)2 is substituted with one or more substituents of oxo (=O), wherein p'' is 1 to 5; The conditions are that A is absent, B is -CH2CH2-, X is -NR1R2, R1 is -CH3, and each of R3, R4, R5, and R6 has carbons at C9 and C6. 10 A C=C bond between carbon and carbon C 12 and C 13 When R2 is a straight-chain C18 alkyl group with one C=C bond, then R2 is different from -CH3, -CH2CH2OH and -CH2CH2NH2.

[0013] In some embodiments, the lipid nanoparticles may comprise at least one compound of formula (I), (I1), (I2), (I3) or (I4) as defined herein, or lipids having the structure of formula (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or lipids having the structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c) as ionizable lipids.

[0014] In some embodiments, the lipid nanoparticles may comprise: (a) about 40 mol% to about 85 mol% of ionizable lipids; (b) about 1 mol% to about 20 mol% of neutral lipids; (c) 0 to about 50 mol% of auxiliary lipids; (d) 0 to about 5 mol% of polymer-conjugated lipids; and (e) 0 to about 10 mol% of hydrophobic components.

[0015] In some embodiments, the lipid nanoparticles may comprise: (a) about 40 mol% to about 85 mol% of ionizable lipids; (b) about 1 mol% to about 20 mol% of neutral lipids; (c) about 1 mol% to about 50 mol% of auxiliary lipids; (d) 0 to about 5 mol% of polymer-conjugated lipids; and (e) 0 to about 10 mol% of hydrophobic components.

[0016] In some embodiments, the lipid nanoparticles may comprise: (a) about 40 mol% to about 85 mol% of ionizable lipids; (b) about 1 mol% to about 20 mol% of neutral lipids; (c) about 1 mol% to about 50 mol% of accessory lipids; (d) about 1 mol% to about 5 mol% of polymer-conjugated lipids; and (e) 0 to about 10 mol% of hydrophobic components.

[0017] In some embodiments, the lipid nanoparticles may comprise: (a) about 40 mol% to about 85 mol% of ionizable lipids; (b) about 1 mol% to about 20 mol% of neutral lipids; (c) about 1 mol% to about 50 mol% of accessory lipids; (d) about 1 mol% to about 5 mol% of polymer-conjugated lipids; and (e) about 0.1 mol% to about 10 mol% of hydrophobic components.

[0018] In some embodiments, the lipid nanoparticles may comprise: (a) about 40 mol% to about 85 mol% of ionizable lipids; (b) about 5 mol% to about 20 mol% of neutral lipids; (c) about 1 mol% to about 50 mol% of accessory lipids; (d) about 1 mol% to about 4 mol% of polymer-conjugated lipids; and (e) about 0.1 to about 10 mol% of hydrophobic components.

[0019] In some embodiments, the lipid nanoparticles may comprise: (a) about 40 mol% to about 85 mol% of ionizable lipids; (b) about 5 mol% to about 10 mol% of neutral lipids; (c) about 1 mol% to about 40 mol% of accessory lipids; (d) about 1 mol% to about 4 mol% of polymer-conjugated lipids; and (e) about 0 to about 5 mol% of hydrophobic components.

[0020] In some embodiments, the lipid nanoparticles may comprise: (a) about 40 mol% to about 85 mol% of ionizable lipids; (b) about 5 mol% to about 10 mol% of neutral lipids; (c) about 1 mol% to about 40 mol% of accessory lipids; (d) about 1 mol% to about 4 mol% of polymer-conjugated lipids; and (e) about 0.1 to about 5 mol% of hydrophobic components.

[0021] According to another aspect, a pharmaceutical composition is provided comprising lipid nanoparticles as defined herein and pharmaceutically acceptable excipients.

[0022] According to another aspect, a method for delivering cargo to cells is provided, which includes contacting the cells with lipid nanoparticles as defined herein, wherein the lipid nanoparticles contain cargo.

[0023] According to another aspect, the use of lipid nanoparticles as defined herein for delivering cargo to cells is provided, wherein the lipid nanoparticles contain cargo.

[0024] According to another aspect, a vaccine comprising lipid nanoparticles as defined herein is provided, wherein the lipid nanoparticles comprise at least one cargo, preferably comprising at least one of a small molecule, an antibody, a polynucleotide, or a polypeptide, and more preferably, comprising at least one nucleic acid such as mRNA.

[0025] According to another approach, a method for vaccinating subjects against an infectious agent is provided, which includes: (i) Exposing subjects to a vaccine as defined herein, and (ii) Triggering an immune response.

[0026] According to another aspect, the use of vaccines, as defined herein, for vaccinating subjects against infectious agents is provided.

[0027] According to another approach, a method for treating a subject's cancer is provided, which includes administering lipid nanoparticles, as defined herein, to the subject, wherein the lipid nanoparticles contain anticancer cargoes or cargoes that trigger an immune response against cancer cells.

[0028] According to another aspect, lipid nanoparticles as defined herein are provided for treating cancer in a subject, wherein the lipid nanoparticles contain anticancer cargoes or cargoes that trigger an immune response against cancer cells.

[0029] According to another aspect, the use of lipid nanoparticles as defined herein for treating cancer in subjects is provided, wherein the lipid nanoparticles contain anticancer cargoes or cargoes that trigger an immune response against cancer cells.

[0030] According to another aspect, the use of lipid nanoparticles as defined herein for the preparation of medicaments for treating cancer in subjects is provided, wherein the lipid nanoparticles contain anticancer cargoes or cargoes that trigger an immune response against cancer cells.

[0031] Details of the various embodiments are set forth in the description below. Other features, objects, and advantages will be apparent from the specification and claims. Brief description of the attached diagram

[0032] Figure 1 A frozen TEM image of LNP-04 (containing spike mRNA) formulated with ionizable lipid 305 is shown.

[0033] Figure 2 A frozen TEM image of LNP-04 (containing spike mRNA) formulated with ionizable lipid 305 is shown.

[0034] Figure 3 A frozen TEM image of LNP-05 (containing spike mRNA) formulated with ionizable lipid 306 is shown.

[0035] Figure 4 A frozen TEM image of LNP-05 (containing spike mRNA) formulated with ionizable lipid 306 is shown.

[0036] Figure 5 Cryo-TEM images of LNP-19 (containing spike mRNA) prepared with ionizable lipid 305, obtained by point-of-care mixing, are shown.

[0037] Figure 6 Frozen TEM images of LNP-19 (containing spike mRNA) prepared with ionizable lipid 305 by instantaneous mixing are shown.

[0038] Figure 7 Frozen TEM images of LNP-20 (containing spike mRNA) formulated with ionizable lipid 305 and squalene, obtained by instantaneous mixing, are shown.

[0039] Figure 8Frozen TEM images of LNP-20 (containing spike mRNA) formulated with ionizable lipid 305 and squalene, obtained by instantaneous mixing, are shown.

[0040] Figure 9 This is a schematic diagram illustrating the on-the-fly preparation of lipid nanoparticles. The lipid solution in a pre-filled syringe (A) is injected (C) into a vial (B) containing an mRNA solution, followed by vigorous shaking (D) to deliver the mRNA (E) encapsulated in LNPs, which is then ready for drug delivery.

[0041] Figure 10 The transfection efficiency of LNP-04 and PTX-COVID-19-B in Huh-7 cells was compared using ELISA. The PTX-COVID-19-B vaccine is an mRNA-LNP vaccine containing mRNA encoding the full-length spike protein of the Covid-19-B virus. In experiments using the spike mRNA-LNP disclosed herein, PTX-COVID-19-B can be used as a positive control. The configuration of PTX-COVID-19-B is described, for example, in WO 2023 / 050003 (see “PTX-B”).

[0042] Figure 11 The transfection efficiency of LNP-05 and PTX-COVID-19-B in Huh-7 cells was compared using ELISA.

[0043] Figure 12 The transfection efficiency of LNP-04, LNP-06, and PTX-COVID-19-B in Huh-7 cells was compared using ELISA.

[0044] Figure 13 The transfection efficiency of LNP-04, LNP-19, LNP-20 and PTX-COVID-19-B in Huh-7 cells was compared using ELISA.

[0045] Figure 14A The figure shows the spike protein-specific IgG response of LNP-04 and LNP-05 measured 14 days after the second intramuscular immunization in mice.

[0046] Figure 14B The figure shows the neutralizing antibody (NAb) response against Wuhan-Hu-1 / D614G of LNP-04 and LNP-05, measured 14 days after the second intramuscular immunization in mice.

[0047] Figure 15 The figure shows the neutralizing antibody (NAb) response against Wuhan-Hu-1 / D614G with different mRNA doses of LNP-04 and LNP-05.

[0048] Figure 16 The image shows antigen-specific IFN levels in spleen cells measured 14 days after a second immunization with LNP-04 and LNP-05 in mice. The level of secretory cells.

[0049] Figure 17 The levels of antigen-specific IL-4 secreting cells in spleen cells were shown 14 days after a second immunization with LNP-04 and LNP-05 in mice.

[0050] Figure 18 The study demonstrated the control of MC38gp tumor growth in mice after immunization with LNP-04-05.

[0051] Figure 19 The conventionally prepared LNP-21 and instant LNP-22 were shown compared with the commercial transfection agent MessengerMAX. TM Comparison of transfection efficiencies. (a) Flow cytometry data; (b) Percentage of transfected cells calculated from flow cytometry data.

[0052] Figures 20A-20B The comparison of LNP-35 and LNP-38 by ELISA is shown. Figure 20A ), LNP-39, LNP-40, LNP-41, LNP-43, LNP-44, LNP-45 and LNP-46( Figure 20B The transfection efficiency of PTX-COVID-19-B in Huh-7 cells was also measured.

[0053] Figures 21A-21B The comparison of LNP-24, LNP-26, LNP-42, and LNP-49 by ELISA is shown. Figure 21A ), LNP-25, LNP-27, LNP-29, LNP-41, LNP-43 and LNP-47( Figure 21B The transfection efficiency of PTX-COVID-19-B in Huh-7 cells was also measured.

[0054] Figure 22 The transfection efficiency of LNP-28, LNP-44, and PTX-COVID-19-B in Huh-7 cells was compared using ELISA.

[0055] Figures 23A-23B The comparison of LNP-31 and LNP-54 by ELISA is shown. Figure 23A LNP-30 and LNP-48 Figure 23BThe transfection efficiency of PTX-COVID-19-B in Huh-7 cells was also measured.

[0056] Figures 24A-24D The transfection efficiency of LNP-21 in different PBMC cell subsets was shown. Figure 24A The transfection efficiency of LNP-21 in PBMCs, as assessed by flow cytometry and gated to different cell subpopulations, and measured by tdTomato expression, is shown. Figure 24B The transfection efficiency of LNP-21 in purified T cells (naïve T cells, or T cells activated with anti-CD3 / anti-CD28 antibody or PMA / ionomycin 24 hours after activating) from donor PBMCs, as measured by tdTomato expression, is shown compared to MessengerMAX™. Figure 24C A schematic diagram showing the transfection efficiency assay in immature / mature monocyte-derived dendritic cells (MDDC) and unpolarized / polarized monocyte-derived macrophages (MDM). Figure 24D The results show the difference between MessengerMax and the results measured by tdTomato expression. Figure 24A The results of LNP-21 transfection efficiency and transfected cell viability in the experiment.

[0057] Figures 25A-25C The effect of LNP on immune activation was demonstrated. Figures 25A-25B This image shows PBMCs from two healthy human donors (donor 1 and donor 2) transfected with 100 ng / well of tdTomato mRNA encapsulated in LNP-21 or MessengerMax (Thermofisher). Supernatant from transfected cells was collected 24 hours post-transfection and processed using LEGENDplex. TM Quantitative determination (Biolegend). Figure 25C The images show LNP-35 and LNP-23 cells containing encapsulated spike mRNA added to the TLR-4 reporter cell line. Luciferase was measured 16 hours later to indicate TLR-4 activation. Cells treated with MPLA (Monophosphoryllipid A) or formulation buffer served as positive and negative controls.

[0058] Figures 26A-26C The in vivo immunogenicity of LNP-35 and LNP-23 was demonstrated. Female C57BL / 6 mice were inoculated twice at weeks 0 and 2 with 5 µg of COVID-19 spike mRNA encapsulated in either LNP-35 or LNP-23. Serum from inoculated mice was collected 10 days after the second inoculation to detect neutralizing antibodies (nAbs) against the Wuhan (D614G) pseudovirus. Figure 26ASpleen cells from mice were used to detect the production of cytokines (IFN-γ and IL-4) via ELISPOT. Figures 26B-26C The numbers at the top of the column represent the geometric mean titer of nAb, or the average number of spot-forming units (SFU) per million input cells.

[0059] Figures 27A-27H The results show the effects of immunization with LNP-04-05 and LNP-06-02 using gp33 or adenoviral peptide (AV) pulses (e.g., Sebzda, E. et al.). J Exp Med (as shown in (1996) 183 (3): 1093-1104), results of in vivo induction of cytolytic T cells and immunogenicity. Figure 27A This diagram illustrates an in vivo assay of cytolytic lymphocytes performed on day 8 post-immunization. Splenocyte target cells were isolated from unimmunized mice, stained with Cell Trace Violet (CTV), and then pulsed with gp33 or AV peptide for 1 hour. Approximately 3 x 10-1 6 Target cells were re-injected intravenously (iv) into inoculated mice, and spleen cells were collected 4 hours post-injection (pi) to measure specific lysis relative to formulation buffer normalization. Figure 27B Showing Figure 27A The experimental results. Figure 27C-27D The percentage of peripheral gp33+ and gp34+ CD8+ T cells in blood samples collected via the saphenous vein on day 7 is shown. RBCs were lysed with ACK lysis buffer and stained with PE-labeled tetramers targeting gp33 or gp34. Figure 27E-27H Serum cytokine / chemokine levels in blood samples collected 6 hours after LNP immunization, as measured by Legendplex, are shown.

[0060] Figure 28A-28K The results show the results of a study evaluating the protective efficacy of the mRNA-LNP vaccine (LNP-58) using a transplantable mouse GBM model expressing EGFRvIII via a tumor re-attack setting. Figure 28A A schematic diagram of the immunization and evaluation timeline is shown. In short, on day 1 of the experiment, genetically and sex-matched transplantable GBM cells (1x10⁻¹²) were... 5The tumor was transplanted into the right striatum of 6-8 week old female recipient mice. Subsequently, the mice were either inoculated with EGFRvIII LNPs as shown or left untreated on days 10, 14, 18, and 25. On day 43, all inoculated (surviving) animals were rechallenged by injecting another tumor dose into the contralateral side of the brain. To ensure the validity of the results, a new untreated control group was also included, providing robust comparisons. Figure 28B The survival rate of mice treated with mRNA encoding EGFRvIII encapsulated in LNPs was shown compared with the untreated control and the luciferase control. Figure 28C The results of mouse weight measurements throughout the study are shown to monitor potential side effects of LNP. Figure 28D The results show the quantitative results of total anti-EGFRvIII IgG in mouse plasma throughout the study (FB = mice injected with formulation buffer; tumor control = transplanted tumors treated with FB). Figure 28E Results are shown for detecting spleen cytotoxic T lymphocytes (CTLs) that specifically recognize EGFRvIII mutations using EGFRvIII dextramer reagent conjugated with PE chromophore. Figure 28F The results show the assay of spleen cytotoxic T lymphocytes (CTLs), which respond to stimulation with EGFRvIII peptides by expressing the activation-inducing marker (AIM) CD69 (determined by flow cytometry). Spleen-derived cells were incubated overnight with EGFRvIII-derived peptides to detect the responsiveness of CTLs to these peptides. Figure 28G-28I The results show the changes in the expression of CTL cell surface markers assessed by flow cytometry on day 21 of the study. Figure 28J The results show the changes in γδ T cell frequency assessed by flow cytometry on day 21 by staining TCRγδ cell surface markers. Figure 28K The results show the changes in the expression of T helper (Th) cell surface markers as assessed by flow cytometry on day 21 of the study. Detailed Implementation

[0061] I. Introduction The following description illustrates exemplary compounds, compositions, methods, parameters, etc. However, it should be understood that the description is not intended to limit the scope of this disclosure, but is provided as a description of exemplary embodiments.

[0062] Delivering therapeutic agents to subjects is crucial for their therapeutic efficacy, and this is often hampered by the limited ability of compounds to reach target cells and tissues. Improving the delivery of such therapeutic agents to target cells in tissues via multiple delivery pathways is essential. Compared to traditional therapies, nucleic acid therapy has become a mainstream approach for treating a wide range of diseases and indications due to its versatility, lower immune response, and higher potency. For example, nucleic acid therapy includes using small interfering RNAs (siRNAs) to reduce the translation of messenger RNA (mRNAs), using mRNA as a means of generating targets of interest, using circular RNAs (oRNAs) that can provide continuous production of peptides or can act as a sponge competing with other RNA molecules, and using viral vectors to provide continuous production of targets of interest. However, some nucleic acids are unstable and easily degraded, therefore they need to be formulated to prevent degradation and facilitate intracellular delivery of nucleic acids.

[0063] This disclosure relates to novel lipid compounds based on glutamic acid or glutaric acid and compositions comprising said lipid compounds, and more specifically, to nanoparticles based on these lipid compounds based on glutamic acid or glutaric acid, which are capable of encapsulating goods such as bioactive agents and therapeutic agents.

[0064] This disclosure also relates to novel lipid nanoparticle compositions that may have improved stability and / or increased efficacy, such as increased immunogenicity (when used in vaccines) and / or facilitate intracellular delivery of bioactive agents and therapeutic agents. In some embodiments, the lipid nanoparticle compositions may have low or reduced toxicity. This disclosure also relates to pharmaceutical compositions comprising such lipid compositions and capable of delivering therapeutically effective amounts of bioactive agents into a patient's cells.

[0065] Examples of bioactive agents include, but are not limited to: (1) proteins, including immunoglobulins; (2) polynucleotides, such as genomic DNA, cDNA or mRNA; (3) antisense polynucleotides; and (4) low molecular weight compounds, whether synthetic or naturally occurring, such as peptide hormones and antibiotics.

[0066] "Lipids" are organic compounds containing fatty acid esters and characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally classified into at least three categories: (1) "simple lipids", which include fats and oils as well as waxes; (2) "complex lipids", which include phospholipids and glycolipids; and (3) "derived lipids", such as steroids.

[0067] "Lipid particles" or "lipid nanoparticles" (i.e., "LNPs") refer to lipid formulations that can be used to deliver goods, such as therapeutic nucleic acids (e.g., mRNA), to target sites of interest (e.g., cells, tissues, organs, etc.). In a preferred embodiment, lipid particles can be used to encapsulate nucleic acids. In a preferred embodiment, lipid nanoparticles can be formed from ionizable lipids, neutral lipids (e.g., phospholipids), polymer-conjugated lipids that can prevent nanoparticle aggregation (e.g., PEG-lipids), and optionally auxiliary lipids (e.g., cholesterol). In some embodiments, therapeutic nucleic acids (e.g., mRNA) can be encapsulated within the lipid portion of the nanoparticles, thereby protecting them from enzymatic degradation. In another preferred embodiment, the lipid nanoparticles may contain another component, such as a hydrophobic component, to improve LNP internalization, immune activation, and / or antibody production.

[0068] Lipid nanoparticles typically contain cholesterol (which contributes to stability and promotes membrane fusion), phospholipids (which provide structure for the LNP bilayer and also facilitate endosome escape), polyethylene glycol (PEG) derivatives (which reduce LNP aggregation and “shield” LNPs from nonspecific endocytosis by immune cells), and ionizable lipids (which complex negatively charged RNA and enhance endosome escape), which together form an LNP-forming composition.

[0069] Lipid nanoparticles can typically have particle sizes (e.g., expressed as average diameter) in the following ranges: 30 nm to 200 nm, 40 nm to 180 nm, 50 nm to 150 nm, 60 nm to 130 nm, 60 nm to 120 nm, 60 nm to 110 nm, 60 nm to 100 nm, 60 nm to 90 nm, 70 nm to 110 nm, 70 nm to 100 nm, 80 nm to 100 nm, 90 nm to 100 nm, 70 nm to 90 nm, 80 nm to 90 nm, or 70 nm to 80 nm. In some embodiments, the particle size can be 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, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm, and is substantially non-toxic. Furthermore, when present in the lipid nanoparticles of this disclosure, the nucleic acids resist degradation by nucleases in aqueous solutions.

[0070] II. Lipids Ionizable lipids This disclosure relates to compounds that are ionizable lipids, and more specifically, to ionizable lipids based on glutamate or glutaric acid. Ionizable lipids may be cationic lipids.

[0071] In some embodiments, the compounds disclosed herein are based on a glutamic acid or glutaric acid core, such as an α-glutamic acid core, a β-glutamic acid core, an α-ketoglutaric acid core, or a β-ketoglutaric acid core: , , , .

[0072] In some embodiments, the compounds disclosed herein are based on a glutamate core, such as an α-glutamate core or a β-glutamate core.

[0073] In some embodiments, the compounds of this disclosure comprise a glutamic acid core. Such a glutamic acid core comprises two ester moieties and a primary, secondary, or tertiary amino group (e.g., -O-CO-CH2CH2CH(NR1R2)-CO-O- or -O-CO-CH2CH(NR1R2)CH2-CO-O-). In some embodiments, the compounds of this disclosure comprise a glutaric acid core. Such a glutaric acid core comprises three ester moieties (e.g., -O-CO-CH2CH2CH(OCOR1)-CO-O- or -O-CO-CH2CH(OCOR1)CH2-CO-O-). The ester bonds exhibit biodegradable properties.

[0074] In some embodiments, the ionizable lipid compound of this disclosure may be a compound of formula (I): (I) Or its pharmaceutically acceptable salt. in A does not exist or is -CH2-; B is -CH2- or -CH2CH2-; Where A does not exist, then B is -CH2CH2-, and when A is -CH2-, then B is -CH2-; X is either -NR1R2 or -O-(C=O)R 1’ ; R1 is H or an optional substituted straight-chain C1-C4 alkyl group; R2 is H, an optionally substituted straight-chain C1-C8 alkyl group, -C(=O)R7, -S(=O)2-R7, or... ; R 1’It is a straight-chain C1-C8 alkyl group that can be optionally substituted; R 20 It is H or an optional substituted straight-chain C1-C8 alkyl group; R 21 It is an optional substituted straight-chain C1-C8 alkyl or -(C=O)-R 22 , where R 22 It is an optional substituted C1-C8 alkyl group; R3 and R4 are independently H, a straight-chain or branched C4-C60 alkyl, a straight-chain or branched C4-C60 alkenyl, or a straight-chain or branched C4-C60 alkynyl, wherein at least one of R3 or R4 is different from H; R5 and R6 are independently straight-chain or branched C4-C60 alkyl, straight-chain or branched C4-C60 alkenyl, or straight-chain or branched C4-C60 alkynyl. R7 is an optionally substituted straight-chain C1-C8 alkyl, optionally substituted straight-chain C2-C8 alkenyl, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group. When any one of R3, R4, R5, and R6 represents an alkenyl group, the alkenyl group independently contains 1 to 6 C=C bonds, each C=C bond independently having an E or Z configuration; When any one of R3, R4, R5, and R6 represents an alkynyl group, the alkynyl group independently contains 1 to 6 C≡C bonds; When R7 represents an alkenyl group, the alkenyl group contains one to two C=C bonds, each C=C bond having an E or Z configuration independently; When any alkyl and / or alkenyl group is substituted, the group is independently substituted by one or more of the following: -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 , -O-(C=O)-CH3, -O-(C=O)-(CH2) p CH3, -O-(C=O)-(CH2) p -R 19 ,-(C=O)-OCH3,-(C=O)-O-(CH2) p CH3、-(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -R19 -(C=O)NH-(CH2) p -R 19 ,-(C=S)-CH3,-O-CH3,-O-(CH2) p CH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p -R 19 -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, optionally substituted 5- to 10-membered heterocyclic groups, C5-C12 aralkyl, C5-C10 aryl, or optionally substituted 5- to 10-membered heteroaryl groups, wherein p and p' are independently 1 to 5; and wherein R 19 It is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5 to 10 heterocyclic groups or optionally substituted 5 to 10 heteroaryl groups; When any 5- to 10-membered heteroaryl and / or 5- to 10-membered heterocyclic group is substituted, the group is independently selected from C1-C4 alkyl, -(CH2) ... p’’ N(CH3)2 is substituted with one or more substituents of oxo (=O), wherein p'' is 1 to 5; The conditions are that A is absent, B is -CH2CH2-, X is -NR1R2, R1 is -CH3, and each of R3, R4, R5, and R6 has carbons at C9 and C6. 10 A C=C bond between carbon and carbon C 12 and C 13 When R2 is a straight-chain C18 alkyl group with one C=C bond, then R2 is different from -CH3, -CH2CH2OH and -CH2CH2NH2.

[0075] In some embodiments, when any group is described as "substituted by one or more substituents," the group may be substituted by, for example, 1 to 6 substituents, preferably 1 to 4 substituents, and more preferably 1 to 3 substituents. In some embodiments, when any group is described as being substituted by one or more substituents, the group may be substituted by 1 or 2 substituents. In some embodiments, when any group is described as being substituted by one or more substituents, the group may be substituted by 1 substituent. In some embodiments, when any group is described as being substituted by one or more substituents, the group may be substituted by 2 substituents. In some embodiments, when any group is described as being substituted by one or more substituents, the group may be substituted by 3 substituents.

[0076] In some embodiments, the ionizable lipid compound of this disclosure may be a compound of formula (I1): (I1) or its pharmaceutically acceptable salt. in R1 is H or an optional substituted straight-chain C1-C4 alkyl group; R2 is H, an optionally substituted straight-chain C1-C8 alkyl group, or -C(=O)R7, -S(=O)2-R7, or... ; R 20 It is H or an optional substituted straight-chain C1-C8 alkyl group; R 21 It is an optional substituted straight-chain C1-C8 alkyl or -(C=O)-R 22 , where R 22 It is an optional substituted C1-C8 alkyl group; R3 and R4 are independently H, a straight-chain or branched C4-C60 alkyl, a straight-chain or branched C4-C60 alkenyl, or a straight-chain or branched C4-C60 alkynyl; wherein at least one of R3 or R4 is different from H; R5 and R6 are independently straight-chain or branched C4-C60 alkyl, straight-chain or branched C4-C60 alkenyl, or straight-chain or branched C4-C60 alkynyl. R7 is an optionally substituted straight-chain C1-C8 alkyl, optionally substituted straight-chain C2-C8 alkenyl, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group. When any one of R3, R4, R5, and R6 represents an alkenyl group, the alkenyl group independently contains 1 to 6 C=C bonds, each C=C bond independently having an E or Z configuration; When any one of R3, R4, R5, and R6 represents an alkynyl group, the alkynyl group independently contains 1 to 6 C≡C bonds; When R7 represents an alkenyl group, the alkenyl group contains one to two C=C bonds, each C=C bond having an E or Z configuration independently; When any alkyl and / or alkenyl group is substituted, the group is independently substituted by one or more of the following: -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 , -O-(C=O)-CH3, -O-(C=O)-(CH2) p CH3, -O-(C=O)-(CH2) p -R 19,-(C=O)-OCH3,-(C=O)-O-(CH2) p CH3、-(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -R 19 -(C=O)NH-(CH2) p -R 19 ,-(C=S)-CH3,-O-CH3,-O-(CH2) p CH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p -R 19 -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, optionally substituted 5- to 10-membered heterocyclic groups, C5-C12 aralkyl, C5-C10 aryl, or optionally substituted 5- to 10-membered heteroaryl groups, wherein p and p' are independently 1 to 5; and wherein R 19 It is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5 to 10 heterocyclic groups or optionally substituted 5 to 10 heteroaryl groups; When any 5- to 10-membered heteroaryl and / or 5- to 10-membered heterocyclic group is substituted, the group is independently selected from C1-C4 alkyl, -(CH2) ... p’’ N(CH3)2 is substituted with one or more substituents of oxo (=O), where p'' is 1 to 5.

[0077] In some embodiments, in compounds of formula (I) or (I1) or pharmaceutically acceptable salts thereof, R1 is H or an optionally substituted straight-chain C1-C4 alkyl group. In some embodiments, R1 is H or an optionally substituted straight-chain C1-C3 alkyl group. In some embodiments, R1 is H or an optionally substituted straight-chain C1-C2 alkyl group. In some embodiments, in compounds of formula (I) or (I1) or pharmaceutically acceptable salts thereof, R1 is H or CH3. In some embodiments, R1 is H.

[0078] In some embodiments, R is in a compound of formula (I) or a pharmaceutically acceptable salt thereof. 1’ It is an optional substituted straight-chain C1-C6 alkyl group. In some embodiments, R 1’ It is an optionally substituted straight-chain C1-C4 alkyl group. In some embodiments, R 1’ It is an optional substituted straight-chain C1-C3 alkyl group. In some embodiments, R 1’ It is an optional substituted straight-chain C2-C4 alkyl group. In some embodiments, R 1’ It is a straight-chain C3-C4 alkyl group that can be optionally substituted.

[0079] In some embodiments, R is in a compound of formula (I) or a pharmaceutically acceptable salt thereof. 1’ It can be substituted with any substituent that can be present on an alkyl group as defined herein. In some embodiments, R 1’ It can be replaced by -NH2, -NHCH3, -N(CH3)2, or -N(CH2CH3)2. In some embodiments, R 1’ It can be replaced by -NHCH3, -N(CH3)2, or -N(CH2CH3)2. In some embodiments, R 1’ It can be replaced by -N(CH3)2.

[0080] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R2 is H or optionally substituted straight-chain C1-C4 alkyl, -C(=O)R7, -S(=O)2-R7, or... .

[0081] In some embodiments, R2 is H, optionally substituted straight-chain C1-C2 alkyl, -C(=O)R7, -S(=O)2-R7, or... .

[0082] In some embodiments, R2 is H, optionally substituted straight-chain C3-C4 alkyl, -C(=O)R7, -S(=O)2-R7, or... .

[0083] In some embodiments, R2 is H, optionally substituted straight-chain C1 alkyl, -C(=O)R7, -S(=O)2-R7, or... .

[0084] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R1 is H and R2 is H.

[0085] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R1 is H and R2 is optionally a substituted straight-chain C1-C8 alkyl, -C(=O)R7, -S(=O)2-R7, or... .

[0086] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R1 is H and R2 is optionally a substituted straight-chain C1-C4 alkyl group, -C(=O)R7, -S(=O)2-R7, or... .

[0087] In some embodiments, in the compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, R1 is an optionally substituted straight-chain C1-C4 alkyl and R2 is an optionally substituted straight-chain C1-C4 alkyl or -C(=O)R7.

[0088] In some embodiments, in the compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, R7 is optionally a substituted straight-chain C1-C8 alkyl, optionally a substituted straight-chain C2-C8 alkenyl, optionally a substituted 5- to 10-membered heterocyclic group or optionally a substituted 5- to 10-membered heteroaryl group.

[0089] In some embodiments, in the compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, R7 is optionally a substituted straight-chain C1-C8 alkyl, optionally a substituted straight-chain C2-C8 alkenyl, optionally a substituted 5- or 6-membered heterocyclic group, or optionally a substituted 5- or 6-membered heteroaryl group.

[0090] In some embodiments, in the compound of formula (I) or (I1) or its pharmaceutically acceptable salt, R7 is optionally a substituted straight-chain C1-C4 alkyl, optionally a substituted straight-chain C2-C4 alkenyl, optionally a substituted 5- to 10-membered heterocyclic group or optionally a substituted 5- to 10-membered heteroaryl group.

[0091] In some embodiments, in the compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, R7 is optionally a substituted straight-chain C1-C4 alkyl, optionally a substituted straight-chain C2-C4 alkenyl, optionally a substituted 5- or 6-membered heterocyclic group, or optionally a substituted 5- or 6-membered heteroaryl group.

[0092] In some implementations, when R7 represents an alkenyl group, the alkenyl group may contain one or two C=C bonds, each C=C bond having an E or Z configuration independently.

[0093] In some embodiments, R is present in a compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof. 20 It is H or an optional substituted straight-chain C1-C8 alkyl group.

[0094] In some embodiments, R is present in a compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof. 21 It is an optional substituted straight-chain C1-C8 alkyl or -(C=O)-R 22 , where R 22 It is an optional substituted C1-C8 alkyl group.

[0095] In some embodiments, R is present in a compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof. 20 It is H or an optional substituted straight-chain C1-C4 alkyl group.

[0096] In some embodiments, R is present in a compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof. 21 It is an optional substituted straight-chain C1-C4 alkyl group or -(C=O)-R 22 , where R 22 It is an optional substituted C1-C4 alkyl group.

[0097] In some embodiments, in a compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, when any alkyl and / or alkenyl groups (e.g., R1, R2) are present... 1’ R2, R7, R 20 R 21 and / or R 22 When the alkyl and / or alkenyl groups in the form are substituted, the group can be independently substituted by one or more of the following: -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 , -O-(C=O)-CH3, -O-(C=O)-(CH2) p CH3, -O-(C=O)-(CH2) p -R 19 ,-(C=O)-OCH3,-(C=O)-O-(CH2) p CH3、-(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -R 19 -(C=O)NH-(CH2) p -R 19,-(C=S)-CH3,-O-CH3,-O-(CH2) p CH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p -R 19 -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, optionally substituted 5- to 10-membered heterocyclic groups, C5-C12 aralkyl, C5-C10 aryl, or optionally substituted 5- to 10-membered heteroaryl groups, wherein p and p' are independently 1 to 5; and wherein R 19 Independently, it is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5- to 10-membered heterocyclic group, or optionally substituted 5- to 10-membered heteroaryl group. In some embodiments, p and p' are independently 1 to 5, 1 to 4, 1 to 3, 1 to 2, or both are 1.

[0098] In some embodiments, in a compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, when any alkyl and / or alkenyl groups (e.g., R1, R2) are present... 1’ R2, R7, R 20 R 21 and / or R 22 When the alkyl and / or alkenyl groups in the form are substituted, the group can be independently substituted by one or more of the following: -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 , -O-(C=O)-CH3, -O-(C=O)-(CH2) p CH3, -O-(C=O)-(CH2) p -R 19 ,-(C=O)-OCH3,-(C=O)-O-(CH2) p CH3、-(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -R 19 -(C=O)NH-(CH2) p -R 19-O-CH3, -O-(CH2) p CH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p -R 19 -(C=O)NH2, optionally substituted 5- to 10-membered heterocyclic groups, C5-C12 aryl, C5-C10 aryl or optionally substituted 5- to 10-membered heteroaryl groups, wherein p and p' are independently 1 to 5; and wherein R 19 Independently, it is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5- to 10-membered heterocyclic group, or optionally substituted 5- to 10-membered heteroaryl group. In some embodiments, p and p' are independently 1 or 3. In some embodiments, p and p' are independently 1 or 2.

[0099] In some embodiments, in a compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, when any alkyl and / or alkenyl groups (e.g., R1, R2) are present... 1’ R2, R7, R 20 R 21 and / or R 22 When the alkyl and / or alkenyl groups in the form are substituted, the group can be independently substituted by one or more of the following: -OH, -(C=O)-(CH2). p -R 19 -O-(C=O)-(CH2) p -R 19 -(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -N(CH3)2、-(C=O)NH-(CH2) p -R 19 , -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p N(CH3)2, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group, wherein p and p' are independently 1 to 5; and wherein R 19Independently, it is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5- to 6-membered heterocyclic groups, or optionally substituted 5- to 10-membered heteroaryl groups. In some embodiments, p and p' are independently 1 to 3. In some embodiments, p and p' are independently 1 or 2.

[0100] In some embodiments, in a compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, when any alkyl and / or alkenyl groups (e.g., R1, R2) are present... 1’ R2, R7, R 20 R 21 and / or R 22 When the alkyl and / or alkenyl groups in the form are substituted, the group can be independently substituted by one or more of the following: -OH, -(C=O)-(CH2). p -R 19 -O-(C=O)-(CH2) p -R 19 -(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -N(CH3)2、-(C=O)NH-(CH2) p -R 19 , -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p N(CH3)2, a 5- to 10-membered heterocyclic group optionally substituted with one C1-C4 alkyl group or a 5- to 10-membered heteroaryl group optionally substituted with one C1-C4 alkyl group, wherein p and p' are independently 1 to 35; and wherein R 19 Independently, it is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, or a 5- to 10-membered heteroaryl group optionally substituted with one C1-C4 alkyl group. In some embodiments, p and p' are independently 1 to 3. In some embodiments, p and p' are independently 1 or 2.

[0101] In some embodiments, in a compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, when any alkyl and / or alkenyl groups (e.g., R1, R2) are present... 1’ R2, R7, R 20 R 21 and / or R22 When the alkyl and / or alkenyl groups in the form are substituted, the group can be independently substituted by one or more of the following: -OH, -(C=O)-(CH2). p -R 19 -O-(C=O)-(CH2) p -R 19 -(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -N(CH3)2、-(C=O)NH-(CH2) p -R 19 -N(CH3)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p N(CH3)2, a 5- to 10-membered heterocyclic group optionally substituted with one C1-C2 alkyl group or a 5- to 10-membered heteroaryl group optionally substituted with one C1-C2 alkyl group, wherein p and p' are independently 1 to 5; and wherein R 19 It is -N(CH3)2, a 5- to 6-membered heterocyclic group optionally substituted with one C1-C2 alkyl group, or a 5- to 6-membered heteroaryl group optionally substituted with one C1-C2 alkyl group.

[0102] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclic groups (e.g., R7 and R...) 19 When the 5- to 10-membered heteroaryl and / or 5- to 10-membered heterocyclic groups are substituted, the group can be independently selected from C1-C4 alkyl groups, -(CH2) groups, etc. p’’ N(CH3)2 is substituted with one or more substituents of oxo (=O), wherein p'' is 1 to 5. In some embodiments, p'' is 1 to 3. In some embodiments, p'' is 1 or 2.

[0103] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclic groups (e.g., R7 and R...) 19 When the 5- to 10-membered heteroaryl and / or 5- to 10-membered heterocyclic group is substituted, the group can be independently substituted by one or more substituents selected from C1-C2 alkyl, -(CH2)2N(CH3)2 and oxo (=O).

[0104] In some embodiments, R is present in a compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof. 20 It is a straight-chain C1-C3 alkyl group, either H or optionally substituted with -N(CH3)2.

[0105] In some embodiments, R is present in a compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof. 21 It is a 5- to 10-membered heterocyclic group that is optionally substituted with -N(CH3)2 or optionally substituted with a straight-chain C1-C3 alkyl group, or -(C=O)-R 22 , where R 22 It is a C1-C3 alkyl group that is optionally substituted with -N(CH3)2.

[0106] In some embodiments, R is present in a compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof. 21 It is a straight-chain C1-C4 alkyl group optionally substituted with -N(CH3)2 or a 5- to 10-membered heterocyclic group optionally substituted with C1-C2 alkyl, or -(C=O)-R 22 , where R 22 It is a C1-C3 alkyl group that is optionally substituted with -N(CH3)2.

[0107] In some embodiments, in the compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, any 5 to 10 heteroaryl group may independently represent furanyl, benzofuranyl, benzimidazolyl, quinolinyl, isoquinolinyl, thiophenyl, pyrroleyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazoleyl, pyrazinyl, pyridinyl, pyridazinyl, or pyrimidinyl. As described above, these groups themselves may be substituted.

[0108] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, the 5- to 10-membered heterocyclic groups may independently represent lactam, morpholino, piperazine, or piperidinyl groups. As mentioned above, these groups themselves may be substituted.

[0109] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, the -NR1R2 group has one of the following structures: , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0110] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, the group -NR1R2 is .

[0111] In some embodiments, in the compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R3 and R4 are independently H, a straight-chain or branched C4-C60 alkyl, a straight-chain or branched C4-C60 alkenyl, or a straight-chain or branched C4-C60 alkynyl, wherein at least one of R3 or R4 is different from H. In some embodiments, R3 and R4 are independently H, a straight-chain or branched C4-C30 alkyl, a straight-chain or branched C4-C30 alkenyl, or a straight-chain or branched C4-C30 alkynyl, wherein at least one of R3 or R4 is different from H. In some embodiments, R3 and R4 are independently H, a straight-chain or branched C6-C25 alkyl, or a straight-chain or branched C6-C25 alkenyl, wherein at least one of R3 or R4 is different from H.

[0112] In some embodiments, in the compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R5 and R6 are independently straight-chain or branched C4-C30 alkyl, straight-chain or branched C4-C30 alkenyl, or straight-chain or branched C4-C30 alkynyl. In some embodiments, R5 and R6 are independently straight-chain or branched C6-C25 alkyl, or straight-chain or branched C6-C25 alkenyl.

[0113] In some embodiments, in the compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R3, R4, R5, and R6 are independently straight-chain or branched C5-C20 alkyl, straight-chain or branched C5-C20 alkenyl, or straight-chain or branched C5-C20 alkynyl. In some embodiments, in the compounds of formula (I) or their pharmaceutically acceptable salts, R3, R4, R5, and R6 are independently straight-chain or branched C6-C20 alkyl, straight-chain or branched C6-C20 alkenyl, or straight-chain or branched C6-C20 alkynyl. In some embodiments, in the compounds of formula (I) or their pharmaceutically acceptable salts, R3, R4, R5, and R6 are independently straight-chain or branched C7-C20 alkyl, straight-chain or branched C7-C20 alkenyl, or straight-chain or branched C7-C20 alkynyl. In some embodiments, in the compound of formula (I) or its pharmaceutically acceptable salt, R3, R4, R5 and R6 are independently straight-chain or branched C8-C20 alkyl, straight-chain or branched C8-C20 alkenyl, or straight-chain or branched C8-C20 alkynyl.

[0114] In some embodiments, in the compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R3, R4, R5, and R6 are independently straight-chain or branched C5-C20 alkyl, or straight-chain or branched C5-C20 alkenyl. In some embodiments, in the compounds of formula (I) or their pharmaceutically acceptable salts, R3, R4, R5, and R6 are independently straight-chain or branched C6-C20 alkyl, or straight-chain or branched C6-C20 alkenyl. In some embodiments, in the compounds of formula (I) or their pharmaceutically acceptable salts, R3, R4, R5, and R6 are independently straight-chain or branched C7-C20 alkyl, or straight-chain or branched C7-C20 alkenyl. In some embodiments, in the compound of formula (I) or its pharmaceutically acceptable salt, R3, R4, R5 and R6 are independently straight-chain or branched C8-C20 alkyl or straight-chain or branched C8-C20 alkenyl groups.

[0115] In some embodiments, in the compound of formula (I) or (I1) or its pharmaceutically acceptable salt, when any one of R3, R4, R5 and R6 is an alkenyl group, the alkenyl group independently contains 1 to 6 C=C bonds, or 1 to 5 C=C bonds, or 1 to 4 C=C bonds, or 1 to 3 C=C bonds, or 1 or 2 C=C bonds.

[0116] In some embodiments, in compounds of formula (I) or (I1) or pharmaceutically acceptable salts thereof, when any one of R3, R4, R5, and R6 is an alkynyl group, the alkynyl group independently comprises 1 to 6 hydroxyl groups. Keys, or 1 to 5 Keys, or 1 to 4 Keys, or 1 to 3 Keys, 1 or 2 key.

[0117] In some embodiments, in the compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, when any one of R3, R4, R5, and R6 is a C8-C20 alkenyl group, the alkenyl group independently comprises one or two C=C bonds, and when any one of R3, R4, R5, and R6 is a C8-C20 ynyl group, the ynyl group independently comprises one or two C=C bonds. key.

[0118] In some embodiments, R3 and R4 are straight-chain C6-C16 alkyl groups in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts. In some embodiments, R3 and R4 are branched C8-C12 alkyl groups.

[0119] In some embodiments, in the compound of formula (I) or (I1) or its pharmaceutically acceptable salt, R3 and R4 are straight-chain C8-C20 alkenyl groups, and each alkenyl group contains one C=C bond.

[0120] In some embodiments, in the compound of formula (I) or (I1) or its pharmaceutically acceptable salt, R3 and R4 are straight-chain C8-C16 alkenyl groups, and each alkenyl group contains one C=C bond.

[0121] In some embodiments, in the compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, R3 is H and R4 is a straight-chain C6-C25 alkenyl group, wherein the alkenyl group contains one C=C bond.

[0122] In some embodiments, in the compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, R4 is H and R3 is a straight-chain C6-C25 alkenyl group, wherein the alkenyl group contains one C=C bond.

[0123] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R5 and R6 are independently straight-chain C8-C20 alkyl groups.

[0124] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R5 and R6 are independently straight-chain C10-C18 alkyl groups.

[0125] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R5 and R6 are straight-chain C6-C16 alkyl groups. In some embodiments, R5 and R6 are branched C8-C12 alkyl groups.

[0126] In some embodiments, in the compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, R5 and R6 are straight-chain C8-C20 alkenyl groups, wherein each alkenyl group contains one C=C bond.

[0127] In some embodiments, in the compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, R5 and R6 are straight-chain C15-C20 alkenyl groups, wherein each alkenyl group contains two C=C bonds.

[0128] In some embodiments, in the compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, R5 and R6 are branched C22-C25 alkenyl groups, wherein each alkenyl group contains one C=C bond.

[0129] In some embodiments, in the compound of formula (I) or (I1) or a pharmaceutically acceptable salt thereof, R5 and R6 are branched C13-C17 alkenyl groups, wherein each alkenyl group contains 3 C=C bonds.

[0130] In some embodiments, any one of R3, R4, R5, and R6 in the compound of formula (I) or (I1) or its pharmaceutically acceptable salt can be a C6-C25 alkenyl group, and the alkenyl group independently contains one, two, or three C=C bonds.

[0131] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R3, R4, R5, and R6 independently have one of the following structures, wherein one of R3 and R4 is different from H: , , , , , , , , , , or .

[0132] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R3 and R4 are both different from H and are the same.

[0133] In some embodiments, R5 and R6 are the same in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts.

[0134] In some embodiments, in compounds of formula (I) or (I1) or their pharmaceutically acceptable salts, R3 and R4 are different from H and R3, R4, R5 and R6 are the same.

[0135] In some embodiments, the ionizable lipid compound of this disclosure may be a compound of formula (I2) or (I3):

[0136] (I2) (I3) Or its pharmaceutically acceptable salt. Among them, R1, R2, R3, R4, R5, R6 and R 1’ As defined in this paper for equations (I) and / or (I1).

[0137] In some embodiments, the ionizable lipid compound of this disclosure may be a compound of formula (I2) or a pharmaceutically acceptable salt thereof, wherein R1 is H or C1-C2 alkyl and R2 is -C(=O)R7; and wherein R3, R4, R5, R6 and R7 are as defined herein with respect to formula (I) and / or (I1).

[0138] In some embodiments, the ionizable lipid compound of this disclosure may be a compound of formula (I3) or a pharmaceutically acceptable salt thereof, wherein R 1’ It is a straight-chain C1-C4 alkyl group substituted with the following group: -(C=O)-(CH2) p -R 19 -O-(C=O)-(CH2) p -R 19 -(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -N(CH3)2、-(C=O)NH-(CH2) p -R 19 -N(CH3)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p N(CH3)2, optionally substituted 5- to 6-membered heterocyclic group or optionally substituted 5- to 6-membered heteroaryl group, wherein p and p' are independently 1 to 5; and wherein R 19 It is -N(CH3)2, optionally substituted 5- to 6-membered heterocyclic group or optionally substituted 5- to 6-membered heteroaryl group; wherein when the 5- to 6-membered heteroaryl group and / or the 5- to 6-membered heterocyclic group is substituted, the group is independently substituted by a substituent selected from C1-C2 alkyl; and wherein R3, R4, R5 and R6 are as defined herein with respect to formula (I) and / or (I1).

[0139] In some embodiments, the ionizable lipid compounds of this disclosure may be compounds of formula (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), or (I1-6): (I1-1) (I1-2) (I1-3) (I1-4) (I1-5) (I1-6) Or its pharmaceutically acceptable salt. Where R1 is H or a straight-chain C1-C4 alkyl group; Where R 10 and R 11 Independently, it is -H, -(C=O)-CH3, or -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 Or -(C=O)-(CH2) p -(C=O)-R 19 ;and Among them, R3, R4, R5, R6, R7, R 19 R 20 and R 21 As defined in this paper for equations (I) and / or (I1), and p is 1 to 5.

[0140] In some implementations, in formulas (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), or (I1-6), p can be independently 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or p can be 1, 2, 3, 4, or 5.

[0141] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-4a): (I1-4a), Or its pharmaceutically acceptable salt. R1 is H or a straight-chain C1-C4 alkyl group; and R3, R4, R5 and R6 are as defined herein with respect to formula (I) and / or (I1).

[0142] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-4a) or a pharmaceutically acceptable salt thereof, wherein R1 is H or -CH3.

[0143] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-4a) or a pharmaceutically acceptable salt thereof, wherein R1 is -CH3.

[0144] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-4b): (I1-4b), Or its pharmaceutically acceptable salt. Where R1 is H or a straight-chain C1-C4 alkyl group; Where R 12 and R 13 Independently, it is a straight-chain C1-C4 alkyl group that is optionally substituted; wherein when the straight-chain alkyl group is substituted, the group is independently substituted with -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2 or a 5-6 heteroaryl group; and wherein R3, R4, R5 and R6 are as defined herein with respect to formula (I) and / or (I1).

[0145] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-4b) or a pharmaceutically acceptable salt thereof, wherein R1 is H or -CH3.

[0146] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-4b) or a pharmaceutically acceptable salt thereof, wherein R1 is -CH3.

[0147] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-4b) or a pharmaceutically acceptable salt thereof, wherein R 12 and R 13 It is independently a straight-chain C1-C4 alkyl group substituted with -NH2, -NHCH3, -N(CH3)2 or -N(CH2CH3)2.

[0148] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-4b) or a pharmaceutically acceptable salt thereof, wherein R 12 and R 13 It is independently a straight-chain C1-C4 alkyl group substituted with -N(CH3)2.

[0149] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5a): (I1-5a), Or its pharmaceutically acceptable salt. Where R1 is H or a straight-chain C1-C4 alkyl group; Where q ranges from 1 to 5; Where R 14 and R'14 Independently represents -H, -CH3, -CH2CH3, or -(CH2). p O-(C=O)CH3, where p is 1 to 5, or R 14 and R' 14 The linkage forms a 6-membered heterocyclic group optionally substituted with -CH3 or -CH2CH3; and wherein R3, R4, R5 and R6 are as defined herein with respect to formula (I) and / or (I1).

[0150] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5a) or a pharmaceutically acceptable salt thereof, wherein R1 is H or -CH3.

[0151] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5a) or a pharmaceutically acceptable salt thereof, wherein R1 is H.

[0152] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5a) or a pharmaceutically acceptable salt thereof, wherein R 14 and R' 14 Independently represents -H, -CH3, -CH2CH3, or -(CH2). p O-(C=O)CH3, where p is 1 to 3, or R 14 and R' 14 The linkage forms a 6-membered heterocyclic group that can be optionally substituted with -CH3 or -CH2CH3.

[0153] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5b): (I1-5b), Or its pharmaceutically acceptable salt. Where X1 is -NH- or -O-; Where R 15 It is a straight-chain C1-C4 alkyl group, -(CH2) p- R 19 Or -(CH2) p N(CH3)-((CH2) p’ -N(CH3)2), where p and p' are independently 1 to 5, and R3, R4, R5, R6 and R 19 As defined in this paper for equations (I) and / or (I1).

[0154] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5b) or a pharmaceutically acceptable salt thereof, wherein R 15 It is -(CH2)2N(CH3)-((CH2)2-N(CH3)2) or -(CH2)p -R 19 , where p is 1 to 3.

[0155] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5b) or a pharmaceutically acceptable salt thereof, wherein R 15 It is -(CH2)2N(CH3)-((CH2)2-N(CH3)2), -(CH2) p -N(CH3)2 or -(CH2) p -R 23 Where p is 1 to 3, and R 23 It is a 5- to 6-membered heteroaryl or 5- to 6-membered heterocyclic group that is optionally substituted with a C1-C2 alkyl group.

[0156] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5b) or a pharmaceutically acceptable salt thereof, wherein R 15 It is -(CH2)2N(CH3)-((CH2)2-N(CH3)2), -(CH2) p -N(CH3)2 or -(CH2) p -R 23 Where p is 1 to 3, and R 23 It is furanyl, phenylthio, pyrroleyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazoleyl, pyrazinyl, pyridinyl, pyridinyl, pyridinyl, piperidinyl, or piperazinyl, wherein R 23 It may be optionally substituted with C1-C2 alkyl groups.

[0157] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5b) or a pharmaceutically acceptable salt thereof, wherein R 15 It is -(CH2)2N(CH3)-((CH2)2-N(CH3)2), -(CH2) p -N(CH3)2 or -(CH2) p -R 23 Where p is 1 to 3, and R 23 It is imidazole or piperidinyl, where R 23 It may be optionally substituted with C1-C2 alkyl groups.

[0158] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5c): (I1-5c), Or its pharmaceutically acceptable salt. Where R 16 It can be optionally modified by straight-chain C1-C4 alkyl groups, oxo (=O) and / or -(CH2). p-N(CH3)2-substituted 5 to 6-membered heteroaryl or 5 to 6-membered heterocyclic group, wherein p is 1 to 3, and wherein R3, R4, R5 and R6 are as defined herein with respect to formula (I) and / or (I1).

[0159] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5c) or a pharmaceutically acceptable salt thereof, wherein R 16 It is a furanyl, phenylthio, pyrrolidinyl, pyrrolydinyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazinyl, pyridinyl, pyridinyl, pyridinyl, piperazinyl, piperidinyl, or pyrimidinyl group optionally substituted with a straight-chain C1-C4 alkyl, oxo (=O) and / or -(CH2)pN(CH3)2, wherein p is 1 to 3.

[0160] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5c) or a pharmaceutically acceptable salt thereof, wherein R 16 It is an imidazolyl or pyrrolidinyl group optionally substituted with C1-C2 alkyl, oxo (=O) and / or -(CH2)pN(CH3)2, wherein p is 1 to 3.

[0161] In some embodiments, the ionizable lipid compound can be a compound of formula (I1-5d): (I1-5d), Or its pharmaceutically acceptable salt. Where R 17 It can be optionally surrounded by straight-chain C1-C4 alkyl groups or -(CH2). p -N(CH3)2-substituted 5 to 6-membered heteroaryl groups, wherein p is 1 to 3; The C=C bonds present in equation (I1-5d) independently have either an E or Z configuration; and R3, R4, R5 and R6 are as defined herein with respect to equation (I) and / or (I1).

[0162] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5d) or a pharmaceutically acceptable salt thereof, wherein R 17 It can be a furanyl, phenylthio, pyrroleyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazinyl, pyridinyl, pyridinyl, pyridinyl, or pyrimidinyl group optionally substituted with a C1-C2 alkyl group.

[0163] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-5d) or a pharmaceutically acceptable salt thereof, wherein R 17 It is an imidazole group.

[0164] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-6a), (I1-6b), or (I1-6c): (I1-6a) (I1-6b) (I1-6c) Or its pharmaceutically acceptable salt. in R 31 It is a straight-chain C1-C4 alkyl group that is optionally substituted with -N(CH3)2; R 32 It is -N(CH3)2 or optionally a 5- to 6-membered heterocyclic group substituted with a straight-chain C1-C4 alkyl group, and z is 1 to 4, and R3, R4, R5 and R6 are as defined herein with respect to formula (I) and / or (I1).

[0165] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-6a) or a pharmaceutically acceptable salt thereof, wherein R 31 It is -(CH2)3-N(CH3)2.

[0166] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-6b) or (I1-6c) or a pharmaceutically acceptable salt thereof, wherein R 32 It is -N(CH3)2 and z is 2 to 4.

[0167] In some embodiments, the ionizable lipid compound may be a compound of formula (I1-6b) or (I1-6c) or a pharmaceutically acceptable salt thereof, wherein R 32 It is a piperazine or piperidinyl group substituted with a C1-C2 alkyl group and z is 3.

[0168] In some implementations, R3 and R4 can be the same in equations (I), (I1), (I2), (I1-1), (I1-2), (I1-3), (I1-4), (I1-4a), (I1-4b), (I1-5), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6), (I1-6a), (I1-6b), or (I1-6c). In some implementations, R5 and R6 can be the same in equations (I), (I1), (I2), (I1-1), (I1-2), (I1-3), (I1-4), (I1-4a), (I1-4b), (I1-5), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6), (I1-6a), (I1-6b), or (I1-6c). In some implementations, in formulas (I), (I1), (I2), (I1-1), (I1-2), (I1-3), (I1-4), (I1-4a), (I1-4b), (I1-5), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6), (I1-6a), (I1-6b), or (I1-6c), R3 and R4 are the same, and R5 and R6 are the same.

[0169] In some embodiments, the ionizable lipid compound may be a compound of formula (I1a): (I1a), Or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18 and n is a number from 2 to 14; and wherein the C=C bond present in formula (I1a) independently has an E or Z configuration.

[0170] In some embodiments, the ionizable lipid compound can be a compound of formula (I2a): (I2a), Or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18 and n is a number from 2 to 14; and wherein the C=C bond present in formula (I2a) independently has an E or Z configuration.

[0171] In some embodiments, the ionizable lipid compound may be a compound of formula (I2b): (I2b), Or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18 and n is a number from 2 to 14; and wherein the C=C bond present in formula (I2b) independently has an E or Z configuration.

[0172] In some embodiments, the ionizable lipid compound may be a compound of formula (I3a): (I3a), Or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18 and n is a number from 2 to 14; and R1 is H or a straight-chain C1-C4 alkyl; and wherein the C=C bond present in formula (I3a) independently has an E or Z configuration.

[0173] In some embodiments, in formula (I3a), R1 is H or C1-C3 alkyl, or R1 is H or C1-C2 alkyl, or R1 is H or -CH3, more preferably, R1 is -CH3.

[0174] In some embodiments, the ionizable lipid compound may be a compound of formula (I3b): (I3b), Or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18; n is a number from 2 to 14; R1 is H or a straight-chain C1-C4 alkyl group; and R 12 and R 13 Independently, it is a straight-chain C1-C4 alkyl group that is optionally substituted; wherein when the straight-chain alkyl group is substituted, the group is independently substituted by -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2 or a 5-6 membered heteroaryl group; and wherein the C=C bond present in formula (I3b) independently has an E or Z configuration.

[0175] In some embodiments, in formula (I3b), R1 is H or C1-C3 alkyl, or R1 is H or C1-C2 alkyl, or R1 is H or -CH3, more preferably, R1 is -CH3.

[0176] In some implementations, in equation (I3b), R 12 and R 13 It is independently a C1-C3 alkyl group, or R 12 and R 13 Independently selected from C1 alkyl, C2 alkyl, and C3 alkyl. In some embodiments, R 12 and R 13 Both can be C3 alkyl groups.

[0177] In some implementations, in equation (I3b), R 12 and R 13 Independently, it is a straight-chain C1-C4 alkyl group substituted with -NH2, -NHCH3, -N(CH3)2 or -N(CH2CH3)2, more preferably substituted with -N(CH3)2.

[0178] In some embodiments, the ionizable lipid compound may be a compound of formula (I4a): (I4a), Or its pharmaceutically acceptable salt, wherein m is a number from 6 to 18; n is a number from 2 to 14; and q is a number from 1 to 4; wherein R 14 and R' 14 Independently represents -H, -CH3, -CH2CH3, or -(CH2). p O-(C=O)CH3, where p is 1 to 5; and the C=C bond present in formula (I4a) independently has an E or Z configuration.

[0179] In some embodiments, in formula (I4a), p is 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or p can be 1, 2, 3, 4, or 5. In some embodiments, in formula (I4a), q is 1 to 4, or 1 to 3, or 1 to 2, or q can be 1, 2, 3, or 4. In some embodiments, in formula (I4a), p is 1 to 3.

[0180] In some embodiments, the ionizable lipid compound may be a compound of formula (I4b): (I4b), Or its pharmaceutically acceptable salt, wherein m is a number from 6 to 18; n is a number from 2 to 14; and R 15 It is -CH3, -(CH2) p CH3, -(CH2) p R 19 Or -(CH2) p N(CH3)-((CH2) p’ -N(CH3)2), where p and p' are independently 1 to 5, and R 19 As defined in this paper for equation (I); and where the C=C bond present in equation (I4b) independently has an E or Z configuration.

[0181] In some embodiments, in formula (I4b), p and p' can be independently 1 to 4, or 1 to 3, or 1 to 2, or p and p' can be independently 1, 2, 3, 4, or 5. In some embodiments, in formula (I4b), p and p' can be independently 1 to 3.

[0182] In some implementations, in equation (I4b), R 15 Preferably, it is -(CH2)2N(CH3)-((CH2)2-N(CH3)2).

[0183] In some embodiments, the ionizable lipid compound may be a compound of formula (I4c): (I4c), Or its pharmaceutically acceptable salt, wherein m is a number from 6 to 18; n is a number from 2 to 14; and R 16 It is a 5-6 member heteroaryl group; and the C=C bond present in formula (I4c) independently has an E or Z configuration.

[0184] In some implementations, in formula (I4c), R 16 It can be furanyl, phenylthio, pyrroleyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazoleyl, pyrazinyl, pyridinyl, pyridinyl, pyridazinyl, or pyrimidinyl. In some embodiments, in formula (I4c), R 16 It is an imidazole group.

[0185] In some embodiments, the ionizable lipid compound may be a compound of formula (I4d): (I4d), Or its pharmaceutically acceptable salt, wherein m is a number from 6 to 18; n is a number from 2 to 14; and R 17 It is a 5-6 membered heteroaryl group; and the C=C bond present in formula (I4d) independently has an E or Z configuration.

[0186] In some implementations, in equation (I4d), R 17 It can be furanyl, phenylthio, pyrroleyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazoleyl, pyrazinyl, pyridinyl, pyridinyl, pyridazinyl, or pyrimidinyl. In some embodiments, in formula (I4d), R 17 It is an imidazole group.

[0187] In some embodiments, the ionizable lipid compound may be a compound of formula (I4e): (I4e), Or its pharmaceutically acceptable salt, wherein m is a number from 6 to 18; n is a number from 2 to 14; and R 18 It is -(CH2) p -R 19 Where p is 1 to 5; and R 19 As defined in this paper for equation (I); and where the C=C bond present in equation (I4e) independently has an E or Z configuration.

[0188] In some implementations, in formula (I4e), R 18 It could be -(CH2) p -R 19Where p is 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or p is 1, 2, 3, 4, or 5; and R 19 As defined in this paper for equation (I).

[0189] In some implementations, in formula (I4e), R 18 It is -(CH2) p -R 19 Where p is 1 to 3, and R 19 As defined herein with respect to equation (I). In some implementations, in equation (I4e), R 18 It is -(CH2)3-(5-6-membered heteroaryl), wherein the 5-6-membered heteroaryl group is, for example, furanyl, phenylthio, pyrroleyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazinyl, pyridinyl, pyridinyl, or pyrimidinyl. In some embodiments, in formula (I4e), R 18 It is -(CH2)3-imidazole.

[0190] In some implementations, in equations (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d), or (I4e), m can independently be an integer from 6 to 18, or m can be an integer from 6 to 17, or 6 to 16, or 6 to 15, or 6 to 14, or 6 to 13, or 6 to 12, or 6 to 11, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or m can be an integer of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. In some implementations, m can be 12.

[0191] In some embodiments, in equations (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d), or (I4e), n can be an integer from 2 to 14 independently, or n can be an integer from 2 to 13, or 2 to 12, or 2 to 11, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or n can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, n can be 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3. In some embodiments, n can be 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4. In some embodiments, n can be 3, 4, or 5. In some implementations, n can be 3 or 5.

[0192] In some embodiments, the ionizable lipid compounds of this disclosure may be selected from, but are not limited to, compounds 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320a, 320b, 321a, 321b, 322, 323, 3... in Table 1. 24, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 358, 359, 360 and 362 or their pharmaceutically acceptable salts.

[0193] In some embodiments, the ionizable lipid compounds of this disclosure may be selected from, but are not limited to, compounds 301, 302, 303, 304, 305, 306, 307, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320a, 320b, 321a, 321b, 322, 323, 324, 3... 25, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 358, 359, 360 and 362 or their pharmaceutically acceptable salts.

[0194] In some embodiments, the ionizable lipid compounds of this disclosure may be selected from, but not limited to, compounds 301, 302, 303, 304, 305, 306, 307, 309, 311, 312, 313, 314, 315, 316, 318, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 347, 350, 351, 352, 353, 355, 358, 359, 360 and 362 of Table 1, or pharmaceutically acceptable salts thereof.

[0195] In some embodiments, the ionizable lipid compounds of this disclosure may be selected from, but not limited to, compounds 305, 313, 314, 315, 316, 317, 318, 320a, 320b, 321a, 321b, 323, 324, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 340, 350, 351 and 360 of Table 1, or pharmaceutically acceptable salts thereof.

[0196] In some embodiments, the ionizable lipid compounds of this disclosure may be selected from, but not limited to, compounds 346, 347, 353, 354, 355 and 359 of Table 1 or pharmaceutically acceptable salts thereof.

[0197] In some embodiments, the ionizable lipid compounds of this disclosure may be selected from, but not limited to, compounds 301, 303, 304, 305, 306, 307, 311, 312, 313, 314, 315, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 347, 350, 351, 352, 353, 355, 358 and 359 of Table 1, or pharmaceutically acceptable salts thereof.

[0198] In some embodiments, the ionizable lipid compounds of this disclosure may be selected from, but not limited to, compounds 305, 306, 307, 313, 314, 315, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 350, 351, 352, 353, 355, 358 and 359 of Table 1, or pharmaceutically acceptable salts thereof.

[0199] In some embodiments, the ionizable lipid compound may be in the form of any enantiomer and / or any diastereomer thereof or any mixture thereof.

[0200] Table 1 Non-limiting examples of ionizable lipid compounds

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209] The cis (Z) or trans (E) stereochemistry drawn in the chemical structures of Table 1 is randomly assigned. In other words, each C=C bond in the chemical structures of Table 1 can independently have a cis or trans configuration. In some embodiments, the compounds in Table 1 may be in the form of a mixture of cis or trans geometric isomers.

[0210] In some embodiments, the ionizable lipid compounds in Table 1 may be in the form of any enantiomer, any diastereomer, any cis or trans geometric isomer, or any mixture thereof.

[0211] As used herein, the term "compound" is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structure depicted or described in relation to the compound. When referring to a characteristic or substituent of a compound, the terms "optional" or "optionally" refer to a characteristic or substituent that may or may not be present. For example, "optionally substituted alkyl" includes "alkyl" and "substituted alkyl" as defined below. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any spatially impractical, synthetically infeasible, and / or inherently unstable substitution or substitution pattern.

[0212] The compounds described herein may have an asymmetric center, a geometric center (e.g., a double bond), or both. Unless a specific stereochemical or isomeric form is specifically indicated, all chiral, diastereomeric, racemic, and geometrical isomeric forms of the structure are intended to be covered. Compounds of this disclosure containing asymmetric substituted atoms can be isolated in an optically active or racemic form. The preparation of optically active forms is known in the art, for example, by resolving the racemic form, by synthesis from optically active starting materials, or by using chiral auxiliaries. Geometrical isomers of alkenes, C=N double bonds, or other types of double bonds may be present in the compounds described herein, and all such stable isomers are included in this disclosure. Specifically, cis and trans geometrical isomers of the compounds of this disclosure may also be present and can be isolated as mixtures of isomers or as separate isomeric forms.

[0213] The compounds described herein also include tautomer forms. Tautomer forms arise from the exchange of single bonds with adjacent double bonds and the accompanying proton migration. Tautomer forms include proton-shift tautomers, which are isomeric protonated states with the same empirical formula and total charge. Examples of proton-shift tautomers include keto-enol pairs, amide-imine pairs, lactam-lactamimide pairs, enamine-imide pairs, and cyclic forms, where the proton can occupy two or more positions in the heterocyclic system, such as 1H- and 3H-imidazolium, 1H-, 2H- and 4H-1,2,4-triazoles, 1H- and 2H-isoindole, and 1H- and 2H-pyrazoles. Tautomer forms can be in equilibrium or spatially locked into one form by appropriate substitution.

[0214] The compounds described herein also include all isotopes of the atoms present in the intermediates or final compounds. "Isotope" refers to atoms having the same atomic number but different mass numbers, caused by different numbers of neutrons in the nucleus. Thus, for example, each individual hydrogen atom present in formula (200) can be considered as... 1 H, 2 H (deuterium) or 3 The presence of H (tritium) atoms is preferred as... 1 H or 2 H exists. Similarly, for example, each individual carbon atom present in equation (200) can be considered as... 12 C 13 C or 14 The presence of C atoms makes it a preferred choice. 12 C exists.

[0215] The compounds or structures and salts disclosed herein can be prepared by conventional methods by combining with solvents or water molecules to form solvates and hydrates.

[0216] neutral lipids In some embodiments, the lipid nanoparticles may also comprise at least one neutral lipid. In some embodiments, the neutral lipid may be a phospholipid or a derivative thereof.

[0217] Examples of phospholipids suitable for use in this disclosure include, but are not limited to: dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearyl-sn-glycerol-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-diacaroyl-sn-glycerol-3-phosphocholine (DBPC), and 1-stearoyl-2-palmitoylphosphatidylcholine. Bases (SPPC), 1,2-dienoyl-sn-glycerol-3-phosphate choline (DEPC), palmitoyl oleoyl phosphatidylcholine (POPC), lysophosphatidylcholine, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoyl phosphatidylcholine, distearate phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof.

[0218] In some embodiments, preferred phospholipids are distearylphosphatidylcholine (DSPC) and dioleoylphosphatidylethanolamine (DOPE).

[0219] In other embodiments, preferred phospholipids are DSPC, DOPC, DMPC, and PE.

[0220] assist lipids In some implementations, the lipid nanoparticles may also contain at least one auxiliary lipid.

[0221] "Assistant lipids" are lipids that enhance transfection (e.g., transfection of lipid nanoparticles containing payload and cargo). Mechanisms by which assistant lipids enhance transfection may include enhanced particle stability and / or enhanced membrane fusion. Assistant lipids include steroids and alkyl resorcinols. Assistant lipids suitable for use in this disclosure include, but are not limited to, cholesterol, 5-heptadecyl resorcinol, cholesterol hemisuccinate, cholesterol, cholesterol ketones, cholesterol ketones, coprosterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, and mixtures thereof.

[0222] In some implementations, cholesterol is the preferred auxiliary lipid.

[0223] Polymer-conjugated lipids In some embodiments, the lipid nanoparticles may also comprise at least one polymer-conjugated lipid.

[0224] In some embodiments, the polymer-conjugated lipids comprise a polymer conjugated to at least one lipid. In some embodiments, the polymer-conjugated lipids may comprise at least one component that reduces particle aggregation, at least one component that reduces the clearance of lipid nanoparticles from the subject's circulation, at least one component that increases the ability of lipid nanoparticles to penetrate the mucus layer, at least one component that reduces the subject's immune response to the administration of lipid nanoparticles, at least one component that alters the membrane fluidity of lipid nanoparticles, at least one component that contributes to the stability of lipid nanoparticles, or any combination thereof. In some embodiments, the lipid nanoparticles may be substantially free of polymer-conjugated lipids. In some embodiments, the lipid nanoparticles may be free of any amount of polymer-conjugated lipids.

[0225] In some embodiments, the polymer present in the polymer-conjugated lipid may comprise at least one polyethylene glycol (PEG), at least one polypropylene glycol (PPG), poly(2-oxazoline) (POZ), at least one polyamide (ATTA), at least one cationic polymer, or any combination thereof.

[0226] In some embodiments, the lipid conjugated with the polymer may be selected from, but is not limited to, at least one of the ionizable lipids, neutral lipids, or auxiliary lipids listed above.

[0227] In some embodiments, the polymer conjugated with at least one lipid is PEG, and the lipid conjugated with the polymer may be referred to as "PEG-lipid". In some embodiments, at least one PEG-lipid may be selected from, but is not limited to, at least one of the following: Siglec-1L-PEG-DSPE, R)-2,3-bis(octadecoxy)propyl-1-(methoxypoly(ethylene glycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, P EG2k-C11, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG click DMG C14, PEG-C12, PEG-C10, N(carbonyl-methoxy polyethylene glycol-2000)-l,2-distearate-sn-glycerol-3-phosphate ethanolamine, Myrj52, mPEG-PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-Distearyl-sn-glycerol-3-phosphate ethanolamine-PEG2000, HPEG-2K-LIPD, folic acid PEG-DSPE, DSPE-PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE-PEG2000 maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE-P EG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearylglycerol-polyethylene glycol, Cl8PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-PEG2000, (R)-2,3-bis(octadecoxy)propyl-1-(methoxypoly(ethylene glycol)2000)propylcarbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

[0228] In some embodiments, preferred polymer-conjugated lipids are polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2-oxazoline) (POZ), polyamide (ATTA), cationic polymers, polysarcosine (Psar), polyglutamic acid (PGA), and 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol (PEG-DMG).

[0229] In some embodiments, the preferred polymer-conjugated lipid is a PEG-lipid selected from PEG-DMG or PEG-DSG.

[0230] In some implementations, the preferred PEG-lipid is PEG2k-DMG.

[0231] In some embodiments, the average molecular weight of the polymeric portion of the polymer-conjugated lipid (e.g., PEG) can be from 500 to 20,000 Daltons. In some embodiments, the molecular weight of the polymer can be about 500 to 20,000, 1,000 to 20,000, 1,500 to 20,000, 2,000 to 20,000, 2,500 to 20,000, 3,000 to 20,000, 3,500 to 20,000, 4,000 to 20,000, 4,500 to 20,000, 5,000 to 20,000, 5,500 to 20,000, 6,000 to 20,000, 6,500 to 20,000, 7,000 to 20,000, 7,500 to 20,000, 8,000 to 20,000, 8,500 to 20,000. 00, 9,000 to 20,000, 9,500 to 20,000, 10,000 to 20,000, 10,500 to 20,000, 11,000 to 20,000, 11,500 to 20,000, 12,000 to 20,000, 12,500 to 20,000, 13,000 to 20,000, 13,500 to 20,000, 14,000 to 20,000, 14,500 to 20,000, 15,000 to 20,000, 15,500 to 20,000, 16,000 to 20,000, 16,500 to 20,000, 17,000 to 20,000 0, 17,500 to 20,000, 18,000 to 20,000, 18,500 to 20,000, 19,000 to 20,000, 19,500 to 20,000, 500 to 19,500, 1,000 to 19,500, 1,500 to 19,500, 2,000 to 19,500, 2,500 to 19,500, 3,000 to 19,500, 3,500 to 19,500, 4,000 to 19,500, 4,500 to 19,500, 5,000 to 19,500, 5,500 to 19,500, 6,000 to 19,500, 6,500 to 19,500 0, 7,000 to 19,500, 7,500 to 19,500, 8,000 to 19,500, 8,500 to 19,500, 9,000 to 19,500, 9,500 to 19,500, 10,000 to 19,500, 10,500 to 19,500, 11,000 to 19,500, 11,500 to 19,500, 12,000 to 19,500, 12,500 to 19,500, 13,000 to 19,500, 13,500 to 19,500, 14,000 to 19,500, 14,500 to 19,500, 15,000 to 19,500, 15,500 to 19,500, 16,000 to 19,500, 16,500 to 19,500, 17,000 to 19,500, 17,500 to 19,500, 18,000 to 19,500, 18,500 to 19,500, 19,000 to 19,500, 1,500 to 19,000, 2, 000 to 19,000, 2,500 to 19,000, 3,000 to 19,000, 3,500 to 19,000, 4,000 to 19,000, 4,500 to 19,000, 5,000 to 19,000, 5,500 to 19,000, 6,000 to 19,000, 6,500 to 19,000 000, 7,000 to 19,000, 7,500 to 19,000, 8,000 to 19,000, 8,500 to 19,000, 9,000 to 19,000, 9,500 to 19,000, 10,000 to 19,000, 10,500 to 19,000, 11,000 to 19,000 11,500 to 19,000, 12,000 to 19,000, 12,500 to 19,000, 13,000 to 19,000, 13,500 to 19,000, 14,000 to 19,000, 14,500 to 19,000, 15,000 to 19,000, 15,500 to 19,000 16,000 to 19,000, 16,500 to 19,000, 17,000 to 19,000, 17,500 to 19,000, 18,000 to 19,000, 18,500 to 19,000, 1,500 to 18,500, 2,000 to 18,500, 2,500 to 18,500 3,000 to 18,500, 3,500 to 18,500, 4,000 to 18,500, 4,500 to 18,500, 5,000 to 18,500, 5,500 to 18,500, 6,000 to 18,500, 6,500 to 18,500, 7,000 to 18,500, 7,500 to 18 500, 8,000 to 18,500, 8,500 to 18,500, 9,000 to 18,500, 9,500 to 18,500, 10,000 to 18,500, 10,500 to 18,500, 11,000 to 18,500, 11,500 to 18,500, 12,000 to 18,500 00, 12,500 to 18,500, 13,000 to 18,500, 13,500 to 18,500, 14,000 to 18,500, 14,500 to 18,500, 15,000 to 18,500, 15,500 to 18,500, 16,000 to 18,500, 16,500 to 18,500, 17,000 to 18,500, 17,500 to 18,500, 18,000 to 18,500, 1,500 to 18,000, 2,000 to 18,000, 2,500 to 18,000, 3,000 to 18,000, 3,500 to 18,000, 4,000 to 18,000 4,500 to 18,000, 5,000 to 18,000, 5,500 to 18,000, 6,000 to 18,000, 6,500 to 18,000, 7,000 to 18,000, 7,500 to 18,000, 8,000 to 18,000, 8,500 to 18,000, 9,000 to 18 ,000, 9,500 to 18,000, 10,000 to 18,000, 10,500 to 18,000, 11,000 to 18,000, 11,500 to 18,000, 12,000 to 18,000, 12,500 to 18,000, 13,000 to 18,000, 13,500 to 1 8,000, 14,000 to 18,000, 14,500 to 18,000, 15,000 to 18,000, 15,500 to 18,000, 16,000 to 18,000, 16,500 to 18,000, 17,000 to 18,000, 17,500 to 18,000, 1,500 to 1 7,500, 2,000 to 17,500, 2,500 to 17,500, 3,000 to 17,500, 3,500 to 17,500, 4,000 to 17,500, 4,500 to 17,500, 5,000 to 17,500, 5,500 to 17,500, 6,000 to 17,500, 6 500 to 17,500, 7,000 to 17,500, 7,500 to 17,500, 8,000 to 17,500, 8,500 to 17,500, 9,000 to 17,500, 9,500 to 17,500, 10,000 to 17,500, 10,500 to 17,500, 11,000 to 17,500, 11,500 to 17,500, 12,000 to 17,500, 12,500 to 17,500, 13,000 to 17,500, 13,500 to 17,500, 14,000 to 17,500, 14,500 to 17,500, 15,000 to 17,500, 15,500 0 to 17,500, 16,000 to 17,500, 16,500 to 17,500, 17,000 to 17,500, 1,500 to 17,000, 2,000 to 17,000, 2,500 to 17,000, 3,000 to 17,000, 3,500 to 17,000, 4,000 to 17,000, 4,500 to 17,000, 5,000 to 17,000, 5,500 to 17,000, 6,000 to 17,000, 6,500 to 17,000, 7,000 to 17,000, 7,500 to 17,000, 8,000 to 17,000, 8,500 to 17,000, 9,0 00 to 17,000, 9,500 to 17,000, 10,000 to 17,000, 10,500 to 17,000, 11,000 to 17,000, 11,500 to 17,000, 12,000 to 17,000, 12,500 to 17,000, 13,000 to 17,000, 13,5 00 to 17,000, 14,000 to 17,000, 14,500 to 17,000, 15,000 to 17,000, 15,500 to 17,000, 16,000 to 17,000, 16,500 to 17,000, 1,500 to 16,500, 2,000 to 16,500, 2,500 To 16,500, 3,000 to 16,500, 3,500 to 16,500, 4,000 to 16,500, 4,500 to 16,500, 5,000 to 16,500, 5,500 to 16,500, 6,000 to 16,500, 6,500 to 16,500, 7,000 to 16,500 7,500 to 16,500, 8,000 to 16,500, 8,500 to 16,500, 9,000 to 16,500, 9,500 to 16,500, 10,000 to 16,500, 10,500 to 16,500, 11,000 to 16,500, 11,500 to 16,500, 12, 000 to 16,500, 12,500 to 16,500, 13,000 to 16,500, 13,500 to 16,500, 14,000 to 16,500, 14,500 to 16,500, 15,000 to 16,500, 15,500 to 16,500, 16,000 to 16,500, 1, 500 to 16,000, 2,000 to 16,000, 2,500 to 16,000, 3,000 to 16,000, 3,500 to 16,000, 4,000 to 16,000, 4,500 to 16,000, 5,000 to 16,000, 5,500 to 16,000, 6,000 to 16,000 00, 6,500 to 16,000, 7,000 to 16,000, 7,500 to 16,000, 8,000 to 16,000, 8,500 to 16,000, 9,000 to 16,000, 9,500 to 16,000, 10,000 to 16,000, 10,500 to 16,000, 11,000 to 16,000, 11,500 to 16,000, 12,000 to 16,000, 12,500 to 16,000, 13,000 to 16,000, 13,500 to 16,000, 14,000 to 16,000, 14,500 to 16,000, 15,000 to 16,000, 1 5,500 to 16,000, 1,500 to 15,500, 2,000 to 15,500, 2,500 to 15,500, 3,000 to 15,500, 3,500 to 15,500, 4,000 to 15,500, 4,500 to 15,500, 5,000 to 15,500, 5,500 to 15 500, 6,000 to 15,500, 6,500 to 15,500, 7,000 to 15,500, 7,500 to 15,500, 8,000 to 15,500, 8,500 to 15,500, 9,000 to 15,500, 9,500 to 15,500, 10,000 to 15,500, 10 ,500 to 15,500, 11,000 to 15,500, 11,500 to 15,500, 12,000 to 15,500, 12,500 to 15,500, 13,000 to 15,500, 13,500 to 15,500, 14,000 to 15,500, 14,500 to 15,500, 1 5,000 to 15,500, 1,500 to 15,000, 2,000 to 15,000, 2,500 to 15,000, 3,000 to 15,000, 3,500 to 15,000, 4,000 to 15,000, 4,500 to 15,000, 5,000 to 15,000, 5,500 to 15,000 ,000, 6,000 to 15,000, 6,500 to 15,000, 7,000 to 15,000, 7,500 to 15,000, 8,000 to 15,000, 8,500 to 15,000, 9,000 to 15,000, 9,500 to 15,000, 10,000 to 15,000, 10 500 to 15,000, 11,000 to 15,000, 11,500 to 15,000, 12,000 to 15,000, 12,500 to 15,000, 13,000 to 15,000, 13,500 to 15,000, 14,000 to 15,000, 14,500 to 15,000, 1 500 to 14,500, 2,000 to 14,500, 2,500 to 14,500, 3,000 to 14,500, 3,500 to 14,500, 4,000 to 14,500, 4,500 to 14,500, 5,000 to 14,500, 5,500 to 14,500, 6,000 to 14,500, 6,500 to 14,500, 7,000 to 14,500, 7,500 to 14,500, 8,000 to 14,500, 8,500 to 14,500, 9,000 to 14,500, 9,500 to 14,500, 10,000 to 14,500, 10,500 to 14,500, 1 1,000 to 14,500, 11,500 to 14,500, 12,000 to 14,500, 12,500 to 14,500, 13,000 to 14,500, 13,500 to 14,500, 14,000 to 14,500, 1,500 to 14,000, 2,000 to 14,000, 2, 500 to 14,000, 3,000 to 14,000, 3,500 to 14,000, 4,000 to 14,000, 4,500 to 14,000, 5,000 to 14,000, 5,500 to 14,000, 6,000 to 14,000, 6,500 to 14,000, 7,000 to 14,000 00, 7,500 to 14,000, 8,000 to 14,000, 8,500 to 14,000, 9,000 to 14,000, 9,500 to 14,000, 10,000 to 14,000, 10,500 to 14,000, 11,000 to 14,000, 11,500 to 14,000, 1 2,000 to 14,000, 12,500 to 14,000, 13,000 to 14,000, 13,500 to 14,000, 1,500 to 13,500, 2,000 to 13,500, 2,500 to 13,500, 3,000 to 13,500, 3,500 to 13,500, 4,000 To 13,500, 4,500 to 13,500, 5,000 to 13,500, 5,500 to 13,500, 6,000 to 13,500, 6,500 to 13,500, 7,000 to 13,500, 7,500 to 13,500, 8,000 to 13,500, 8,500 to 13,500 9,000 to 13,500, 9,500 to 13,500, 10,000 to 13,500, 10,500 to 13,500, 11,000 to 13,500, 11,500 to 13,500, 12,000 to 13,500, 12,500 to 13,500, 13,000 to 13,500, 1 500 to 13,000, 2,000 to 13,000, 2,500 to 13,000, 3,000 to 13,000, 3,500 to 13,000, 4,000 to 13,000, 4,500 to 13,000, 5,000 to 13,000, 5,500 to 13,000, 6,000 to 13,000000, 6,500 to 13,000, 7,000 to 13,000, 7,500 to 13,000, 8,000 to 13,000, 8,500 to 13,000, 9,000 to 13,000, 9,500 to 13,000, 10,000 to 13,000, 10,500 to 13,000, 1 1,000 to 13,000, 11,500 to 13,000, 12,000 to 13,000, 12,500 to 13,000, 1,500 to 12,500, 2,000 to 12,500, 2,500 to 12,500, 3,000 to 12,500, 3,500 to 12,500, 4,000 To 12,500, 4,500 to 12,500, 5,000 to 12,500, 5,500 to 12,500, 6,000 to 12,500, 6,500 to 12,500, 7,000 to 12,500, 7,500 to 12,500, 8,000 to 12,500, 8,500 to 12,500 9,000 to 12,500, 9,500 to 12,500, 10,000 to 12,500, 10,500 to 12,500, 11,000 to 12,500, 11,500 to 12,500, 12,000 to 12,500, 1,500 to 12,000, 2,000 to 12,000, 2,5 00 to 12,000, 3,000 to 12,000, 3,500 to 12,000, 4,000 to 12,000, 4,500 to 12,000, 5,000 to 12,000, 5,500 to 12,000, 6,000 to 12,000, 6,500 to 12,000, 7,000 to 12,000 0, 7,500 to 12,000, 8,000 to 12,000, 8,500 to 12,000, 9,000 to 12,000, 9,500 to 12,000, 10,000 to 12,000, 10,500 to 12,000, 11,000 to 12,000, 11,500 to 12,000, 1, 500 to 11,500, 2,000 to 11,500, 2,500 to 11,500, 3,000 to 11,500, 3,500 to 11,500, 4,000 to 11,500, 4,500 to 11,500, 5,000 to 11,500, 5,500 to 11,500, 6,000 to 11,500 00, 6,500 to 11,500, 7,000 to 11,500, 7,500 to 11,500, 8,000 to 11,500, 8,500 to 11,500, 9,000 to 11,500, 9,500 to 11,500, 10,000 to 11,500, 10,500 to 11,500, 11,000 to 11,500, 1,500 to 11,000, 2,000 to 11,000, 2,500 to 11,000, 3,000 to 11,000, 3,500 to 11,000, 4,000 to 11,000, 4,500 to 11,000, 5,000 to 11,000, 5,500 to 11,000 00, 6,000 to 11,000, 6,500 to 11,000, 7,000 to 11,000, 7,500 to 11,000, 8,000 to 11,000, 8,500 to 11,000, 9,000 to 11,000, 9,500 to 11,000, 10,000 to 11,000, 10,5 00 to 11,000, 1,500 to 10,500, 2,000 to 10,500, 2,500 to 10,500, 3,000 to 10,500, 3,500 to 10,500, 4,000 to 10,500, 4,500 to 10,500, 5,000 to 10,500, 5,500 to 10,500 0, 6,000 to 10,500, 6,500 to 10,500, 7,000 to 10,500, 7,500 to 10,500, 8,000 to 10,500, 8,500 to 10,500, 9,000 to 10,500, 9,500 to 10,500, 10,000 to 10,500, 1,500 To 10,000, 2,000 to 10,000, 2,500 to 10,000, 3,000 to 10,000, 3,500 to 10,000, 4,000 to 10,000, 4,500 to 10,000, 5,000 to 10,000, 5,500 to 10,000, 6,000 to 10,000 6,500 to 10,000, 7,000 to 10,000, 7,500 to 10,000, 8,000 to 10,000, 8,500 to 10,000, 9,000 to 10,000, 9,500 to 10,000, 1,500 to 9,500, 2,000 to 9,500, 2,500 to 9,500 0, 3,000 to 9,500, 3,500 to 9,500, 4,000 to 9,500, 4,500 to 9,500, 5,000 to 9,500, 5,500 to 9,500, 6,000 to 9,500, 6,500 to 9,500, 7,000 to 9,500, 7,500 to 9,500, 8,0 00 to 9,500, 8,500 to 9,500, 9,000 to 9,500, 1,500 to 9,000, 2,000 to 9,000, 2,500 to 9,000, 3,000 to 9,000, 3,500 to 9,000, 4,000 to 9,000, 4,500 to 9,000, 5,000 to 9,000, 5,500 to 9,000, 6,000 to 9,000, 6,500 to 9,000, 7,000 to 9,000, 7,500 to 9,000, 8,000 to 9,000, 8,500 to 9,000, 1,500 to 8,500, 2,000 to 8,500, 2,500 to 8,500 3,000 to 8,500, 3,500 to 8,500, 4,000 to 8,500, 4,500 to 8,500, 5,000 to 8,500, 5,500 to 8,500, 6,000 to 8,500, 6,500 to 8,500, 7,000 to 8,500, 7,500 to 8,500, 8,000 To 8,500, 1,500 to 8,000, 2,000 to 8,000, 2,500 to 8,000, 3,000 to 8,000, 3,500 to 8,000, 4,000 to 8,000, 4,500 to 8,000, 5,000 to 8,000, 5,500 to 8,000, 6,000 to 8,000 00, 6,500 to 8,000, 7,000 to 8,000, 7,500 to 8,000, 1,500 to 7,500, 2,000 to 7,500, 2,500 to 7,500, 3,000 to 7,500, 3,500 to 7,500, 4,000 to 7,500, 4,500 to 7,500, 5, 000 to 7,500, 5,500 to 7,500, 6,000 to 7,500, 6,500 to 7,500, 7,000 to 7,500, 1,500 to 7,000, 2,000 to 7,000, 2,500 to 7,000, 3,000 to 7,000, 3,500 to 7,000, 4,000 to 7,000, 4,500 to 7,000, 5,000 to 7,000, 5,500 to 7,000, 6,000 to 7,000, 6,500 to 7,000, 1,500 to 6,500, 2,000 to 6,500, 2,500 to 6,500, 3,000 to 6,500, 3,500 to 6,500 4,000 to 6,500, 4,500 to 6,500, 5,000 to 6,500, 5,500 to 6,500, 6,000 to 6,500, 1,500 to 6,000, 2,000 to 6,000, 2,500 to 6,000, 3,000 to 6,000, 3,500 to 6,000, 4,0 00 to 6,000, 4,500 to 6,000, 5,000 to 6,000, 5,500 to 6,000, 1,500 to 5,500, 2,000 to 5,500, 2,500 to 5,500, 3,000 to 5,500, 3,500 to 5,500, 4,000 to 5,500, 4,500 to 5,500500, 5,000 to 5,500, 1,500 to 5,000, 2,000 to 5,000, 2,500 to 5,000, 3,000 to 5,000, 3,500 to 5,000, 4,000 to 5,000, 4,500 to 5,000, 1,500 to 4,500, 2,000 to 4,500, 2,500 to 4,500, 3,000 to 4,500, 3,500 to 4,500, 4,000 to 4,500, 1,500 Up to 4,000, 2,000 to 4,000, 2,500 to 4,000, 3,000 to 4,000, 3,500 to 4,000, 1,500 to 3,500, 2,000 to 3,500, 2,500 to 3,500, 3,000 to 3,500, 1,500 to 3,000, 2,000 to 3,000, 2,500 to 3,000, 1,500 to 2,500, 2,000 to 2,500, and 1,500 to 2,000 Daltons.

[0232] Hydrophobic components In some embodiments, the lipid nanoparticles may also comprise another type of lipid, referred to herein as a "hydrophobic component." A hydrophobic component is defined as a component incorporated into the lipid bilayer during the formation of the lipid nanoparticles due to its hydrophobicity. In some embodiments, the hydrophobic component may be selected from cardiolipin, squalene, vitamin A and its derivatives, β-carotene, solanine A, and α-tocopherol.

[0233] III. Lipid Nanoparticles Overview Generally, lipid nanoparticles can be characterized as small solid or semi-solid particles having an outer lipid layer with a hydrophilic outer surface exposed to a non-lipid nanoparticle environment, an internal space that can be aqueous (vesicle-like) or non-aqueous (micelle-like), and at least one hydrophobic intermembrane space. The lipid nanoparticle membrane can be layered or non-layered and can contain 1, 2, 3, 4, 5, or more layers. In some embodiments, the lipid nanoparticles may contain cargo or payload located within the internal space of the lipid nanoparticle, in its intermembrane space, on its outer surface, or any combination thereof.

[0234] This disclosure also provides lipid nanoparticles containing cargo or payload. As used herein, the terms "cargo" or "payload" can refer to one or more molecules or structures contained in lipid nanoparticles for delivery to or into cells or tissues. Non-limiting examples of cargo may include nucleic acids, polypeptides, peptides, proteins, liposomes, markers, tags, small chemical molecules, large biomolecules, and any combination or fragment thereof. In originator constructs and benchmark constructs, the construct region containing or encoding cargo or payload is referred to as the "cargo region" or "payload region".

[0235] In some embodiments, the lipid nanoparticles comprise at least one ionizable lipid of the present disclosure discussed in Part II, such as ionizable lipids of formulas (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b), or (I1-6c), or more specifically, the compounds in Table 1. Without wishing to be bound by any theory, the diester bonds of lipids provide biodegradability; these ester bonds are stable at physiological pH but can be enzymatically hydrolyzed within tissues and cells. The lengths of the R3, R4, R5, and R6 groups in formulas (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b), or (I1-6c) can also be adjusted to achieve the desired zeta potential, particle size, or film rigidity.

[0236] In some embodiments, the lipid nanoparticles may comprise any lipid described in this disclosure. In some embodiments, the lipid may be any cationic lipid described in this disclosure. In some embodiments, the lipid nanoparticles may comprise neutral lipids. In some embodiments, the neutral lipid may be a phospholipid or a derivative thereof. In some embodiments, the lipid may be any phospholipid described in this disclosure. In some embodiments, the lipid may be any cholesterol derivative described in this disclosure. In some embodiments, the polymer (e.g., PEG) may be conjugated to at least one lipid. In some embodiments, PEG-lipids may be used in the lipid nanoparticles and may be any PEG-lipid conjugate described in this disclosure.

[0237] In some embodiments, lipid nanoparticles can be characterized by their shape. In some embodiments, lipid nanoparticles may include substantially spherical lipid nanoparticles. In some embodiments, lipid nanoparticles may include substantially rod-shaped (i.e., cylindrical) lipid nanoparticles. In some embodiments, lipid nanoparticles may include substantially disc-shaped lipid nanoparticles. In some embodiments, lipid nanoparticles may include mixtures of such different shapes of lipid nanoparticles.

[0238] In some implementations, as used herein, the term "nanoparticle" refers to any particle with a size ranging from 10 to 1000 nm. Nanoparticles can be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265 nm. 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000 nm.

[0239] In some embodiments, a population of lipid nanoparticles, such as a population of lipid nanoparticles produced from the same formulation, can be characterized by measuring the uniformity of the size, shape, or mass of the particles within the population. In some embodiments, uniformity can be expressed as the polydispersity index (PI) of the population. In some embodiments, uniformity can be expressed as the variability (Đ) of the population. The terms “polydispersity index” and “variability” are understood herein to be equivalent and can be used interchangeably. In some embodiments, a population of lipid nanoparticles produced from a given formulation may have a PI of about 0.1 to 1. In some embodiments, a population of lipid nanoparticles produced from a given formulation may have a PI of less than about 1, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, or less than about 0.1. In some embodiments, the population of lipid nanoparticles generated from a given formulation may have a PI of about 0.1 to 1, 0.1 to 0.8, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.2 to 1, 0.2 to 0.8, 0.2 to 0.6, 0.2 to 0.4, 0.4 to 1, 0.4 to 0.8, 0.4 to 0.6, 0.6 to 1, 0.6 to 0.8, and 0.8 to 1.In some embodiments, the lipid nanoparticles may have a PI range of approximately 0.01 to 0.3, 0.02-0.3, 0.03-0.3, 0.04-0.3, 0.05-0.3, 0.06-0.3, 0.07-0.3, 0.08-0.3, 0.09-0.3, 0.1-0.3, 0.11-0.3, 0.12-0.3, 0.13-0.3, 0.14-0.3, 0.15-0.3, 0.16-0.3, 0.17-0.3, 0.18-0.3, 0.19-0.3, 0.2-0.3, 0.21-0.3, 0.22-0.3, 0.23-0.3, 0.24-0.3. 3, 0.25-0.3, 0.26-0.3, 0.27-0.3, 0.28-0.3, 0.29-0.3, 0.01 to 0.25, 0.02-0.25, 0.03-0.25, 0.04-0.25, 0.05-0.25, 0.06-0.25, 0.07-0.25, 0.08-0.25, 0.09-0.25, 0.1-0.25, 0.11-0.25, 0.12-0.25, 0.13-0.25, 0.14-0.25, 0.15-0.25, 0.16-0.25, 0.17-0.25, 0.18-0.25, 0.19-0.25, 0.2 -0.25, 0.21-0.25, 0.22-0.25, 0.23-0.25, 0.24-0.25, 0.01 to 0.2, 0.02-0.2, 0.03-0.2, 0.04-0.2, 0.05-0.2, 0.06-0.2, 0.07-0.2, 0.08-0.2, 0.09-0.2, 0.1-0.2, 0.11-0.2, 0.12-0.2, 0.13-0.2, 0.14-0.2, 0.15-0.2, 0.16-0.2, 0.17-0.2, 0.18-0.2, 0.19-0.2, 0.01 to 0.15, 0.02-0.15, 0.0 3-0.15, 0.04-0.15, 0.05-0.15, 0.06-0.15, 0.07-0.15, 0.08-0.15, 0.09-0.15, 0.1-0.15, 0.11-0.15, 0.12-0.15, 0.13-0.15, 0.14-0.15, 0.01 to 0.1, 0.02-0.1, 0.03-0.1, 0.04-0.1, 0.05-0.1, 0.06-0.1, 0.07-0.1, 0.08-0.1, 0.09-0.1, 0.01 to 0.05, 0.02-0.05, 0.03-0.05 or 0.04-0.05.

[0240] In some embodiments, the total molar percentage of lipids in the LNP is from about 10% to about 95%, for example, from about 10% to about 20%, from about 21% to about 30%, from about 31% to about 40%, from about 41% to about 50%, from about 51% to about 60%, from about 61% to about 70%, from about 71% to about 80%, from about 81% to about 90%, or from about 91% to about 95%.

[0241] preparation In some embodiments, lipids of this disclosure (which include at least the ionizable lipids disclosed herein) may be incorporated into lipid nanoparticles (LNPs). In some embodiments, the lipid nanoparticles may comprise at least one ionizable lipid, at least one neutral lipid, at least one cofactor lipid, at least one polymer-conjugated lipid, or any combination thereof, wherein the neutral lipid, cofactor lipid, and polymer-conjugated lipid are as defined herein. In some embodiments, the LNP may comprise at least one ionizable lipid, at least one neutral lipid, and at least one cofactor lipid. In some embodiments, the LNP may comprise at least one ionizable lipid, at least one neutral lipid, and at least one polymer-conjugated lipid. In some embodiments, the LNP may comprise at least one neutral lipid, at least one cofactor lipid, and at least one polymer-conjugated lipid. In some embodiments, the LNP may comprise at least one ionizable lipid and at least one neutral lipid. In some embodiments, the LNP may comprise at least one ionizable lipid and at least one cofactor lipid. In some embodiments, the LNP may comprise at least one ionizable lipid and at least one polymer-conjugated lipid. In some embodiments, the LNP may comprise at least one neutral lipid and at least one cofactor lipid. In some embodiments, the LNP may comprise at least one neutral lipid and at least one polymer-conjugated lipid. In some embodiments, the LNP may comprise at least one auxiliary lipid and at least one polymer-conjugated lipid. In some embodiments, the LNP may comprise at least one ionizable lipid. In some embodiments, the LNP may comprise at least one neutral lipid. In some embodiments, the LNP may comprise at least one auxiliary lipid. In some embodiments, the LNP may comprise at least one polymer-conjugated lipid. In some embodiments, the lipid nanoparticle may comprise at least one ionizable lipid, at least one neutral lipid, at least one auxiliary lipid, at least one polymer-conjugated lipid, and at least one hydrophobic component, wherein the ionizable lipid, neutral lipid, auxiliary lipid, polymer-conjugated lipid, and hydrophobic component are as defined herein.

[0242] In some implementations, lipid nanoparticles may also contain adjuvants, cell-targeting components, fat-soluble vitamins, immunomodulatory substances, or components that promote drug absorption.

[0243] In some implementations, the adjuvant may be squalene, the cell-targeting component may be cardiolipin, the fat-soluble vitamin may be vitamin A or E, the immunomodulatory substance may be solanine, and the component that promotes drug absorption may be caffeine.

[0244] In some embodiments, the total molar percentage of ionizable lipids in the lipid nanoparticles, such as lipids having the structure of formula (I), or lipids having the structure of formula (I1), (I2), (I3), or (I4), or lipids having the structure of formula (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d), or (I4e), or lipids having the structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b), or (I1-6c), or compounds in Table 1, may be 0.1%. mol% to 100 mol%. In some embodiments, the lipid nanoparticles contain ionizable lipids, such as lipids having the structure of formula (I), or lipids having the structure of formula (I1), (I2), (I3) or (I4), or lipids having the structure of formula (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or lipids having the structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1... The lipid nanoparticles, or the compounds in Table 1, have a total molar percentage of 10%-95%, for example, about 10% to about 20%, about 21% to about 30%, about 31% to about 40%, about 41% to about 50%, about 51% to about 60%, about 61% to about 70%, about 71% to about 80%, about 81% to about 90%, or about 91% to about 95%. In some embodiments, the lipid nanoparticles contain about 40 mol% to 100 mol% of at least one ionizable lipid. In some embodiments, the lipid nanoparticles contain about 20 mol% to 60 mol% of at least one ionizable lipid. In some embodiments, the lipid nanoparticles comprise about 50 mol% to 85 mol% of at least one ionizable lipid. In some embodiments, the lipid nanoparticles comprise less than about 20 mol% of at least one ionizable lipid. In some embodiments, the lipid nanoparticles comprise more than about 50 mol% or about 70 mol% of at least one ionizable lipid. In some embodiments, the lipid nanoparticles comprise about 95 mol% or less of at least one ionizable lipid.In some embodiments, the lipid nanoparticles comprise amounts of less than or equal to about 95 mol%, 90 mol%, 85 mol%, 80 mol%, 75 mol%, 70 mol%, 65 mol%, 60 mol%, 55 mol%, 50 mol%, 45 mol%, 40 mol%, 35 mol%, 30 mol%, 25 mol%, 20 mol%, 15 mol%, 10 mol%, and 5 mol%. In some embodiments, the lipid nanoparticles comprise amounts of more than or equal to about 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, 70 mol%, 75 mol%, 80 mol%, 85 mol%, 90 mol%, and 95 mol%.

[0245] In some embodiments, the lipid nanoparticles contain ionizable lipids, such as lipids having the structure of formula (I), or lipids having the structure of formula (I1), (I2), (I3), or (I4), or lipids having the structure of formula (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d), or (I4e), or lipids having the structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5). Lipids with structures of (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b), or (I1-6c), or compounds in Table 1, having a total molar percentage of about 30% to 100%, about 40% to 100%, about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, or about 30% to about 60%.

[0246] In some embodiments, the lipid nanoparticles contain ionizable lipids, such as lipids having the structure of formula (I), or lipids having the structure of formula (I1), (I2), (I3), or (I4), or lipids having the structure of formula (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d), or (I4e), or lipids having the structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I4c), (I4d), or (I4e). Lipids of the structures 1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or compounds in Table 1, having a total molar percentage of about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, or about 40% to about 60%.

[0247] In some embodiments, the lipid nanoparticles contain ionizable lipids, such as lipids having the structure of formula (I), or lipids having the structure of formula (I1), (I2), (I3), or (I4), or lipids having the structure of formula (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d), or (I4e), or lipids having the structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I4c), (I4d), or (I4e). Lipids of the structures 1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or compounds in Table 1, having a total molar percentage of about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, or about 50% to about 60%.

[0248] In some embodiments, the lipid nanoparticles contain ionizable lipids, such as lipids having the structure of formula (I), or lipids having the structure of formula (I1), (I2), (I3), or (I4), or lipids having the structure of formula (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d), or (I4e), or lipids having the structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I4c), (I4d), or (I4e). Lipids of the structures 1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or compounds in Table 1, having a total molar percentage of about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, or about 55% to about 60%.

[0249] In some embodiments, the lipid nanoparticles contain ionizable lipids, such as lipids having the structure of formula (I), or lipids having the structure of formula (I1), (I2), (I3), or (I4), or lipids having the structure of formula (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d), or (I4e), or lipids having the structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1- 3) lipids with the structure of (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b), or (I1-6c), or compounds in Table 1, having a total molar percentage of about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 75%, about 60% to about 70%, or about 60% to about 65%.

[0250] In some embodiments, the lipid nanoparticles contain ionizable lipids, such as lipids having the structure of formula (I), or lipids having the structure of formula (I1), (I2), (I3), or (I4), or lipids having the structure of formula (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d), or (I4e), or lipids having the structure of formula (I), (I1), (I2), (I3), (I1-1), or (I1-2). Lipids with structures of (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or compounds in Table 1, having a total molar percentage of about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, or about 65% to about 70%.

[0251] In some embodiments, the lipid nanoparticles contain ionizable lipids, such as lipids having the structure of formula (I), or lipids having the structure of formula (I1), (I2), (I3), or (I4), or lipids having the structure of formula (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d), or (I4e), or lipids having the structure of formula (I), (I1), (I2), (I3), (I1-1), (I4b), (I4c), (I4d), or (I4e). Lipids with structures of I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b), or (I1-6c), or compounds in Table 1, having a total molar percentage of about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, or about 70% to about 75%.

[0252] In some embodiments, the lipid nanoparticles contain ionizable lipids, such as lipids having the structure of formula (I), or lipids having the structure of formula (I1), (I2), (I3), or (I4), or lipids having the structure of formula (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d), or (I4e), or lipids having the structure of formula (I), (I1), (I2), (I3), (I4c), (I4d), or (I4e). Lipids with structures of (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b), or (I1-6c), or compounds in Table 1, having a total molar percentage of about 75% to about 90%, about 75% to about 85%, or about 75% to about 80%.

[0253] In some embodiments, the lipid nanoparticles contain ionizable lipids, such as lipids having the structure of formula (I), or lipids having the structure of formula (I1), (I2), (I3), or (I4), or lipids having the structure of formula (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d), or (I4e), or lipids having the structure of formula (I), (I1), (I2), (I3), (I4c), (I4d), or (I4e). The total molar percentage of lipids with the structures of (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b), or (I1-6c), or the compounds in Table 1, is about 80% to about 90% or about 80% to about 85%.

[0254] In some embodiments, the total molar percentage of ionizable lipids in the lipid nanoparticles, such as lipids having the structure of formula (I), or lipids having the structure of formula (I1), (I2), (I3) or (I4), or lipids having the structure of formula (I1a), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or lipids having the structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or the compounds in Table 1, is about 85% to about 90%.

[0255] In some embodiments, the molar percentage of ionizable lipids in the lipid nanoparticles can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target molar percentage.

[0256] In some implementations, the variability of the transfer vehicle between batches can be less than 15%, less than 10%, or less than 5%.

[0257] In some embodiments, when the lipid nanoparticles contain at least one neutral lipid, the neutral lipid may be present in the lipid nanoparticles in an amount from about 0.1 mol% to 100 mol%. In some embodiments, the lipid nanoparticles contain at least one neutral lipid in an amount of up to 35 mol%, or up to 30 mol%, or up to 25 mol%, or up to 20 mol%, or about 0.1 mol% to about 35 mol%, or about 5 mol% to about 35 mol%, or about 0.1 mol% to about 20 mol%. In some embodiments, the lipid nanoparticles contain at least one neutral lipid in an amount of about 5 mol% to 25 mol%. In some embodiments, the lipid nanoparticles contain less than about 5 mol%. In some embodiments, the lipid nanoparticles contain more than about 25 mol% or about 35 mol%. In some embodiments, the lipid nanoparticles contain at least one neutral lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticles comprise at least one neutral lipid in amounts of about 95 mol%, 90 mol%, 85 mol%, 80 mol%, 75 mol%, 70 mol%, 65 mol%, 60 mol%, 55 mol%, 50 mol%, 45 mol%, 40 mol%, 35 mol%, 30 mol%, 25 mol%, 20 mol%, 15 mol%, 10 mol%, and 5 mol%. In some embodiments, the lipid nanoparticles comprise at most about 10 mol% of at least one neutral lipid.

[0258] In some embodiments, the total molar percentage of neutral lipids in the lipid nanoparticles is about 5% to about 20%, about 5% to about 18%, about 5% to about 16%, about 5% to about 14%, about 5% to about 12%, about 5% to about 10%, or about 5% to about 8%.

[0259] In some embodiments, the total molar percentage of neutral lipids in the lipid nanoparticles is about 7% to about 20%, about 7% to about 18%, about 7% to about 16%, about 7% to about 14%, about 7% to about 12%, about 7% to about 10%, or about 7% to about 8%.

[0260] In some embodiments, the total molar percentage of neutral lipids in the lipid nanoparticles is about 9% to about 20%, about 9% to about 18%, about 9% to about 16%, about 9% to about 14%, about 9% to about 12%, or about 9% to about 10%.

[0261] In some embodiments, the total molar percentage of neutral lipids in the lipid nanoparticles is about 11% to about 20%, about 11% to about 18%, about 11% to about 16%, about 11% to about 14%, or about 11% to about 12%.

[0262] In some embodiments, the total molar percentage of neutral lipids in the lipid nanoparticles is about 13% to about 20%, about 13% to about 18%, about 13% to about 16%, or about 13% to about 14%.

[0263] In some embodiments, the total molar percentage of neutral lipids in the lipid nanoparticles is about 15% to about 20% or about 17% to about 18%.

[0264] In some embodiments, the neutral lipid molar percentage of the lipid nanoparticles may be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target molar percentage.

[0265] In some embodiments, when the lipid nanoparticles contain at least one auxiliary lipid, the auxiliary lipid may be present in the lipid nanoparticles in an amount from about 0.1 mol% to 100 mol%. In some embodiments, the lipid nanoparticles contain up to about 50 mol% of at least one auxiliary lipid. In some embodiments, the lipid nanoparticles contain about 20 mol% to 45 mol% of at least one auxiliary lipid. In some embodiments, the lipid nanoparticles contain about 25 mol% to 55 mol% of at least one auxiliary lipid. In some embodiments, the lipid nanoparticles contain less than about 20 mol% of at least one auxiliary lipid. In some embodiments, the lipid nanoparticles contain more than about 45 mol% or about 55 mol% of at least one auxiliary lipid. In some embodiments, the lipid nanoparticles contain about 95 mol% or less of at least one auxiliary lipid. In some embodiments, the lipid nanoparticles comprise at least one auxiliary lipid in amounts less than or equal to about 95 mol%, 90 mol%, 85 mol%, 80 mol%, 75 mol%, 70 mol%, 65 mol%, 60 mol%, 55 mol%, 50 mol%, 45 mol%, 40 mol%, 35 mol%, 30 mol%, 25 mol%, 20 mol%, 15 mol%, 10 mol%, 5 mol%, and 1 mol%. In some embodiments, the lipid nanoparticles comprise at least one auxiliary lipid in amounts greater than or equal to about 1 mol%, 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, 70 mol%, 75 mol%, 80 mol%, 85 mol%, 90 mol%, and 95 mol%.

[0266] In some embodiments, the molar percentage of the auxiliary lipids in the lipid nanoparticles can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target molar percentage.

[0267] In some embodiments, the total molar percentage of auxiliary lipids in the lipid nanoparticles is from about 1% to about 50%, for example, from about 1% to about 45%, from about 1% to about 40%, from about 1% to about 35%, from about 1% to about 30%, from about 1% to about 25%, from about 1% to about 20%, from about 1% to about 15%, from about 1% to about 10%, or from about 1% to about 5%.

[0268] In some embodiments, the total molar percentage of assisting lipids in the lipid nanoparticles is about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, or about 5% to about 10%.

[0269] In some embodiments, the total molar percentage of assisting lipids in the lipid nanoparticles is about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, or about 10% to about 15%.

[0270] In some embodiments, the total molar percentage of assisting lipids in the lipid nanoparticles is about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, or about 15% to about 20%.

[0271] In some embodiments, the total molar percentage of auxiliary lipids in the lipid nanoparticles is about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, or about 20% to about 25%.

[0272] In some embodiments, the total molar percentage of auxiliary lipids in the lipid nanoparticles is about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, or about 25% to about 30%.

[0273] In some embodiments, the total molar percentage of auxiliary lipids in the lipid nanoparticles is about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, or about 30% to about 35%.

[0274] In some embodiments, the total molar percentage of accessory lipids in the lipid nanoparticles is about 35% to about 50%, about 13% to about 45%, or about 35% to about 40%. In some embodiments, the total molar percentage of accessory lipids in the lipid nanoparticles is about 40% to about 50%, or about 40% to about 45%. In some embodiments, the total molar percentage of accessory lipids in the lipid nanoparticles is about 45% to about 50%.

[0275] In some embodiments, when the lipid nanoparticles comprise at least one polymer-conjugated lipid, the polymer-conjugated lipid may be present in the lipid nanoparticles in an amount from about 0.1 mol% to 100 mol%. In some embodiments, the lipid nanoparticles comprise about 95 mol% or less of at least one polymer-conjugated lipid. In some embodiments, the lipid nanoparticles comprise up to 15 mol% or about 0.5 mol% to 15 mol% of at least one polymer-conjugated lipid. In some embodiments, the lipid nanoparticles comprise about 15 mol% to 40 mol% of at least one polymer-conjugated lipid. In some embodiments, the lipid nanoparticles comprise less than about 0.1 mol% of at least one polymer-conjugated lipid. In some embodiments, the lipid nanoparticles comprise up to about 5 mol% of at least one polymer-conjugated lipid. In some embodiments, the lipid nanoparticles comprise at least one polymer-conjugated lipid in amounts less than or equal to about 95 mol%, 90 mol%, 85 mol%, 80 mol%, 75 mol%, 70 mol%, 65 mol%, 60 mol%, 55 mol%, 50 mol%, 45 mol%, 40 mol%, 35 mol%, 30 mol%, 25 mol%, 20 mol%, 15 mol%, 10 mol%, and 5 mol%. In some embodiments, the lipid nanoparticles comprise at least one polymer-conjugated lipid in amounts greater than or equal to about 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, 70 mol%, 75 mol%, 80 mol%, 85 mol%, 90 mol%, and 95 mol%.

[0276] In some embodiments, the molar percentage of the polymer-conjugated lipids in the lipid nanoparticles can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target molar percentage.

[0277] In some embodiments, the total molar percentage of polymer-conjugated lipids in the lipid nanoparticles is from about 0.1% to about 10%, for example, from about 0.1% to about 9%, from about 0.1% to about 8%, from about 0.1% to about 7%, from about 0.1% to about 6%, from about 0.1% to about 5%, or from about 0.1% to about 4%, from about 0.1% to about 3%, from about 0.1% to about 2%, or from about 0.1% to about 1%.

[0278] In some embodiments, the total molar percentage of polymer-conjugated lipids in the lipid nanoparticles is from about 0.5% to about 10%, for example, from about 0.5% to about 9%, from about 0.5% to about 8%, from about 0.5% to about 7%, from about 0.5% to about 6%, from about 0.5% to about 5%, or from about 0.5% to about 4%, from about 0.5% to about 3%, from about 0.5% to about 2%, or from about 0.5% to about 1%.

[0279] In some embodiments, the total molar percentage of polymer-conjugated lipids in the lipid nanoparticles is about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, or about 1% to about 2%.

[0280] In some embodiments, the total molar percentage of polymer-conjugated lipids in the lipid nanoparticles is about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, or about 2% to about 3%.

[0281] In some embodiments, the total molar percentage of the polymer-conjugated lipids in the lipid nanoparticles is about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, or about 3% to about 4%.

[0282] In some embodiments, the total molar percentage of polymer-conjugated lipids in the lipid nanoparticles is about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, or about 4% to about 5%.

[0283] In some embodiments, the total molar percentage of polymer-conjugated lipids in the lipid nanoparticles is about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 5% to about 7%, or about 5% to about 6%.

[0284] In some embodiments, the total molar percentage of polymer-conjugated lipids in the lipid nanoparticles is about 6% to about 10%, about 6% to about 9%, about 6% to about 8%, or about 6% to about 7%. In some embodiments, the total molar percentage of polymer-conjugated lipids in the lipid nanoparticles is about 7% to about 10%, about 7% to about 9%, or about 7% to about 8%. In some embodiments, the total molar percentage of polymer-conjugated lipids in the lipid nanoparticles is about 8% to about 10%, or about 8% to about 9%. In some embodiments, the total molar percentage of PEG-lipids in the lipid nanoparticles is about 9% to about 10%.

[0285] In some embodiments, the lipid nanoparticles may comprise a hydrophobic component. In some embodiments, the total molar percentage of the hydrophobic component in the lipid nanoparticles is at most about 20%, or at most about 15%, or at most about 10%, or about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 5%. In some embodiments, the total molar percentage of the hydrophobic component in the lipid nanoparticles is about 3% to about 20%, about 3% to about 15%, about 3% to about 10%, or about 3% to about 5%. In some embodiments, the total molar percentage of the hydrophobic component in the lipid nanoparticles is at most about 5%.

[0286] In some embodiments, the total molar percentage of the hydrophobic component in the lipid nanoparticles is about 5% to about 20%, about 5% to about 15%, or about 5% to about 10%.

[0287] In some embodiments, the total molar percentage of the hydrophobic component in the lipid nanoparticles is about 7% to about 20%, about 7% to about 15%, or about 7% to about 10%.

[0288] In some embodiments, the total molar percentage of the hydrophobic component in the lipid nanoparticles is about 9% to about 20%, about 9% to about 15%, or about 9% to about 10%.

[0289] In some embodiments, the total molar percentage of the hydrophobic component in the lipid nanoparticles is about 11% to about 20% or about 11% to about 15%.

[0290] In some embodiments, the total molar percentage of the hydrophobic component in the lipid nanoparticles is about 13% to about 20% or about 13% to about 15%.

[0291] In some embodiments, the total molar percentage of the hydrophobic component in the lipid nanoparticles is from about 15% to about 20% or from about 15% to about 20%.

[0292] In some embodiments, the total molar percentage of the hydrophobic component in the lipid nanoparticles is from about 17% to about 20% or from about 17% to about 20%.

[0293] In some embodiments, the total molar percentage of the hydrophobic component in the lipid nanoparticles is from about 19% to about 20% or from about 19% to about 20%.

[0294] In some embodiments, the lipid nanoparticles comprise about 30-85 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., phospholipid), about 1-50 mol% of at least one accessory lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid).

[0295] In some embodiments, at least one ionizable lipid is at least one compound having a structure of formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d), or (I4e) as defined herein, or a structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b), or (I1-6c), or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof.

[0296] In some embodiments, the lipid nanoparticles comprise about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., phospholipid), about 30-40 mol% of at least one accessory lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid).

[0297] In some embodiments, the lipid nanoparticles comprise about 35-45 mol% of at least one ionizable lipid, about 25-35 mol% of at least one neutral lipid (e.g., phospholipid), about 20-30 mol% of at least one accessory lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid).

[0298] In some embodiments, the lipid nanoparticles comprise about 45-65 mol% of at least one ionizable lipid, about 5-10 mol% of at least one neutral lipid (e.g., phospholipid), about 25-40 mol% of at least one accessory lipid (e.g., cholesterol), and about 0.5-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid).

[0299] In some embodiments, the lipid nanoparticles comprise about 40-60 mol% of at least one ionizable lipid, about 5-15 mol% of at least one neutral lipid (e.g., phospholipid), about 35-45 mol% of at least one accessory lipid (e.g., cholesterol), and about 0.5-3 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid).

[0300] In some embodiments, the lipid nanoparticles comprise about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., phospholipid), about 15-50 mol% of at least one accessory lipid (e.g., cholesterol), and about 0.01-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid).

[0301] In some embodiments, the lipid nanoparticles comprise about 10-75 mol% of at least one ionizable lipid, about 0.5-50 mol% of at least one neutral lipid (e.g., phospholipid), about 5-60 mol% of at least one accessory lipid (e.g., cholesterol), and about 0.1-20 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid).

[0302] In some embodiments, the lipid nanoparticles comprise about 50-65 mol% of at least one ionizable lipid, about 3-15 mol% of at least one neutral lipid (e.g., phospholipid), about 30-40 mol% of at least one accessory lipid (e.g., cholesterol), and about 0.5-2 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid).

[0303] In some embodiments, the lipid nanoparticles comprise about 50-85 mol% of at least one ionizable lipid, about 3-15 mol% of at least one neutral lipid (e.g., phospholipid), about 1-40 mol% of at least one accessory lipid (e.g., cholesterol), and about 0.5-2 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid).

[0304] In some embodiments, the lipid nanoparticles comprise about 25-75 mol% of at least one ionizable lipid, about 0.1-15 mol% of at least one neutral lipid (e.g., phospholipid), about 5-50 mol% of at least one accessory lipid (e.g., cholesterol), and about 0.5-20 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid).

[0305] In some embodiments, the lipid nanoparticles comprise about 50-65 mol% of at least one ionizable lipid, about 5-10 mol% of at least one neutral lipid (e.g., phospholipid), about 25-35 mol% of at least one accessory lipid (e.g., cholesterol), and about 5-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid).

[0306] In some embodiments, the lipid nanoparticles comprise about 20-60 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., phospholipid), about 25-55 mol% of at least one accessory lipid (e.g., cholesterol), and about 0.5-15 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid).

[0307] In some embodiments, the lipid nanoparticles comprise about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., phospholipid), about 18.5-48.5 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 0-20 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0308] In some embodiments, the lipid nanoparticles comprise about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., phospholipid), about 18.5-48.5 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0309] In some embodiments, the lipid nanoparticles comprise about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., phospholipid), about 18.5-48.5 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 0-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0310] In some embodiments, the lipid nanoparticles comprise about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., phospholipid), about 18.5-48.5 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0311] In some embodiments, the lipid nanoparticles comprise about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., phospholipid), about 30-45 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 0-20 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0312] In some embodiments, the lipid nanoparticles comprise about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., phospholipid), about 30-45 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0313] In some embodiments, the lipid nanoparticles comprise about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., phospholipid), about 30-45 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 0-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0314] In some embodiments, the lipid nanoparticles comprise about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., phospholipid), about 30-45 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0315] In some embodiments, the lipid nanoparticles comprise about 50-85 mol% of at least one ionizable lipid, about 5-20 mol% of at least one neutral lipid (e.g., phospholipid), about 1-40 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 0-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0316] In some embodiments, the lipid nanoparticles comprise about 50-85 mol% of at least one ionizable lipid, about 5-20 mol% of at least one neutral lipid (e.g., phospholipid), about 1-40 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 0.1-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0317] In some embodiments, the lipid nanoparticles comprise about 50-85 mol% of at least one ionizable lipid, about 5-20 mol% of at least one neutral lipid (e.g., phospholipid), about 1-40 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 0.1-5 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0318] In some embodiments, the lipid nanoparticles comprise about 50-85 mol% of at least one ionizable lipid, about 5-20 mol% of at least one neutral lipid (e.g., phospholipid), about 1-40 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, β-carotene, solanine A, and / or α-tocopherol), wherein the at least one ionizable lipid has a structure or formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d), or (I4e) as defined herein. At least one compound having the structure of (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b), or (I1-6c), or at least one compound listed in Table 1 herein, or a pharmaceutically acceptable salt thereof.

[0319] In some embodiments, the lipid nanoparticles comprise about 50-85 mol% of at least one ionizable lipid, about 5-20 mol% of at least one neutral lipid (e.g., phospholipid), about 1-40 mol% of at least one accessory lipid (e.g., cholesterol), about 1-5 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 1-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, β-carotene, solanine A, and / or α-tocopherol), wherein the at least one ionizable lipid has a structure or formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d), or (I4e) as defined herein. At least one compound having the structure of (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b), or (I1-6c), or at least one compound listed in Table 1 herein, or a pharmaceutically acceptable salt thereof.

[0320] In some embodiments, the lipid nanoparticles comprise about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., phospholipid), about 30-45 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, β-carotene, solanine A, and / or α-tocopherol), wherein the at least one ionizable lipid has a structure or formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d), or (I4e) as defined herein. At least one compound having the structure of (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b), or (I1-6c), or at least one compound listed in Table 1 herein, or a pharmaceutically acceptable salt thereof.

[0321] In some embodiments, the lipid nanoparticles comprise about 40-60 mol% of at least one ionizable lipid, about 5-10 mol% of at least one phospholipid or phospholipid derivative described herein, about 30-45 mol% of at least one cholesterol or cholesterol derivative described herein, about 1-4 mol% of at least one PEG-lipid described herein, and about 0-5 mol% of squalene, cardiolipin, solanine A, vitamin A, retinol, β-carotene, and / or α-tocopherol, wherein the at least one ionizable lipid has a structure or formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d), or (I4e) as defined herein. At least one compound having the structure of (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or at least one compound listed in Table 1 herein, or a pharmaceutically acceptable salt thereof.

[0322] In some embodiments, the lipid nanoparticles comprise about 40-60 mol% of at least one ionizable lipid, about 5-10 mol% of DSPC, about 30-45 mol% of cholesterol, about 1-4 mol% of PEG-DMG, and about 0-5 mol% of squalene, cardiolipin, vitamin A, retinol, β-carotene, solanine A, and / or α-tocopherol, wherein the at least one ionizable lipid has a structure or formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d), or (I4e) as defined herein. At least one compound having the structure of (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or at least one compound listed in Table 1 herein, or a pharmaceutically acceptable salt thereof.

[0323] In some embodiments, the lipid nanoparticles comprise about 50-85 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-20 mol% of at least one neutral lipid (e.g., phospholipid), about 1-40 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0324] In some embodiments, the lipid nanoparticles comprise about 50-85 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-20 mol% of at least one neutral lipid (e.g., phospholipid), about 1-40 mol% of at least one accessory lipid (e.g., cholesterol), about 1-5 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 1-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0325] In some embodiments, the lipid nanoparticles comprise about 35-55 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-25 mol% of at least one neutral lipid (e.g., phospholipid), about 30-45 mol% of at least one accessory lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., PEG-lipid), and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol).

[0326] In some embodiments, the lipid nanoparticles comprise about 40-60 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-10 mol% of at least one phospholipid or phospholipid derivative described herein, about 30-45 mol% of at least one cholesterol or cholesterol derivative described herein, about 1-4 mol% of at least one PEG-lipid described herein, and about 0-5 mol% of squalene, cardiolipin, solanine A, vitamin A, retinol, β-carotene, and / or α-tocopherol.

[0327] In some embodiments, the lipid nanoparticles comprise about 40-60 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-10 mol% of DSPC, about 30-45 mol% of cholesterol, about 1-4 mol% of PEG-DMG, and about 0-5 mol% of squalene, cardiolipin, vitamin A, retinol, beta-carotene, solanine A, and / or α-tocopherol.

[0328] In some embodiments, the lipid nanoparticles may comprise about 40 mol% to about 100 mol% of ionizable lipids, 0 to about 10 mol% of neutral lipids, 0 to about 50 mol% of auxiliary lipids, 0 to about 5 mol% of polymer-conjugated lipids, and 0 to about 5 mol% of other lipids. The hydrophobic component is mol% of the ionizable lipid having a structure of formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d) or (I4e) as defined herein, or at least one compound having a structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, wherein mol% is based on the total lipids present in the nanoparticles.

[0329] In some embodiments, the lipid nanoparticles may comprise about 40 mol% to about 99 mol% of ionizable lipids, about 1 mol% to about 10 mol% of neutral lipids, 0 to about 50 mol% of accessory lipids, 0 to about 5 mol% of polymer-conjugated lipids, and 0 to about 5 mol% of other lipids. The hydrophobic component is mol% of the ionizable lipid having a structure of formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d) or (I4e) as defined herein, or at least one compound having a structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, wherein mol% is based on the total lipids present in the nanoparticles.

[0330] In some embodiments, the lipid nanoparticles may comprise about 40 mol% to about 60 mol% of ionizable lipids, about 1 mol% to about 10 mol% of neutral lipids, about 1 mol% to about 50 mol% of accessory lipids, 0 to about 5 mol% of polymer-conjugated lipids, and 0 to about 5 mol% of other lipids. The hydrophobic component is mol% of the ionizable lipid having a structure of formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d) or (I4e) as defined herein, or at least one compound having a structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, wherein mol% is based on the total lipids present in the nanoparticles.

[0331] In some embodiments, the lipid nanoparticles may comprise about 40 mol% to about 85 mol% of ionizable lipids, about 1 mol% to about 10 mol% of neutral lipids, about 1 mol% to about 50 mol% of accessory lipids, 0 to about 5 mol% of polymer-conjugated lipids, and 0 to about 5 mol% of other lipids. The hydrophobic component is mol% of the ionizable lipid having a structure of formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d) or (I4e) as defined herein, or at least one compound having a structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, wherein mol% is based on the total lipids present in the nanoparticles.

[0332] In some embodiments, the lipid nanoparticles may comprise about 40 mol% to about 85 mol% of ionizable lipids, about 1 mol% to about 10 mol% of neutral lipids, about 1 mol% to about 50 mol% of accessory lipids, about 1 mol% to about 5 mol% of polymer-conjugated lipids, and 0 to about 5 mol% of other components. The hydrophobic component is mol% of the ionizable lipid having a structure of formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d) or (I4e) as defined herein, or at least one compound having a structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, wherein mol% is based on the total lipids present in the nanoparticles.

[0333] In some embodiments, the lipid nanoparticles may comprise about 40 mol% to about 85 mol% of ionizable lipids, about 1 mol% to about 10 mol% of neutral lipids, about 1 mol% to about 50 mol% of accessory lipids, about 1 mol% to about 5 mol% of polymer-conjugated lipids, and about 0.1 mol% to about 5 mol% of other lipids. The hydrophobic component is mol% of the ionizable lipid having a structure of formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d) or (I4e) as defined herein, or at least one compound having a structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, wherein mol% is based on the total lipids present in the nanoparticles.

[0334] In some embodiments, the lipid nanoparticles may comprise about 40 mol% to about 85 mol% of ionizable lipids, about 5 mol% to about 10 mol% of neutral lipids, about 1 mol% to about 45 mol% of accessory lipids, about 1 mol% to about 4 mol% of polymer-conjugated lipids, and about 0.1 mol% to about 5 mol% of other lipids. The hydrophobic component is mol% of the ionizable lipid having a structure of formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d) or (I4e) as defined herein, or at least one compound having a structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, wherein mol% is based on the total lipids present in the nanoparticles.

[0335] In some embodiments, the lipid nanoparticles may comprise about 40 mol% to about 85 mol% of ionizable lipids, about 5 mol% to about 10 mol% of neutral lipids, about 1 mol% to about 40 mol% of accessory lipids, about 1 mol% to about 40 mol% of polymer-conjugated lipids, and 0.1 mol% to about 5 mol% of other lipids. The hydrophobic component is mol% of the ionizable lipid having a structure of formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d) or (I4e) as defined herein, or at least one compound having a structure of formula (I), (I1), (I2), (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, wherein mol% is based on the total lipids present in the nanoparticles.

[0336] In some embodiments, the lipid nanoparticles may comprise about 40 mol% to about 85 mol% of ionizable lipids, about 5 mol% to about 10 mol% of phospholipids as neutral lipids, about 1 mol% to about 40 mol% of sterols as co-lipids, about 1 mol% to about 4 mol% of PEG-lipids as polymer-conjugated lipids, and 1 mol% to about 5 mol% of squalene, cardiolipin, α-tocopherol, solanine A, vitamin A, or combinations thereof as hydrophobic components, wherein the ionizable lipids have a structure or formula (I), (I1), (I1a), (I2), (I2a), (I2b), (I3), (I3a), (I3b), (I4), (I4a), (I4b), (I4c), (I4d), or (I4e) as defined herein. At least one compound having the structure of (I3), (I1-1), (I1-2), (I1-3), (I1-4), (I1-5), (I1-6), (I1-4a), (I1-4b), (I1-5a), (I1-5b), (I1-5c), (I1-5d), (I1-6a), (I1-6b) or (I1-6c), or at least one compound listed in Table 1 herein, or a pharmaceutically acceptable salt thereof, wherein mol% is based on the total lipids present in the nanoparticles.

[0337] Encapsulation In some embodiments, lipid nanoparticles may completely or partially encapsulate the cargo or payload. In some embodiments, substantially 0% of the cargo present in the final formulation is exposed to the environment outside the lipid nanoparticles (i.e., the cargo is completely encapsulated). In some embodiments, the cargo is associated with the lipid nanoparticles but is at least partially exposed to the environment outside the lipid nanoparticles. In some embodiments, the lipid nanoparticles can be characterized by the percentage of cargo that is not exposed to the environment outside the lipid nanoparticles, for example, encapsulation efficiency. For clarity, approximately 100% encapsulation efficiency refers to a lipid nanoparticle formulation in which substantially all cargo is completely encapsulated by the lipid nanoparticles, while approximately 0% encapsulation efficiency refers to lipid nanoparticles in which substantially no cargo is encapsulated, such as lipid nanoparticles in which cargo is bound to the outer surface of the lipid nanoparticles. In some embodiments, the lipid nanoparticles may have an encapsulation efficiency of less than about 100%, less than about 95%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. In some embodiments, the lipid nanoparticles may have a content of about 90% to 100%, 80% to 100%, 70% to 100%, 60% to 100%, 50% to 100%, 40% to 100%, 30% to 100%, 20% to 100%, 10% to 100%, 80% to 90%, 70% to 90%, 60% to 90%, 50% to 90%, 40% to 90%, 30% to 90%, 20% to 90%, 10% to 90%, 70% to 80%, 60% to Encapsulation rates of 80%, 50% to 80%, 40% to 80%, 30% to 80%, 20% to 80%, 10% to 80%, 60% to 70%, 50% to 70%, 40% to 70%, 30% to 70%, 20% to 70%, 10% to 70%, 40% to 50%, 30% to 50%, 20% to 50%, 10% to 50%, 30% to 40%, 20% to 40%, 10% to 40%, 20% to 30%, 10% to 30%, and 10% to 20%.

[0338] In some embodiments, the weight ratio of lipid nanoparticles (including all lipids) to the cargo or payload is from about 100:1 to about 1:1, for example, from about 100:1 to about 90:1, from about 89:1 to about 80:1, from about 79:1 to about 70:1, from about 69:1 to about 60:1, from about 59:1 to about 50:1, from about 49:1 to about 40:1, from about 39:1 to about 30:1, from about 29:1 to about 20:1, from about 19:1 to about 10:1 and from about 9:1 to about 1:1.

[0339] In some embodiments, the lipid nanoparticles further comprise a starting or baseline construct having at least one cargo or payload. The cargo or payload can be a small molecule, antibody, polynucleotide, or peptide. The cargo or payload can contain at least one nucleic acid, such as mRNA. The cargo or payload can be any DNA, RNA, or peptide described herein.

[0340] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having at least one cargo or payload, said cargo or payload being an RNA-encoding construct.

[0341] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having at least one cargo or payload, said cargo or payload being non-coding RNA.

[0342] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having at least one cargo or payload, said cargo or payload being oRNA.

[0343] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having at least one cargo or payload, said cargo or payload being mRNA.

[0344] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having at least one cargo or payload, said cargo or payload being personalized vaccine mRNA. Non-limiting examples include 3-GP.

[0345] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having at least one cargo or payload, said cargo or payload being Covid-19 vaccine mRNA. A non-limiting example is the coronavirus spike protein.

[0346] In some embodiments, at least one RNA compound comprises a functional RNA, wherein the RNA causes at least one change in a cell, tissue, organ, and / or organism. The change in state may include, but is not limited to, altering the expression level of a polypeptide, altering the translation level of a nucleic acid, altering the expression level of a nucleic acid, altering the amount of a polypeptide present in a cell, tissue, organ, and / or organism, altering the genetic sequence of a cell, tissue, organ, and / or organism, adding nucleic acids to a target genome, removing nucleic acids from a target genome, altering physiological activity in a cell, tissue, organ, and / or organism, or any combination thereof.

[0347] In some embodiments, the lipid nanoparticles comprise a starting or reference construct having at least one cargo or payload, said cargo or payload being DNA.

[0348] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having two cargoes or payloads, said cargoes or payloads being DNA. The DNA can be the same DNA or different DNA. As a non-limiting example, the DNA is the same. As a non-limiting example, the DNA is different. As a non-limiting example, the DNA is different but encodes the same payload or cargo. As a non-limiting example, the DNA is different fragments of a larger payload or cargo (e.g., the heavy or light chain of an antibody), which can be assembled together using natural systems or synthetic methods known in the art to produce a functional peptide (e.g., an antibody).

[0349] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having three cargoes or payloads, said cargoes or payloads being DNA. The DNAs may be the same DNA or different DNAs. As a non-limiting example, the DNAs are the same. As a non-limiting example, the DNAs are different. As a non-limiting example, two DNAs are the same and one DNA is different. As a non-limiting example, the first DNA is different from the second and third DNAs. As a non-limiting example, the first, second, and third DNAs are all different. As a non-limiting example, the first DNA is different from the second and third DNAs, but they all encode the same payload or cargo. As a non-limiting example, the first DNA is different from the second and third DNAs, but the second and third DNAs encode the same payload or cargo.

[0350] In some embodiments, the lipid nanoparticles comprise a starting or reference construct having at least one cargo or payload, said cargo or payload being a polypeptide.

[0351] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having two cargoes or payloads, said cargoes or payloads being peptides. The peptides may be the same peptide or different peptides. As a non-limiting example, the peptides are the same. As a non-limiting example, the peptides are different. As a non-limiting example, the peptides are different fragments of a larger payload or cargo (e.g., the heavy or light chain of an antibody), which can be assembled together using natural systems or synthetic methods known in the art to produce a functional peptide (e.g., an antibody).

[0352] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having three cargoes or payloads, said cargoes or payloads being peptides. The peptides may be the same peptide or different peptides. As a non-limiting example, the peptides are the same. As a non-limiting example, the peptides are different. As a non-limiting example, two peptides are the same and one peptide is different. As a non-limiting example, the first peptide is different from the second and third peptides. As a non-limiting example, the first, second, and third peptides are all different. As a non-limiting example, the first peptide is different from the second and third peptides, but they all encode the same payload or cargo. As a non-limiting example, the first peptide is different from the second and third peptides, but the second and third peptides encode the same payload or cargo.

[0353] In some embodiments, the lipid nanoparticles comprise a starting or reference construct having at least one cargo or payload, said cargo or payload being a peptide.

[0354] In some embodiments, lipid nanoparticles comprise a starting or baseline construct having two cargoes or payloads, said cargoes or payloads being peptides. The peptides can be the same peptide or different peptides. As a non-limiting example, the peptides are identical. As a non-limiting example, the peptides are different. As a non-limiting example, the peptides are different fragments of a larger payload or cargo (e.g., the heavy or light chain of an antibody) that can be assembled together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., an antibody).

[0355] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having three cargoes or payloads, said cargoes or payloads being peptides. The peptides may be the same peptide or different peptides. As a non-limiting example, the peptides are the same. As a non-limiting example, the peptides are different. As a non-limiting example, two peptides are the same and one peptide is different. As a non-limiting example, the first peptide is different from the second and third peptides. As a non-limiting example, the first, second, and third peptides are all different. As a non-limiting example, the first peptide is different from the second and third peptides, but they all encode the same payload or cargo. As a non-limiting example, the first peptide is different from the second and third peptides, but the second and third peptides encode the same payload or cargo.

[0356] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having at least one cargo or payload, said cargo or payload being RNA.

[0357] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having two cargoes or payloads, said cargoes or payloads being RNA. The RNAs may be the same RNA or different RNAs. As a non-limiting example, the RNAs are the same. As a non-limiting example, the RNAs are different. As a non-limiting example, the RNAs are different but encode the same payload or cargo. As a non-limiting example, the RNAs are different fragments of a larger payload or cargo (e.g., the heavy or light chain of an antibody), which can be assembled together using natural systems or synthetic methods known in the art to produce a functional peptide (e.g., an antibody).

[0358] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having three cargoes or payloads, said cargoes or payloads being RNA. The RNAs may be the same or different RNAs. As a non-limiting example, the RNAs are the same. As a non-limiting example, the RNAs are different. As a non-limiting example, two RNAs are the same and one RNA is different. As a non-limiting example, the first RNA is different from the second and third RNAs. As a non-limiting example, the first, second, and third RNAs are all different. As a non-limiting example, the first RNA is different from the second and third RNAs, but they all encode the same payload or cargo. As a non-limiting example, the first RNA is different from the second and third RNAs, but the second and third RNAs encode the same payload or cargo.

[0359] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having two cargoes or payloads, one of which is RNA and the other is DNA. The RNA and DNA may encode the same peptide or polypeptide, or they may encode different peptides or polypeptides. As a non-limiting example, the RNA and DNA may encode the same peptide or polypeptide. As a non-limiting example, the RNA and DNA may encode different peptides or polypeptides. As a non-limiting example, the RNA and DNA may be different fragments of a larger payload or cargo (e.g., the heavy or light chain of an antibody), which may be assembled together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., an antibody).

[0360] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having two cargoes or payloads, one of which is RNA and the other is a peptide. The RNA may encode the same peptide as the peptide cargo / payload, or it may encode a different peptide. As a non-limiting example, the RNA encodes the same peptide. As a non-limiting example, the RNA encodes a different peptide. As a non-limiting example, the RNA and peptide are different fragments of a larger payload or cargo (e.g., the heavy or light chain of an antibody), which can be assembled using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., an antibody).

[0361] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having two cargoes or payloads, one of which is RNA and the other is a polypeptide. The RNA may encode the same polypeptide as the polypeptide cargo / payload, or it may encode a different polypeptide. As a non-limiting example, the RNA encodes the same polypeptide. As a non-limiting example, the RNA encodes a different polypeptide. As a non-limiting example, the RNA and polypeptide are different fragments of a larger payload or cargo (e.g., the heavy or light chain of an antibody), which can be assembled together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., an antibody).

[0362] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having two cargoes or payloads, one of which is DNA and the other is a peptide. The DNA may encode the same peptide as the peptide cargo / payload, or the DNA may encode different peptides. As a non-limiting example, the DNA encodes the same peptide. As a non-limiting example, the DNA encodes different peptides. As a non-limiting example, the DNA and peptide are different fragments of a larger payload or cargo (e.g., the heavy or light chain of an antibody), which can be assembled together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., an antibody).

[0363] In some embodiments, the lipid nanoparticles comprise a starting or baseline construct having two cargoes or payloads, one cargo or payload being DNA and the other being a polypeptide. The DNA may encode the same polypeptide as the polypeptide cargo / payload, or the DNA may encode different polypeptides. As a non-limiting example, the DNA encodes the same polypeptide. As a non-limiting example, the DNA encodes different polypeptides. As a non-limiting example, the DNA and polypeptide are different fragments of a larger payload or cargo (e.g., the heavy or light chain of an antibody), which can be assembled together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., an antibody).

[0364] Preparation method The lipid nanoparticles described herein can be formed using techniques known in the art. As a non-limiting example, mixing an organic solution containing lipids with an acidic aqueous solution containing a starting or reference construct in a microfluidic channel results in the formation of a targeted system (lipid nanoparticles and a reference construct). Another technique may involve turbulent mixing in a T-junction, where the flow rates of the solution of the starting or reference construct and the lipid solution are substantially the same.

[0365] In some embodiments, the lipid nanoparticle formulation can be prepared by the method described in International Publication No. WO2008103276, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the lipid nanoparticle formulation can be prepared as described in International Publication No. WO2019131770, the contents of which are incorporated herein by reference in their entirety.

[0366] In some embodiments, lipid nanoparticle formulations can be prepared by the method described in International Publication No. WO2020237227, the contents of which are incorporated herein by reference in their entirety.

[0367] In some embodiments, lipid nanoparticle formulations can be prepared using the methods and apparatus described in US20240181406A1, the contents of which are incorporated herein by reference in their entirety.

[0368] In another embodiment, the lipid nanoparticle formulation can be prepared using the FDmiX mixing system developed by FDX Fluid Dynamix GmbH (see https: / / www.fdx.de / en / fdmix / , the contents of which are incorporated herein by reference in their entirety).

[0369] In some embodiments, lipid nanoparticle formulations can be prepared using a so-called “on-demand” mixing method as described herein (see Examples). This involves preparing a cargo (e.g., mRNA) solution (which can be freeze-dried or lyophilized) and a lipid solution separately. The lipid and mRNA solutions are mixed, followed by vigorous shaking, which provides mRNA encapsulated in LNPs, ready for use / administration. On-demand mixing can produce mRNA-LNPs of comparable quality and efficacy to mRNA-LNPs obtained by conventional methods (conventional mRNA-LNPs). The manufacture of conventional mRNA-LNPs is a sensitive process, typically involving specialized equipment and analytical techniques. mRNA-LNPs are sensitive to minute variations in the manufacturing process. Long-term storage of mRNA-LNPs requires cryogenic storage facilities. Even so, the long-term stability of mRNA-LNPs may not be guaranteed. On the other hand, in on-demand mixing, the LNPs are not pre-manufactured prior to administration. The mRNA and lipid solutions can be manufactured, filled, and released using conventional small molecule drug manufacturing equipment. Both the mRNA and lipid solutions are stable under freezing conditions.

[0370] IV. Cargo and Payload This disclosure also provides compositions or constructs comprising the lipid nanoparticles of this disclosure, wherein the lipid nanoparticles may comprise, encode, or conjugate to a cargo or payload to produce a construct. In some embodiments, the cargo or payload is or encodes a bioactive molecule, such as, but not limited to, a therapeutic protein. As used herein, the term “bioactive” refers to the characteristic of any substance that is active in a biological system and particularly in an organism. For example, a substance that has a biological effect on an organism when administered to that organism is considered bioactive. In some embodiments, the cargo or payload is or encodes one or more proteins, peptides, or other factors that are prophylactically or therapeutically active. As a non-limiting example, the cargo or payload may be or encode a substance that enhances tumor-killing activity in cancer, such as, but not limited to, TRAIL or tumor necrosis factor (TNF). As another non-limiting example, the cargo or payload may be or encode a substance suitable for treating conditions such as: muscular dystrophy (e.g., the cargo or payload is or encodes dystrophin); cardiovascular disease (e.g., the cargo or payload is or encodes SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc.); neurodegenerative diseases (e.g., the cargo or payload is or encodes NGF, BDNF, GDNF, NT-3, etc.); chronic pain (e.g., the cargo or payload is or encodes GlyRa). l) Enkephalin or glutamate decarboxylase (e.g., the cargo or payload is or encodes GAD65, GAD67, or another isoform)); lung diseases (e.g., the cargo or payload is or encodes CFTR); hemophilia (e.g., the cargo or payload is or encodes factor VIII or factor IX); tumor formation (e.g., the cargo or payload is or encodes PTEN, ATM, ATR, EGFR, ERBB2, ERBB3, ERBB4, Notch1, Notch2, Notch3, Notch4, AKT, AKT2, AKT3, HIF, HI Fla, HIF3a, Met, HRG, Bcl2, PPARα, PPARγ, WT1 (Wilms tumor), FGF receptor family members (5 members: 1, 2, 3, 4, 5), CDKN2a, APC, RB (retinoblastoma), MEN1, VHL, BRCA1, BRCA2, AR (androgen receptor), TSG101, IGF, IGF receptor, Igfl (4 variants), Igf2 (3 variants), Igfl receptor, Igf2 receptor, Bax, Bcl2, caspase family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12), Kras, Ape;Age-related macular degeneration (e.g., cargo or payload is or encodes Aber, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin D, Vldlr); schizophrenia (e.g., neuroregulatory protein (Nrgl), Erb4 (neuroregulatory protein receptor), complex protein-1 (Cplxl), Tph1 tryptophan hydroxylase, Tph2 tryptophan hydroxylase 2, axonin 1, GSK3, GSK3a, GSK3b, 5-HIT (Slc6a4), COMT, DRD (Drdla), SLC6A3, DAOA, DTNBPI, Dao (Daol)); trinucleotide repeat disorders (e.g., HTT (Huntington's disease), SBMA / SMAXI / AR (Kennedy's disease), FXN / X25 (Friedreich's ataxia), ATX3 (Machado-Joseph's disease)). (Dx), ATXNI and ATXN2 (spinocerebellar ataxia), DMPK (myotonic dystrophy), Atrophin-1 and Atnl (DRPLA disease), CBP (Creb-BP-global instability), VLDLR (Alzheimer's disease), Atxn7, Atxn10); Fragile X syndrome (e.g., cargo or payload is or encodes FMR2, FXRI, FXR2, mGLUR5), secretase-related diseases (e.g., cargo or payload is or encodes APH-1 (α and β), presenilin (Psenl), nigra (Ncstn), PEN-2); ALS (e.g., cargo or payload is or encodes SOD1, ALS2, STEX, FUS, TARD BP, VEGF) (VEGF-a, VEGF-b, VEGF-c)); Autism (e.g., cargo or payload is or encodes Mecp2, BZRAP1, MDGA2, Sema5A, axonin 1); Alzheimer's disease (e.g., cargo or payload is or encodes E1, CHIP, UCH, UBB, Tau, LRP, PICALM, clusterin, PS1, SORL1, CR1, Vldlr, Ubal, Uba3, CHIP28 (Aqpl, aquaporin 1), Uchll, Uchl3, APP);Inflammation (e.g., cargo or payload is or encodes IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-171), IL-11-23, Cx3crl, ptpn22, TNFα, NOD2 / CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3crl); Parkinson's disease (e.g., X-synuclein, DJ-1, LRRK2, Parkin, PINK1); blood disorders and coagulation disorders, such as anemia, naked lymphocyte syndrome, bleeding disorders, hemophagocytic lymphohistiocytosis, hemophilia A, hemophilia B, hemorrhage. Disorders), leukopenia and leukemia, sickle cell anemia and thalassemia (e.g., cargo or payload is or coded CRAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSNI, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT, TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP, RFX5, TBXA2R, P2RX1, P2X1, HF1, CFH, HUS, MCFD2, FANCA, FAC A, FA1, FA, FA A. FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCDI, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BR1PI, BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596, PRF1, HP LH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3, F8, FSC, PI, ATT, F5, ITGB2, CD18, LCAMB, LAD, EIF2B 1. EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4, HBB, HBA2, HBB, HBD, LCRB, HBA1);B-cell non-Hodgkin's lymphoma or leukemia (e.g., cargo or payload is or is coded as BCL7A, BCL7, ALI, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1AI, 1KI, LYF1, HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AFIO, ARHGEF12, LARG, KIAA0382, CALM, CL) TH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPMI, NUP214, D9S46E, CAN, CAIN, RUNXI, CBFA2, AML1, WH SC1LI, NSD3, FLT3, AF1Q, NPMI, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF1Q, CALM, CLTH, ARL11, ARLTS1, P 2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPNII, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABLI, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN); Inflammatory and immune-related diseases and conditions. (For example, cargo or payload is or is coded as KIR3DL1, NKAT3, NKB1, AMB11, K1R3DS1, IFNG, CXCL12, TNFRSF6, APT1, FAS, CD95, ALPS1A, IL2RG, SCIDX1, SCIDX, IMD4, CCL5, SCYA5, D17S136E, TCP228, IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5), CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSFS, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI);Inflammation (e.g., cargo or payload is or encodes IL-10, IL-1 (IL-IA, IL-IB), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-171), IL-11-23, Cx3crl, ptpn22, TNFα, NOD2 / CARD15 for IBD, IL-6, IL-12) (IL-12a, IL-12b), CTLA4, Cx3cII), JAK3, JAKL, DCLREIC, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDAXI, SCIDAX, IMD4); metabolic, liver, kidney and protein diseases and conditions (e.g., cargo or payload is or is coded TTR, PALB, APOA1, APP, AAA, CVAP, ADI, GSN, FGA, LYZ, TTR, PALB, KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988, CFTR, ABCC7, CF, MRP7, SLC2A2, GLUT2, G 6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM, TCF1, HNF1A, MODY3, SCOD1, SCOL, CTNNB1, PDGFRL, PDGRL, PRLTS, AX1NI, AXIN, CTNNB1, TP53, P53, LFS 1. IGF2R, MPRI, MET, CASP8, MCH5, UMOD, HNFJ, FJHN, MCKD2, ADMCKD2, PAH, PKU1, QDPR, DHPR, PTS, FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63);Musculoskeletal disorders and conditions (e.g., cargo or payload is or is coded as DMD, BMD, MYF6, LMNA, LMN1, EMD2, FPLD, CMDIA, HGPS, LGMDIB, LMNA, LMNI, EMD2, FPLD, CMDIA, FSHMD1A, FSHD1A, FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2) G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDCIC, LGMD21, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, LRP5, BMNDl, LRP7, LR3, OPPG, VBCH2, CLC N7, CLC7, OPTA2, OSTMI, GL, TCIRG1, TIRC7, OC116, OPTB1, VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1);Neurological and neuronal diseases and conditions (e.g., cargo or payload is or encodes SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c), APP, AAA, CVAP, ADI, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65LI, NOS3, PLAU, URK, ACE, DCPI, ACEI, MPO, PAC1PI, PAXIPIL, PTIP, A2M, BLMH, BMH, PSEN1, AD3, Mecp2, BZRAP1, MDGA2, Sema5A, axonin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2, FMR2, FXR1, FXR2, mGLUR5, HD, IT15) PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17, NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJI, PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN, PARK2, PDJ, DBH, NDUFV2, MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16, MRX79, X-synuclein, DJ-1, neuromodulatory protein-1 (Nrgl), Erb4 (neural regulatory protein receptor), complex protein-1 (Cplxl), Tphl tryptophan hydroxylase, Tph2 (tryptophan hydroxylase 2), axonin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), CONT, DRD (Drdla), SLC6A, DAOA, DTNBP1, Dao (Daol), APH-1 (α and β), presenilin (Psenl), nifedipine (Ncstn), PEN-2, Nosl, Parpl, Natl, Nat2, HTT, SBMA / SMAX1 / AR, FXN / X25, ATX3, TXN, ATXN2, DMPK, Atrophin-1, Atnl, CBP, VLDLR, Atxn7 and AtxnlO);And eye diseases and conditions (e.g., Aber, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin-D, Vldlr, Ccr2, CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYAI, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47). HSF4, CTM, HSF4, CTM, MIP, AQPO, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG 3. CCL, CRYAA, CRYAI, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1, APOA1, TGFBI, CSD2, CDG G1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1SI, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, C FD, KERA, CNA2, MYOC, TIGR, GLCIA, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1BI, GLC3A, OPA1, NTG, NP G, CYP1BI, GLC3A, CRB1, RP12, CRX, CORD2, CRD, RPGRIPI, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3, ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD and VMD2). ;

[0371] In some implementations, the cargo or payload is or encodes a factor that can influence cell differentiation. As a non-limiting example, expression of one or more of Oct4, Klf4, Sox2, c-Myc, L-Myc, dominant-negative p53, Nanog, Glisl, Lin28, TFIID, mir-302 / 367, or other miRNAs can lead to cells becoming induced pluripotent stem (iPS) cells.

[0372] In some embodiments, the cargo or payload is or encodes a factor for transdifferentiating cells. Non-limiting examples of factors include: GATA4, Tbx5, Mef2C, Myocd, Hand2, SRF, Mespl, SMARCD3 for cardiomyocytes; Ascii, Nurl, LmxlA, Bm2, Mytll, NeuroDl, FoxA2 for nerve cells; and one or more of Hnf4a, Foxal, Foxa2, or Foxa3 for hepatocytes.

[0373] polypeptides, proteins and peptides The lipid nanoparticles of this disclosure may comprise, encode, or be conjugated to cargo or payload, which is a polypeptide, protein, or peptide. As used herein, the term "polypeptide" generally refers to a polymer of amino acids linked by peptide bonds and includes both "protein" and "peptide". The polypeptides of this disclosure include all polypeptides, proteins, and / or peptides known in the art. Non-limiting classes of polypeptides include antigens, antibodies, antibody fragments, cytokines, peptides, hormones, enzymes, oxidants, antioxidants, synthetic polypeptides, and chimeric polypeptides.

[0374] As used herein, the term "peptide" generally refers to a shorter polypeptide of about 50 amino acids or fewer. A peptide having only two amino acids may be called a "dipeptide." A peptide having only three amino acids may be called a "tripeptide." A polypeptide generally refers to a polypeptide having about 4 to about 50 amino acids. Peptides can be obtained by any method known to those skilled in the art. In some embodiments, peptides can be expressed in a culture. In some embodiments, peptides can be obtained by chemical synthesis (e.g., solid-phase peptide synthesis).

[0375] In some embodiments, the lipid nanoparticles of this disclosure may comprise, encode, or be conjugated to cargo or payload, which is a simple protein that, upon hydrolysis, yields amino acids and occasionally small carbohydrates. Non-limiting examples of simple proteins include albumin, albuminoids, globulins, glutenin, histones, and protamine.

[0376] In some embodiments, the lipid nanoparticles of this disclosure may comprise, encode, or be conjugated to cargo or payload, said cargo or payload being a conjugated protein, which may be a simple protein associated with a non-protein. Non-limiting examples of conjugated proteins include glycoproteins, hemoglobin, lecithin, nucleoproteins, and phosphoproteins.

[0377] In some embodiments, the lipid nanoparticles of this disclosure may comprise, encode, or be conjugated to cargo or payload, said cargo or payload being a derived protein derived from a simple protein or conjugated protein by chemical or physical means. Non-limiting examples of derived proteins include denatured proteins and peptides.

[0378] In some implementations, the polypeptide, protein, or peptide may be unmodified.

[0379] In some embodiments, the polypeptide, protein, or peptide may be modified. Types of modification include, but are not limited to, phosphorylation, glycosylation, acetylation, ubiquitination / SUMOylation, methylation, palmitoylation, quinoneation, amidation, myristoylation, pyrrolidone carboxylation, hydroxylation, phosphopanyl thioethylamineation, isopreneation, GPI anchoring, oxidation, ADP-ribosylation, sulfation, S-nitrosylation, citrullination, nitration, γ-carboxyglutamateation, formylation, hydroxyputrescine-lysineation (Hypusine), topaquinone (TPQ)ation, bromination, lysine topaquinone (LTQ), tryptophan topaquinone (TTQ), iodination, and cysteine ​​tryptophan quinone (CTQ). In some aspects, the polypeptide, protein, or peptide may be modified by post-transcriptional modifications that can affect its structure, subcellular localization, and / or function.

[0380] In some implementations, peptides, proteins, or polypeptides can be modified using phosphorylation. Phosphorylation, or the addition of a phosphate group to a serine, threonine, or tyrosine residue, is one of the most common forms of protein modification. Protein phosphorylation plays an important role in the fine-tuning of signal transduction cascades within cells.

[0381] In some implementations, peptides, proteins, or polypeptides may be modified using ubiquitination, which is the covalent attachment of ubiquitin to a target protein. Ubiquitination-mediated protein turnover has been shown to play a role in driving the cell cycle and in intracellular signaling pathways independent of protein degradation.

[0382] In some embodiments, polypeptides, proteins, or peptides can be modified using acetylation and methylation, which can play a role in regulating gene expression. As a non-limiting example, acetylation and methylation can mediate the formation of chromatin domains (e.g., euchromatin and heterochromatin), which has an impact on mediating gene silencing.

[0383] In some implementations, peptides, proteins, or glycosylation can be used to modify them. Glycosylation is the attachment of one of a large number of glycan groups and is a modification that occurs in about half of all proteins, playing a role in biological processes, including but not limited to embryonic development, cell division, and regulation of protein structure. The two main types of protein glycosylation are N-glycosylation and O-glycosylation. For N-glycosylation, the glycan is attached to asparagine, while for O-glycosylation, the glycan is attached to serine or threonine.

[0384] In some implementations, peptides, proteins, or polypeptides can be modified using SUMOylation. SUMOylation involves adding SUMO (small ubiquitin-like modifier) ​​to a protein and is a post-translational modification similar to ubiquitination.

[0385] Antibody As used herein, the term "antibody" is referred to in the broadest sense and specifically covers a variety of embodiments, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies), and antibody fragments (e.g., diabody), provided they exhibit the desired biological activity (e.g., "functionality"). Antibodies are primarily amino acid-based molecules; they are monomeric or multimeric polypeptides containing at least one amino acid region derived from a known or parental antibody sequence and at least one amino acid region derived from a non-antibody sequence. Antibodies may contain one or more modifications (including, but not limited to, the addition of a sugar moiety, a fluorescent moiety, a chemical tag, etc.). For the purposes of this document, "antibody" may contain variable heavy and light chain domains and an Fc region.

[0386] The cargo or payload may contain or encode polypeptides that form one or more functional antibodies.

[0387] In some embodiments, the cargo or payload may contain or encode a polypeptide that forms or functions as any antibody, including but not limited to antibodies known in the art and / or commercially available antibodies that can be used for therapeutic, diagnostic, or research purposes. Furthermore, the cargo or payload may contain or encode such antibodies or fragments of antibodies, such as, but not limited to, variable domains or complementarity-determining regions (CDRs).

[0388] As used herein, the term "natural antibody" refers to a heterotetrameric glycoprotein typically about 150,000 Daltons in size, composed of two identical light (L) chains and two identical heavy (H) chains. Genes encoding the antibody heavy and light chains are known, and the segments comprising each gene have been well characterized and described (Matsuda, F. et al., 1998. The Journal of Experimental Medicine. 188(11); 2151-62; and Li, A. et al., 2004. Blood. 103 (12:4602-9, each of which is incorporated herein by reference in its entirety). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isoforms. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domains of the light chain are aligned with the first constant domain of the heavy chain, and the light chain... The variable domains of the light chain are aligned with those of the heavy chain. As used herein, the term "light chain" refers to the antibody component from any vertebrate species, which, based on the amino acid sequence of its constant domain, is assigned to one of two distinct types (called κ and λ). Depending on the amino acid sequence of the constant domain of the antibody heavy chain, antibodies can be assigned to different classes. There are five major classes of complete antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0389] As used herein, the term "variable domain" refers to a specific antibody domain present on both the heavy and light chains of an antibody, the sequence of which varies considerably between antibodies, and the domain is used for the binding and specificity of each specific antibody to its specific antigen. Variable domains contain hypervariable regions. As used herein, the term "hypervariable region" refers to a region within a variable domain containing amino acid residues responsible for antigen binding. The amino acids present within the hypervariable region determine the structure of the complementarity-determining region (CDR), which becomes part of the antibody's antigen-binding site. As used herein, the term "CDR" refers to an antibody region containing a structure complementary to its target antigen or epitope. Other portions of the variable region that do not interact with the antigen are called framework (FW) regions. The antigen-binding site (also called the antigen-binding site or paratope) contains the amino acid residues required to interact with a specific antigen. The exact residues that make up the antigen-binding site are usually elucidated by cocrystallographic analysis with the antigen to which they are bound, but can also be evaluated using computation based on comparisons with other antibodies (Strohl, WR Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, pp. 47-54, the contents of which are incorporated herein by reference in their entirety). Determining the residues that make up the CDR can include using a numbering scheme, including but not limited to those cited by Kabat [Wu, TT et al., 1970, JEM, 132(2):211-50 and Johnson, G. et al., 2000, Nucleic Acids Res.28(1): 214-8, each of which is incorporated herein by reference in its entirety], Chothia [Chothia and Lesk, J.Mol.Biol.196, 901 (1987); Chothia et al., Nature 342, 877 (1989) and Al-Lazikani, B. et al., 1997, J. Mol.Biol.273(4):927-48, each of which is incorporated herein by reference in its entirety], and Lefranc (Lefranc, MP et al., 2005, Immunome The numbering scheme taught by Res. 1:3) and Honegger (Honegger, A. and Pluckthun, A. 2001. J. Mol. Biol. 309(3): 657-70, the contents of which are incorporated herein by reference in their entirety).

[0390] Both the VH and VL domains have three canonical receptors (CDRs). The VL CDRs are referred to herein as CDR-L1, CDR-L2, and CDR-L3, in the order that appears as the variable domain peptide moves from the N-terminus to the C-terminus. The VH CDRs are referred to herein as CDR-H1, CDR-H2, and CDR-H3, in the order that appears as the variable domain peptide moves from the N-terminus to the C-terminus. Except for CDR-H3, each CDR possesses a preferred canonical structure, while CDR-H3 contains amino acid sequences whose sequence and length can be highly variable among antibodies, resulting in diverse three-dimensional structures within the antigen-binding domain. In some cases, the CDR-H3 of a group of related antibodies can be analyzed to assess antibody diversity.

[0391] Various methods for determining CDR sequences are known in the art and can be applied to known antibody sequences. The system described by Kabat, also known as “Kabat numbering,” “Kabat numbering,” “Kabat definition,” and “Kabat marker,” provides a definitive residue numbering system applicable to any variable domain of an antibody and provides precise residue boundaries defining the three CDRs for each chain. (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991), the contents of which are incorporated herein by reference in their entirety). Kabat CDRs contain residues at approximately positions 24–34 (CDR1), approximately positions 50–56 (CDR2), and approximately positions 89–97 (CDR3) in the light chain variable domain, and residues at approximately positions 31–35 (CDR1), approximately positions 50–65 (CDR2), and approximately positions 95–102 (CDR3) in the heavy chain variable domain. Chothia and colleagues discovered that, despite significant differences at the amino acid sequence level, certain sub-regions of the Kabat CDR employ nearly identical peptide skeleton structures. (Chothia et al. (1987) J. Mol. Biol. 196: 901-917; and Chothia et al. (1989) Nature 342: 877-883, each of which is incorporated herein by reference in its entirety). These CDRs may be referred to as “Chothia CDRs,” “Chothia numbers,” or “according to Chothia numbers,” and contain residues at approximately positions 24–34 (CDR1), 50–56 (CDR2), and 89–97 (CDR3) in the light chain variable domain, and residues at approximately positions 26–32 (CDR1), 52–56 (CDR2), and 95–102 (CDR3) in the heavy chain variable domain. Mol.Biol.196:901-917 (1987). The system described by MacCallum, also known as “according to MacCallum numbering” or “MacCallum numbering”, contains residues at approximately positions 30-36 (CDR1), approximately positions 46-55 (CDR2), and approximately positions 89-96 (CDR3) in the light chain variable domain, and residues at approximately positions 30-35 (CDR1), approximately positions 47-58 (CDR2), and approximately positions 93-101 (CDR3) in the heavy chain variable domain.(MacCallum et al. ((1996) J. Mol. Biol. 262(5): 732-745), the contents of which are incorporated herein by reference in their entirety). The system described by AbM, also known as “by AbM number” or “AbM number”, contains residues at approximately positions 24-34 (CDR1), approximately positions 50-56 (CDR2), and approximately positions 89-97 (CDR3) in the light chain variable domain, and residues at approximately positions 26-35 (CDR1), approximately positions 50-58 (CDR2), and approximately positions 95-102 (CDR3) in the heavy chain variable domain. Alternatively, the IMGT (International Immunogenetic Information System) numbering of variable regions can be used. This is the numbering of residues in the variable heavy or light chains of immunoglobulins according to the IIMGT method (Lefranc, M.-P., "The IMGT unique numbering for immunoglobulins, T cell Receptors and Ig-like domains", The Immunologist, 7, 132-136 (1999), which is incorporated herein by reference in its entirety). As used herein, "IMGT sequence numbering" or "numbering according to IMGT" refers to the numbering of the sequence encoding the variable region according to IMGT. For heavy chain variable domains, when numbered according to IMGT, the range of hypervariable regions is amino acid positions 27 to 38 of CDR1, amino acid positions 56 to 65 of CDR2, and amino acid positions 105 to 117 of CDR3. For the variable domains of the light chain, when numbered according to IMGT, the range of the hypervariable region is amino acid positions 27 to 38 of CDR1, amino acid positions 56 to 65 of CDR2, and amino acid positions 105 to 117 of CDR3.

[0392] In some embodiments, the cargo or payload may contain or encode antibodies generated using methods known in the art, such as, but not limited to, immunization and display techniques (e.g., phage display, yeast display, and ribosome display), hybridoma techniques, and cDNA sequences of heavy and light chain variable regions selected from hybridomas or other sources.

[0393] In some embodiments, the cargo or payload may contain or encode antibodies developed using any naturally occurring or synthetic antigen. As used herein, an "antigen" is an entity that induces or triggers an immune response in an organism. An immune response is characterized by the reaction of an organism's cells, tissues, and / or organs to the presence of a foreign entity. Such an immune response typically results in the production of one or more antibodies against the foreign entity, such as an antigen or a portion thereof. As used herein, "antigen" also refers to a binding partner displaying a specific antibody or binder from a library.

[0394] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of cells (or clones), meaning that the individual antibodies comprising the population are identical and / or bind to the same epitopes, except for possible variants that may arise during the production of the monoclonal antibody, which are typically present in small quantities. This contrasts with polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), where each monoclonal antibody targets a single determinant on the antigen.

[0395] The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the production of the antibody through any particular method. Monoclonal antibodies in this article include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies.

[0396] As used herein, the term "humanized antibody" refers to a chimeric antibody comprising a minimum portion derived from one or more non-human (e.g., mouse) antibody sources, with the remainder derived from one or more human immunoglobulin sources. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody) in which residues from the hypervariable region of the receptor antibody are replaced by residues from the hypervariable region of an antibody (donor antibody) from a non-human species, such as a mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and / or ability.

[0397] In some embodiments, the cargo or payload may include or encode an antibody mimic. As used herein, the term "antibody mimic" refers to any molecule that mimics the function or action of an antibody and binds specifically and with high affinity to its molecular target. In some embodiments, the antibody mimic may be a monobody designed to incorporate a fibronectin type III (Fn3) domain as a protein scaffold. In some embodiments, the antibody mimic may be those known in the art, including but not limited to affibody molecules, affilin, affitin, anticalin, avimer, centyrin, DARPINS™, fynomer, Kunitz domains, and domain peptides. In other embodiments, the antibody mimic may include one or more non-peptide regions.

[0398] Antibody fragments and variants In some embodiments, the cargo or payload may contain or encode an antibody fragment comprising an antigen-binding region from a full-length antibody. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, biantibodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of the antibody produces two identical antigen-binding fragments, referred to as "Fab" fragments, each having a single antigen-binding site. A residual "Fc" fragment is also produced, its name reflecting its tendency to crystallize. Pepsin treatment produces an F(ab')2 fragment having two antigen-binding sites and still capable of cross-linking the antigen. The compounds and / or compositions of this disclosure may contain one or more of these fragments.

[0399] In some embodiments, the Fc region may be a modified Fc region, wherein the Fc region may have a single amino acid substitution compared to the corresponding sequence of the wild-type Fc region, wherein the single amino acid substitution produces an Fc region having more desirable properties than the wild-type Fc region. Non-limiting examples of Fc properties that can be altered by a single amino acid substitution include binding properties or response to pH conditions.

[0400] As used herein, the term "Fv" refers to an antibody fragment containing the smallest segment required to form a complete antigen-binding site on the antibody. These regions consist of a dimer of a tightly non-covalently associated heavy chain variable domain and a light chain variable domain. Fv fragments can be generated by proteolytic cleavage, but are highly unstable. Recombinant methods known in the art for generating stable Fv fragments typically involve either inserting a flexible linker between the light and heavy chain variable domains to form a single-chain Fv (scFv) or introducing a disulfide bond between the heavy and light chain variable domains.

[0401] As used herein, the term "single-chain Fv" or "scFv" refers to a fusion protein of VH and VL antibody domains, wherein these domains are linked together by a flexible peptide linker to form a single polypeptide chain. In some embodiments, the Fv peptide linker enables the scFv to form the structure required for antigen binding. In some embodiments, scFvs are used in conjunction with phage display, yeast display, or other display methods, wherein they can be expressed in conjunction with surface members (e.g., phage coat proteins) and used to identify high-affinity peptides to a given antigen.

[0402] As used herein, the term "antibody variant" refers to a modified antibody (as opposed to the natural or initiating antibody) or a biomolecule (e.g., an antibody mimic) that is structurally and / or functionally similar to the natural or initiating antibody. The amino acid sequence, composition, or structure of an antibody variant may be altered compared to a natural antibody. Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.

[0403] Multispecific antibodies In some embodiments, the cargo or payload may be, or may encode, an antibody that binds to more than one epitope. As used herein, the terms "multibody" or "multispecific antibody" refer to an antibody in which two or more variable regions bind different epitopes. Epitopes may be on the same or different targets. In some embodiments, a multispecific antibody is a "bispecific antibody" that recognizes two different epitopes on the same or different antigens.

[0404] In some embodiments, the multispecific antibodies can be prepared using the methods described in BIOATLA® and in International Patent Publication WO201109726, the contents of which are incorporated herein by reference in their entirety. First, a homologous, naturally occurring antibody library is generated by any method known in the art (i.e., mammalian cell surface display), and then multispecific antibodies that specifically bind to two or more target antigens are screened using FACSAria or another screening method. In some embodiments, the identified multispecific antibodies are further evolved by any method known in the art to generate a set of modified multispecific antibodies. These modified multispecific antibodies are screened for binding to the target antigens. In some embodiments, the multispecific antibodies can be further optimized by screening the evolved modified multispecific antibodies to obtain optimized or desired characteristics.

[0405] In some embodiments, multispecific antibodies can be prepared using the methods described in BIOATLA® and U.S. Publication No. US20150252119, the contents of which are incorporated herein by reference in their entirety. In one approach, the variable domains of two parent antibodies (where the parent antibodies are monoclonal antibodies) are evolved using any method known in the art in a manner that allows a single light chain to be functionally complementary to the heavy chains of two different parent antibodies. Another approach requires the evolution of the heavy chain of a single parent antibody to recognize a second target antigen. A third approach involves the evolution of the light chain of the parent antibody to recognize a second target antigen. International Publication WO2012009026 describes peptide evolution methods and, by way of non-limiting examples, includes comprehensive positional evolution (CPE), combinatorial protein synthesis (CPS), comprehensive positional insertion (CPI), comprehensive positional deletion (CPD), or any combination thereof. The contents of International Publication WO2012009026 are incorporated herein by reference in their entirety. The Fc region of the multispecific antibody described in US Publication US20150252119 can be generated using the knife-in-hole approach or any other method that allows the Fc domain to form heterodimers. The resulting multispecific antibody can be further evolved to obtain improved characteristics or properties, such as binding affinity to the target antigen.

[0406] Bispecific antibodies In some embodiments, the cargo or payload may be, or may encode, a bispecific antibody. As used herein, the term "bispecific antibody" refers to an antibody capable of binding to two different antigens. Such antibodies typically contain regions derived from at least two different antibodies. For example, a bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein composed of fragments from two different monoclonal antibodies, thus allowing BsAb to bind to two different types of antigens.

[0407] In some cases, cargo or payload may be, or may encode, a bispecific antibody containing antigen-binding regions from two different anti-tau antibodies. For example, such a bispecific antibody may contain binding regions from two different antibodies.

[0408] The bispecific antibody framework may include any of the frameworks described in the following literature: Riethmuller, G., 2012. Cancer Immunity. 12: 12-18; Marvin, JS et al., 2005. Acta Pharmacologica Sinica. 26(6): 649-58; and Schaefer, W. et al., 2011. PNAS. 108(27): 11187-92, the contents of which are incorporated herein by reference in their entirety.

[0409] A new generation of BsMAb has been developed, which are called "trifunctional bispecific" antibodies. These antibodies consist of two heavy chains and two light chains, each derived from two different antibodies. The two Fab regions (arms) target two antigens, and the Fc region (foot) contains the two heavy chains and forms a third binding site.

[0410] Of the two complementary sites at the top of the variable domain in a bispecific antibody, one complementary site targets the target antigen while the other targets a T lymphocyte antigen such as CD3. In the case of a trifunctional antibody, the Fc region can additionally bind to cells expressing Fc receptors, such as macrophages, natural killer (NK) cells, or dendritic cells. In summary, the targeted cell establishes a connection with one or two cells of the immune system, which subsequently destroys the targeted cell.

[0411] Other types of bispecific antibodies have been designed to overcome certain problems, such as short half-life, immunogenicity, and side effects caused by cytokine release. These include chemically linked Fabs consisting only of Fab regions, and various types of bivalent and trivalent single-chain variable fragments (scFvs) that mimic the variable domains of two antibodies in fusion proteins. Among these newer forms, the most well-developed are bispecific T-cell adaptor (BiTE) and mAb2 antibodies, which are antibodies engineered to contain Fcab antigen-binding fragments instead of the Fc constant region.

[0412] Using molecular genetics, two scFvs can be engineered to tandem into a single polypeptide, called a “tandem scFv” (tascFv), the two scFvs separated by an adapter domain. TascFvs have been found to have poor solubility and require refolding when produced in bacteria, or they can be manufactured in mammalian cell culture systems, which avoids the refolding requirement but may result in low yields. Constructing tascFvs using the genes of two different scFvs has yielded “bispecific single-stranded variable fragments” (bis-scFvs). Only two tascFvs have been developed by commercial companies and entered the clinical stage; both are bispecific agents, in active early development by Micromet for an oncology indication, and described as “bispecific T-cell connectors (BiTEs).” Blinatumomab is an anti-CD19 / anti-CD3 bispecific tascFv that enhances T-cell responses against B-cell non-Hodgkin lymphoma in phase II. MT110 is a bispecific anti-EP-CAM / anti-CD3 tascFv that enhances T cell responses to solid tumors in phase I. Affimed is also investigating a bispecific tetravalent "TandAb".

[0413] In some implementations, the cargo or payload may be, or may encode, an antibody containing a single antigen-binding domain. These molecules are very small, with a molecular weight approximately one-tenth that observed for full-size mAbs. Other antibodies may include “nanobodies,” derived from the antigen-binding variable heavy chain region (VHH) of heavy chain antibodies (lacking light chains) found in camels and llamas.

[0414] The technology disclosed and claimed in Memorial Sloan-Kettering Cancer Center PCT Publication WO2014144573 (the contents of which are incorporated herein by reference in their entirety) is a polymerization technique for preparing dimer multispecific binders (e.g., fusion proteins containing antibody components) that have improved properties over multispecific binders without dimerization capability.

[0415] In some cases, the cargo or payload may be, or may encode, a tetravalent bispecific antibody (such as TetBiAb disclosed and claimed in PCT Publication WO2014144357, the contents of which are incorporated herein by reference in their entirety). TetBiAb is characterized by a second pair of Fab fragments with a second antigen specificity attached to the C-terminus of the antibody, thus providing a molecule that is bivalent for each of the two antigens. The tetravalent antibody is produced through genetic engineering by covalently linking the antibody heavy chain to a Fab light chain, which associates with its corresponding, co-expressed Fab heavy chain.

[0416] In some aspects, the cargo or payload may be, or may encode, a biosynthetic antibody as described in U.S. Patent No. 5,091,513 (the contents of which are incorporated herein by reference in their entirety). Such an antibody may comprise one or more amino acid sequences constituting regions serving as biosynthetic antibody binding sites (BABS). These sites include 1) non-covalently associated or disulfide-bonded synthetic VH and VL dimers; 2) VH-VL or VL-VH single chains, wherein VH and VL are attached via polypeptide linkers; or 3) individual VH or VL domains. The binding domain comprises linked CDR and FR regions, which may be derived from individual immunoglobulins. Biosynthetic antibodies may also comprise other polypeptide sequences that function as attachment sites, for example, enzymes, toxins, binding sites, or immobilization media or radioactive atoms. Methods for producing biosynthetic antibodies, methods for designing BABS having any specificity that can be triggered by antibody production in vivo, and methods for producing analogues thereof are disclosed.

[0417] In some embodiments, the cargo or payload may be, or may encode, an antibody having an antibody receptor framework taught in U.S. Patent No. 8,399,625. Such antibody receptor frameworks may be particularly suitable for receiving a CDR from an antibody of interest. In some cases, a CDR from an anti-tau antibody known in the art or developed according to the methods presented herein may be used.

[0418] Miniaturized antibodies In some implementations, the cargo or payload may be, or can encode, a “miniaturized” antibody. One of the best examples of mAb miniaturization is Trubion Pharmaceuticals’ Small Modular Immunoplasmic Pulses (SMIPs). These molecules, which can be monovalent or bivalent, are recombinant single-chain molecules containing a VL, a VH antigen-binding domain, and one or two constant “effective” domains, all of which are linked by a linker domain. It is presumed that such molecules can provide the claimed advantage of increased tissue or tumor penetration through fragments while retaining the immune effector function conferred by the constant domains. At least three “miniaturized” SMIPs are in clinical development. TRU-015, an anti-CD20 SMIP developed in collaboration with Wyeth, is the most advanced project and has progressed to Phase II for rheumatoid arthritis (RA). Early attempts in systemic lupus erythematosus (SLE) and B-cell lymphoma were ultimately terminated. Trubion and Facet Biotechnology are collaborating on TRU-016, an anti-CD37 SMIP for the treatment of CLL and other lymphoid tumors, which is in Phase II. Wyeth has received approval for its anti-CD20 SMIP SBI-087 for the treatment of autoimmune diseases, including RA, SLE, and possibly multiple sclerosis, although these projects are still in the very early stages of clinical testing.

[0419] Double antibody In some embodiments, the cargo or payload may be or may encode a biantibody. As used herein, the term "biantibody" refers to a small antibody fragment having two antigen-binding sites. A biantibody contains a heavy chain variable domain VH linked to a light chain variable domain VL within the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with a complementary domain of the other chain, resulting in two antigen-binding sites.

[0420] Biantibodies are functional bispecific single-chain antibodies (bscAbs). These bivalent antigen-binding molecules consist of non-covalent dimers of scFvs and can be produced in mammalian cells using recombinant methods (see, for example, Mack et al., Proc. Natl. Acad. Sci., 92: 7021-7025, 1995). A small number of biantibodies have entered clinical development. In a study sponsored by the Beckman Institute in City of Hope (Clinicaltrials.gov NCT00647153), the iodine-123-labeled biantibody form of the anti-CEA chimeric antibody cT84.66 was evaluated for preoperative immunoscintigraphy in colorectal cancer.

[0421] Unibody In some implementations, the cargo or payload may be, or can be encoded as, a “monomer” in which the hinge region has been removed from the IgG4 molecule. While IgG4 molecules are unstable and can undergo light-heavy chain heterodimer exchange with each other, the absence of the hinge region completely prevents heavy chain-heavy chain pairing, leaving highly specific monovalent light / heavy heterodimers while retaining the Fc region to ensure stability and half-life in vivo. This configuration minimizes the risk of immune activation or oncogenic growth because the interaction between IgG4 and the FcR is weak, and the monovalent antibody does not promote the formation of intracellular signaling complexes. However, these views are largely supported by laboratory evidence rather than clinical evidence. Other antibodies may be “miniaturized” antibodies, compressed to 100 kDa.

[0422] Intracellular antibodies In some embodiments, the cargo or payload may be or may encode an intracellular antibody. The term "intracellular antibody" refers to an antibody form that is not secreted from the cell from which it is produced, but rather targets one or more intracellular proteins. Intracellular antibodies can be used to influence numerous cellular processes, including but not limited to intracellular transport, transcription, translation, metabolic processes, proliferation signaling, and cell division. In some embodiments, the methods of this disclosure may include intracellular antibody-based therapies. In some such embodiments, the variable domain sequences and / or CDR sequences disclosed herein may be incorporated into one or more constructs for intracellular antibody-based therapies. For example, intracellular antibodies may target one or more glycosylated intracellular proteins, or may modulate the interaction between one or more glycosylated intracellular proteins and another protein.

[0423] Intracellular antibodies targeting intracellular targets were first described more than two decades ago (Biocca, Neuberger and Cattaneo EMBO J. 9: 101-108, 1990, the contents of which are incorporated herein by reference in their entirety). Intracellular expression of intracellular antibodies in different compartments of mammalian cells allows for the blocking or modulation of the function of endogenous molecules (Biocca, et al., EMBO J. 9: 101-108, 1990; Colby et al., Proc.Natl.Acad.Sci.USA101:17616-21, 2004, the contents of which are incorporated herein by reference in their entirety). Intracellular antibodies can alter protein folding, protein-protein, protein-DNA, and protein-RNA interactions, and protein modifications. They can induce phenotypic knockout and act as neutralizers by directly binding to target antigens, translocating their intracellular transport, or inhibiting their binding to binding partners. They have been widely used as research tools and are gradually becoming therapeutic molecules for treating human diseases such as viral diseases, cancer, and misfolding diseases. The rapidly growing biomarket for recombinant antibodies offers intracellular antibodies with enhanced binding specificity, stability, and solubility, as well as lower immunogenicity, making them suitable for therapeutic use.

[0424] In some implementations, intracellular antibodies offer advantages over interfering RNA (iRNA); for example, iRNA has been shown to perform a variety of nonspecific functions, while intracellular antibodies have been demonstrated to have high specificity and affinity for target antigens. Furthermore, as proteins, intracellular antibodies have a much longer active half-life than iRNA. Therefore, when the active half-life of intracellular target molecules is long, gene silencing via iRNA may produce effects slowly, while the effects of intracellular antibody expression can be almost instantaneous. Finally, intracellular antibodies can be designed to block certain binding interactions of specific target molecules without affecting other binding interactions.

[0425] Intracellular antibodies are typically single-stranded variable fragments (scFvs) expressed by recombinant nucleic acid molecules and engineered to be retained within cells (e.g., in the cytoplasm, endoplasmic reticulum, or periplasm). Intracellular antibodies can be used to, for example, deactivate the function of proteins to which they bind. The expression of intracellular antibodies can also be modulated by using an inducible promoter in a nucleic acid expression vector containing the intracellular antibody. Intracellular antibodies can be generated using methods known in the art, such as those disclosed and reviewed in the following literature: Marasco et al., 1993 Proc. Natl. Acad. Sci. USA, 90: 7889-7893; Chen et al., 1994, Hum. Gene Ther. 5: 595-601; Chen et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 5932-5936; Maciejewski et al., 1995, Nature Med., 1: 667-673; Marasco, 1995, Immunotech, 1: 1-19; Mhashilkar, et al., 1995, EMBOJ. 14: 1542-51; Chen et al., 1996, Hum. Gene Therap., 7: 1515-1525; Marasco, Gene Ther. 4:11-15, 1997; Rondon and Marasco, 1997, Annu. Rev. Microbiol. 51: 257-283; Cohen, et al., 1998, Oncogene 17: 2445-56; Proba et al., 1998, J.Mol.Biol.275:245-253; Cohen et al., 1998, Oncogene 17:2445-2456; Hassanzadeh, et al., 1998, FEBS Lett.437:81-6; Richardson et al., 1998, Gene Ther.5:635-44; Ohage and Steipe, 1999,J. Mol. Biol. 291:1119-1128; Ohage et al., 1999, J. Mol. Biol. 291: 1129-1134; Wirtz and Steipe, 1999, Protein Sci. 8: 2245-2250; Zhu et al., 1999, J. Immunol.Methods 231:207-222; Arafat et al., 2000, Cancer Gene Ther. 7:1250-6; der Maur et al., 2002, J. Biol. Chem. 277:45075-85; Mhashilkar et al., 2002, Gene Ther. 9:307-19; and Wheeler et al., 2003, FASEB J. 17: 1733-5; and the references cited therein). Specifically, intracellular antibodies against CCR5 have been produced by Steinberger et al., 2000, Proc. Natl. Acad. Sci. USA 97:805-810. For general information, see Marasco, WA, 1998, "Intrabodies: Basic Research and Clinical Gene Therapy Applications," Springer: New York; and for reviews on scFv, see Pluckthun in "The Pharmacology of Monoclonal Antibodies," 1994, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315; each of these articles is incorporated herein by reference in its entirety.

[0426] Sequences derived from donor antibodies can be used to develop intracellular antibodies. Intracellular antibodies are typically recombinantly expressed within cells as single-domain fragments, such as isolated VH and VL domains, or as single-chain variable fragment (scFv) antibodies. For example, intracellular antibodies are often expressed as a single polypeptide to form a single-chain antibody containing variable domains of heavy and light chains linked by flexible linker polypeptides. Intracellular antibodies typically lack disulfide bonds and are able to regulate the expression or activity of target genes through their specific binding activity. Single-chain antibodies can also be expressed as single-chain variable region fragments linked to a light chain constant region.

[0427] As is known in the art, intracellular antibodies can be engineered into recombinant polynucleotide vectors to encode subcellular transport signals at their N- or C-terminus, thereby allowing high-concentration expression in the subcellular compartment where the target protein resides. For example, intracellular antibodies targeting the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and optionally a C-terminal ER retention signal. Intracellular antibodies intended to function in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are attached to intracellular antibodies to tether them to the cytoplasmic side of the plasma membrane. Intracellular antibodies can also be targeted to function in the cytosol. For example, cytosol-based intracellular antibodies are used to isolate factors in the cytosol, thereby preventing them from being transported to their native cellular target sites.

[0428] Intracellular antibody expression presents several technical challenges. In particular, the protein conformation, folding, and structural stability of newly synthesized intracellular antibodies are affected by the reducing conditions of the intracellular environment.

[0429] Intracellular antibodies can be promising therapeutic agents for misfolded diseases, including tau diseases, prion diseases, Alzheimer's disease, Parkinson's disease, and Huntington's disease, because they possess the ability to specifically recognize different conformations of proteins (including pathological isoforms) with virtually unlimited specificity, and because they can target potential aggregation sites (both intracellular and extracellular). These molecules can function as neutralizers against amyloid by inhibiting amyloid aggregation, and / or as molecular diverters of intracellular transport by redirecting protein pathways away from their potential aggregation sites.

[0430] Larger antibody (maxibody) In some implementations, the cargo or payload may be or may encode a large antibody (a bivalent scFV fused to the amino terminus of the Fc (CH2-CH3 domain)) encoding IgG.

[0431] Chimeric antigen receptor (CAR) In some embodiments, the cargo or payload may be or may encode a chimeric antigen receptor (CAR) that, when transduced into immune cells (e.g., T cells and NK cells), can redirect the immune cells to fight a target (e.g., tumor cells) that expresses a molecule recognized by the extracellular target portion of the CAR.

[0432] As used herein, the term "chimeric antigen receptor (CAR)" refers to a synthetic receptor that mimics the TCR on the surface of T cells. Generally, a CAR consists of an extracellular targeting domain, a transmembrane domain / region, and an intracellular signaling / activation domain. In a standard CAR receptor, the components—the extracellular targeting domain, the transmembrane domain, and the intracellular signaling / activation domain—are linearly constructed as a single fusion protein. The extracellular region contains the targeting domain / part of the receptor that recognizes a specific tumor antigen or other tumor cell surface molecules (e.g., scFv). The intracellular region may contain the signaling domain of the TCR complex (e.g., the CD3ζ signaling region), and / or one or more co-stimulatory signaling domains, such as those from CD28, 4-1BB (CD137), and OX-40 (Cd134). For example, first-generation CARs only have a CD3ζ signaling domain. To increase T cell persistence and proliferation, a co-stimulatory intracellular domain is added, resulting in second-generation CARs with a CD3ζ signaling domain plus one co-stimulatory signaling domain, and third-generation CARs with a CD3ζ signaling domain plus two or more co-stimulatory signaling domains. When expressed by T cells, the CAR confers antigen specificity to the T cells determined by the CAR's extracellular targeting portion. In some aspects, one or more elements, such as homing and suicide genes, can be added to develop more powerful and safer CAR structures (so-called fourth-generation CARs).

[0433] In some implementations, the extracellular targeting domain is connected to the intracellular signaling domain via a hinge (also called a spatial domain or spacer region) and a transmembrane region. The hinge connects the extracellular targeting domain to the transmembrane domain, which traverses the cell membrane and connects to the intracellular signaling domain. Due to the size of the target protein bound by the targeting moiety and the size and affinity of the targeting domain itself, the hinge may need to be modified to optimize the potency of CAR-transformed cells against cancer cells. When the targeting moiety recognizes and binds to the target cell, the intracellular signaling domain generates an activation signal for the CAR T cell, which is further amplified by a “second signal” from one or more intracellular co-stimulatory domains. Once activated, the CAR T cell can destroy the target cell.

[0434] In some implementations, the CAR can be split into two parts, each connected to a dimerization domain, such that input conditions triggering dimerization promote the assembly of a fully functional receptor. Wu and Lim reported a split CAR in which the extracellular CD19 binding domain and the intracellular signaling element are separated and connected to the FKBP domain and FRB. (FKBP-rapamycin-binding T2089L mutant) domains, which heterodimerize in the presence of the rapamycin analog AP21967. The split receptor assembles in the presence of AP21967 and binds to specific antigens to activate T cells (Wu et al., Science, 2015, 625(6258): aab4077, the contents of which are incorporated herein by reference in their entirety).

[0435] In some implementations, the CAR can be designed as an inducible CAR with a Tet-On induction system incorporated into the CD19 CAR construct. The CD19 CAR is activated only in the presence of doxycycline (Dox). Sakemura reported that Tet-CD19CAR T cells, in the presence of Dox, exhibited equivalent cytotoxicity to CD19+ cell lines and equivalent cytokine production and proliferation upon CD19 stimulation compared to conventional CD19CAR T cells (Sakemura et al., CancerImmuno. Res., 2016, Jun 21, Epub; the contents of which are incorporated herein by reference in their entirety). This dual system provides greater flexibility for the on / off switching of CAR expression in transduced T cells.

[0436] In some embodiments, the cargo or payload may be or may encode a first-generation, second-generation, third-generation, or fourth-generation CAR. In some embodiments, the cargo or payload may be or may encode a complete CAR construct consisting of an extracellular domain, a hinge and transmembrane domain, and an intracellular signaling region. In other embodiments, the cargo or payload may be or may encode components of a complete CAR construct, including an extracellular targeting portion, a hinge region, a transmembrane domain, an intracellular signaling domain, one or more co-stimulatory domains, and other additional elements that improve the structure and function of the CAR, including but not limited to leader sequences, homing elements, and safety switches, or combinations of these components.

[0437] In some implementations, the cargo or payload may be or may encode a modulotropic CAR. A reversible on / off switching mechanism allows for the management of acute toxicity caused by excessive CAR-T cell proliferation. Ligand-conferred CAR modulation can effectively counteract tumor escape caused by antigen loss, prevent functional exhaustion caused by tonic signaling induced by prolonged antigen exposure, and improve the persistence of CAR-expressing cells in vivo. Modulotropic CARs can be used to downregulate CAR expression to limit on-target tissue toxicity caused by tumor lysis syndrome. Downregulating CAR expression after achieving antitumor efficacy can prevent (1) on-target offtumor toxicity caused by antigen expression in normal tissues; and (2) antigen-independent activation in vivo.

[0438] Extracellular targeting domain / part In some implementations, the extracellular target moiety of the CAR can be any substance that recognizes and binds to a given target molecule (e.g., a neoantigen on tumor cells) with high specificity and affinity. The target moiety can be an antibody or a variant thereof that specifically binds to the target molecule on tumor cells, or a peptide aptamer selected from a random sequence library based on its ability to bind to the target molecule on tumor cells, or a variant or fragment thereof that can bind to the target molecule on tumor cells, or an antigen recognition domain from a natural T-cell receptor (TCR) (e.g., the CD4 extracellular domain that recognizes HIV-infected cells), or an exogenous recognition component such as a cytokine that leads to the recognition of a target cell with a cytokine receptor, or a natural ligand of the receptor.

[0439] In some embodiments, the targeting domain of the CAR can be an Ig NAR, a Fab fragment, a Fab' fragment, an F(ab)'2 fragment, an F(ab)'3 fragment, an Fv, a single-stranded variable fragment (scFv), a bis-scFv, (scFv)2, a minibody, a biantibody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a monomer, a nanobody, or an antigen-binding region derived from an antibody that specifically recognizes a target molecule (e.g., a tumor-specific antigen (TSA)). In one embodiment, the targeting portion is an scFv antibody. When the scFv domain is expressed on the surface of a CAR T cell and subsequently binds to a target protein on a cancer cell, it is able to hold the CAR T cell near the cancer cell and trigger T cell activation. scFv can be generated using conventional recombinant DNA techniques, which are discussed in this disclosure.

[0440] In some implementations, the targeting portion of the CAR construct can be an aptamer, such as a peptide aptamer that specifically binds to a target molecule of interest. The peptide aptamer can be selected from a random sequence library based on its ability to bind to the target molecule of interest.

[0441] In some implementations, the targeting portion of the CAR construct can be a natural ligand of the target molecule, or a variant and / or fragment thereof capable of binding to the target molecule. In some aspects, the targeting portion of the CAR can be a receptor for the target molecule; for example, full-length human CD27, as a CD70 receptor, can be fused in a frame-compliant manner with the CD3ζ signaling domain to form a CD27 chimeric receptor, serving as an immunotherapeutic agent for CD70-positive malignancies.

[0442] In some implementations, the targeting portion of the CAR can recognize tumor-specific antigens (TSA), such as cancer neoantigens that are restricted to expression on tumor cells.

[0443] As a non-limiting example, the CAR disclosed herein may include an extracellular targeting domain capable of binding to a tumor-specific antigen selected from 5T4, 707-AP, A33, AFP (alpha-fetoprotein), AKAP-4 (kinase-anchored protein 4), ALK, α5β1-integrin, androgen receptor, annexin II, α-actin-4, ART-4, B1, B7H3, B7H4, BAGE (B melanoma antigen), BCMA, BCR-ABL fusion protein, β-catenin, BKT antigen, BTAA, CA-I (carbonic anhydrase I), CA50 (cancer antigen 50), CA125, CA15-3, CA195, CA242, calreticulin, CAIX. (Carbonic anhydrase), CAMEL (cytotoxic T lymphocyte recognition antigen on melanoma), CAM43, CAP-1, caspase-8 / m, CD4, CD5, CD7, CD19, CD20, CD22, CD23, CD25, CD27 / m, CD28, CD30, CD33, CD34, CD36, CD38, CD40 / CD154, CD41, CD44v6, CD44v7 / 8, CD45, CD49f, CD56, CD68, KP1, CD74, CD79a / CD79b, CD103, CD123, CD133, CD138, CD171, cdc27 / m, CDK4 (cyclin-dependent kinase 4), CDKN2A, CDS, CEA (Carcinoembryonic antigen), CEACAM5, CEACAM6, Chromogranin, c-Met, c-Myc, coa-1, CSAp, CT7, CT10, Cyclophilin B, Cyclin B1, Cytosine kinase, Cytokeratin, DAM-10, DAM-6, Dek-can fusion protein, Desmin, DEPDC1 (containing DEP domain protein 1), E2A-PRL, EBNA, EGF-R (epidermal growth factor receptor), EGP-1 (epidermal glycoprotein-1) (TROP-2), EGP-2, EGP-40, EGFR (epidermal growth factor receptor), EGFRvIII, EF-2, ELF2M, EMMPRIN, EpCAM (epidermal cell adhesion molecule), EphA2, Epstein-Barr virus antigen, Erb (ErbB1; ErbB3; ErbB4), ETA (epidermal tumor antigen), ETV6-AML1 fusion protein, FAP (fibroblast activating protein), FBP (folate binding protein), FGF-5, folate receptor, FOS-associated antigen 1, fucose GM1, G250, GAGE ​​(GAGE-1);GAGE-2), galactoglobulin, GD2 (gangliosides), GD3, GFAP (glial fibrillary acidic protein), GM2 (carcinoembryonic antigen immunogenicity-1; OFA-I-1), GnT-V, Gp100, H4-RET, HAGE (helicase antigen), HER-2 / neu, HIF (hypoxia-inducible factor), HIF-1, HIF-2, HLA-A2, HLA-A; 0201-R170I, HLA-Al l, HMWMAA, Hom / Mel-40, HSP70-2M (heat shock protein 70), HST-2, HTgp-175, hTERT (or hTRT), human papillomavirus-E6 / human papillomavirus-E7 and E6, iCE (immune capture EIA), IGF-1R, IGH-IGK, IL-2R, IL-5, ILK (integrin-linked kinase), IMP3 (insulin-like growth factor II mRNA-binding protein 3), IRF4 (interferon regulatory factor 4), KDR (kinase insertion domain receptor), KIAA0205, KRAB-zinc finger protein (KID)-3, KID31, KSA (17-1A), K-ras, LAGE, LCK, LDLR / FUT (LDLR-fucosyltransferase AS fusion protein), LeY (Lewis Y), MAD-CT-1, MAGE (tyrosinase, melanoma-associated antigen) (MAGE-1);MAGE-3), melan-A tumor antigen (MART), MART-2 / Ski, MC1R (melanocortin 1 receptor), MDM2, mesothelin, MPHOSPH1, MSA (muscle-specific actin), mTOR (mammalian target of rapamycin), MUC-1, MUC-2, MUM-1 (melanoma-associated antigen (mutant) 1), MUM-2, MUM-3, myosin / m, MYL-RAR, NA88-A, n-acetylglucosamine transferase, neo-PAP, NF-KB (nuclear factor-κB), neurofilaments, NSE (neuron-specific enolase), Notch receptor, NuMa, N-Ras, NY-BR-1, NY-CO-1, NY-ESO-1, tumor suppressor M, OS-9, OY-TES1, p53 mutant, p190 minor bcr-abl (p190 minor bcr-abl), pl5(58), pl85erbB2, pl80erbB-3, PAGE (prostate-related genes), PAP (prostate acid phosphatase), PAX3, PAX5, PDGFR (platelet-derived growth factor receptor), cytochrome P450 (PIPA) involved in piperidine and pyrrolidine utilization, Pml-RARα fusion protein, PR-3 (proteinase 3), PSA (prostate-specific antigen), PSM, PSMA (prostate stem cell antigen), PRAME (melanoma preferentially expressed antigen), PTPRK, RAGE (renal tumor antigen), Raf (A-Raf, B-Raf, and C-Raf), Ras, receptor tyrosine kinase, RCAS1, RGSS, ROR1 (receptor tyrosine kinase-like orphan receptor 1), RU1, RU2, SAGE, SART-1, SART-3, SCP-1, SDCCAG16, SP-17 (sperm protein 17), src family, SSX (Synosal sarcoma X breakpoint)-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, STAT-3, STAT-5, STAT-6, STEAD, Stn, survivin, syk-ZAP70, TA-90 (Mac-2 binding protein / cyclophilin C-related protein), TAAL6, TACSTD1 (tumor-associated calcium signaling protein 1), TACSTD2, TAG-72-4, TAGE, TARP (T cell receptor γ variable reading frame protein), TEL / AML1 fusion protein, TEM1, TEM8 (endothelial sialic acid protein or CD248), TGFβ, TIE2, TLP, TMPRSS2 ETS fusion gene, TNF-receptor (TNF-α receptor, TNF-β receptor);Or TNF-γ receptor), transferrin receptor, TPS, TRP-1 (tyrosine-associated protein 1), TRP-2, TRP-2 / INT2, TSP-180, VEGF receptor, WNT, WT-1 (Wilson's blastoma antigen), and XAGE.

[0444] In some implementations, the cargo or payload may be or may encode a CAR containing a universal immune receptor having a targeting portion capable of binding a labeled antigen.

[0445] In some implementations, the cargo or payload may be or may be encoded with a CAR, which contains a targeting portion capable of binding pathogen antigens.

[0446] In some implementations, the cargo or payload may be or may be encoded as a CAR, which contains a targeting portion capable of binding to non-protein molecules such as tumor-associated glycolipids and carbohydrates.

[0447] In some embodiments, the cargo or payload may be or may encode a CAR containing a targeting portion capable of binding to components in the tumor microenvironment, including proteins expressed in various tumor stromal cells, including tumor-associated macrophages (TAMs), immature monocytes, immature dendritic cells, immunosuppressive CD4+CD25+ regulatory T cells (Tregs), and MDSCs.

[0448] In some implementations, the cargo or payload may be or may be encoded as a CAR, which includes a targeting portion of a TCR capable of binding to cell surface adhesion molecules, surface molecules of inflammatory cells present in autoimmune diseases, or inducing autoimmunity. As a non-limiting example, the targeting portion of this disclosure may be an scFv antibody that recognizes tumor-specific antigens (TSA), such as scFvs of antibodies SS, SS1 and HN1 that specifically recognize and bind to human mesothelin, scFvs of GD2 antibodies, CD19 antigen-binding domains, NKG2D ligand-binding domains, human anti-mesothelin scFvs, anti-CS1 binders, anti-BCMA binding domains, anti-CD19 scFv antibodies, GFRα4 antigen-binding fragments, anti-CLL-1 (C-type lectin-like molecule 1) binding domains, CD33 binding domains, GPC3 (phosphatidylinositol proteoglycan-3) binding domains, GFRα4 (glycosylphosphatidylinositol (GPI) linked GDNF family α-receptor 4 cell surface receptor) binding domains, CD123 binding domains, anti-ROR1 antibodies or fragments thereof, scFvs specifically targeting GPC-3, scFvs targeting CSPG4, and scFvs targeting folate receptor α.

[0449] Intracellular signal transduction domains The intracellular domain of a CAR fusion peptide, upon binding to its target molecule, signals to immune effector cells, activating at least one normal effector function of the immune effector cells, including cytolytic activity (e.g., cytokine secretion) or co-operational activity. Therefore, the intracellular domain comprises the "intracellular signal transduction domain" of the T cell receptor (TCR).

[0450] In some cases, the entire intracellular signal transduction domain can be used. In others, a truncated portion of the intracellular signal transduction domain can be used instead of the complete chain, as long as it transduces the functional signal.

[0451] In some implementations, the intracellular signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from the TCR CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In one instance, the intracellular signaling domain is the CD3ζ (CD3zeta) signaling domain.

[0452] In some implementations, the intracellular region also includes one or more co-stimulatory signaling domains that provide additional signals to immune effector cells. These co-stimulatory signaling domains, combined with signaling domains, can further improve the expansion, activation, memory, persistence, and tumor eradication efficiency of CAR-engineered immune cells (e.g., CAR T cells). In some cases, the co-stimulatory signaling region contains one, two, three, or four cytoplasmic domains of one or more intracellular signaling and / or co-stimulatory molecules. The co-stimulatory signaling domains can be intracellular / cytoplasmic domains of co-stimulatory molecules, including but not limited to CD2, CD7, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS (CD278), GITR (glucocorticoid-induced tumor necrosis factor receptor), LFA-1 (lymphocyte function-associated antigen-1), LIGHT, NKG2C, and B7-H3. In one example, the co-stimulatory signaling domain is derived from the cytoplasmic domain of CD28. In another instance, the costimulatory signaling domain is derived from the cytoplasmic domain of 4-1BB (CD137). In yet another instance, the costimulatory signaling domain may be an intracellular domain of GITR as taught in U.S. Patent No. 9,175,308, the contents of which are incorporated herein by reference in their entirety.

[0453] In some embodiments, the intracellular region may include a functional signaling domain selected from the following proteins: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation proteins (SLAMs) such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10, SLAMF6, SLAMF7, activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, IL-15Ra, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD4 9f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, NKD2C SLP76, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, CD270 (HVEM), GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, DAP 10. TRIM, ZAP70, killer immunoglobulin receptors (KIRs) such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3 and KIR2DP1;Lectin-associated NK cell receptors, such as Ly49, Ly49A, and Ly49C.

[0454] In some embodiments, the intracellular signal transduction domain of this disclosure may include a signal transduction domain derived from JAK-STAT. In other embodiments, the intracellular signal transduction domain of this disclosure may include a signal transduction domain derived from DAP-12 (death-associated protein 12) (Topfer et al., Immunol., 2015, 194: 3201-3212; and Wang et al., Cancer Immunol., 2015, 3: 815-826). DAP-12 is a key signal transduction receptor in NK cells. Activation signals mediated by DAP-12 play an important role in triggering NK cell cytotoxic responses against certain tumor cells and virus-infected cells. The cytoplasmic domain of DAP12 contains the immune receptor tyrosine activation motif (ITAM). Therefore, CARs containing DAP12-derived signal transduction domains can be used for adoptive transfer of NK cells.

[0455] Transmembrane domain In some embodiments, the CAR may include a transmembrane domain. As used herein, the term "transmembrane domain (TM)" broadly refers to an amino acid sequence of about 15 residues in length that spans the plasma membrane. The transmembrane domain may contain at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid residues and span the plasma membrane. In some embodiments, the transmembrane domain may be derived from a natural or synthetic source. The transmembrane domain of the CAR may be derived from any natural membrane-binding or transmembrane protein. For example, the transmembrane region may be derived from the T cell receptor α chain, β chain or ζ chain, CD3ε, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152 or CD154 (i.e., a transmembrane region containing at least the foregoing).

[0456] Optionally, the transmembrane domains of this disclosure can be synthetic. In some aspects, the synthetic sequences may primarily contain hydrophobic residues, such as leucine and valine.

[0457] In some implementations, the transmembrane domain may be selected from the CD8α transmembrane domain, CD4 transmembrane domain, CD28 transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, and human IgG4 Fc region.

[0458] In some implementations, the CAR may include an optional hinge region (also called a spacer region). The hinge sequence is a short amino acid sequence that contributes to the flexibility of the extracellular targeting domain, allowing the target-binding domain to move away from the effector cell surface to achieve proper cell / cell contact, target binding, and effector cell activation. The hinge sequence may be located between the targeting portion and the transmembrane domain. The hinge sequence can be any suitable sequence derived from or obtained from any suitable molecule. The hinge sequence may be derived from the hinge region of immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4), i.e., all or part of the sequence located between the CHI and CH2 domains of immunoglobulins (e.g., the IgG4 Fc hinge), or the extracellular regions of type 1 membrane proteins such as CD8α, CD4, CD28, and CD7, which may be wild-type sequences or derivatives. Some hinge regions contain the immunoglobulin CH3 domain or both the CH3 and CH2 domains. In some embodiments, the hinge region may be obtained from modification with IgG1, IgG2, IgG3 or IgG4, which contains one or more amino acid residues (e.g., 1, 2, 3, 4 or 5 residues) that are replaced by amino acid residues different from those present in the unmodified hinge.

[0459] In some embodiments, the CAR may include one or more linkers between any of the CAR domains. The linker length can be 1-30 amino acids. In this respect, the linker length can be 1, 2, 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, or 30 amino acids. In other embodiments, the linker may be flexible.

[0460] In some implementations, components including the targeting portion, transmembrane domain, and intracellular signaling domain can be constructed within a single fusion peptide. The fusion peptide can be a payload.

[0461] In some implementations, the cargo or payload may be or may encode a CD19-specific CAR targeting different B-cell malignancies and a HER2-specific CAR targeting sarcoma, glioblastoma, and advanced HER2-positive lung malignancies.

[0462] Tandem CAR In some implementations, the CAR may be a tandem chimeric antigen receptor (TanCAR) capable of targeting two, three, four, or more tumor-specific antigens. In some aspects, the CAR is a bispecific TanCAR containing two targeting domains that recognize two different TSAs on tumor cells. A bispecific TanCAR may be further defined as containing an extracellular region comprising a targeting domain specific to a first tumor antigen (e.g., an antigen recognition domain) and a targeting domain specific to a second tumor antigen (e.g., an antigen recognition domain). In other aspects, the CAR is a multispecific TanCAR containing three or more targeting domains arranged in tandem. The length of the spacer region between the targeting domains in the TanCAR may be from about 5 to about 30 amino acids, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 amino acids.

[0463] Split CAR In some implementations, the CAR component, comprising a targeting moiety, a transmembrane domain, and an intracellular signaling domain, can be split into two or more parts, such that it depends on multiple input conditions that facilitate the assembly of a fully functional receptor. As a non-limiting example, a split CAR consists of two parts assembled in a small molecule-dependent manner; one part of the receptor is characterized by an extracellular antigen-binding domain (e.g., scFv), and the other part has an intracellular signaling domain, such as the CD3ζ intracellular domain.

[0464] In other respects, the split portion of the CAR system can be further modified to enhance signaling. As a non-limiting example, the second part of the cytoplasmic fragment can be anchored to the plasma membrane by incorporating a transmembrane domain (e.g., the CD8α transmembrane domain) into the construct. Additional extracellular domains, such as those mediating homodimerization, can also be added to the second part of the CAR system. These modifications can increase receptor export activity, i.e., T cell activation.

[0465] In some implementations, the two parts of the split-component CAR system contain heterodimerization domains that conditionally interact upon binding to the heterodimerizing small molecule. Thus, the receptor component assembles into a complete system in the presence of the small molecule, which can then be activated via antigen conjugation. Any known heterodimerizing component can be incorporated into the split-component CAR system. Other small molecule-dependent heterodimerization domains can also be used, including but not limited to gibberellin-induced dimerization systems (GID1-GAI), trimethoprim-SLF-induced dimerization of ecDHFR and FKBP, and ABA (abscisic acid)-induced dimerization of PP2C and PYL domains. The dual regulation of inducible assembly (e.g., ligand-dependent dimerization) and degradation (e.g., destabilizing domain-induced CAR degradation) in split-component CAR systems can provide greater flexibility to control the activity of CAR-modified T cells.

[0466] Switchable CAR In some implementations, the CAR can be a switchable CAR, a controllable CAR that can be transiently activated in response to a stimulus (e.g., a small molecule). In such a CAR design, a system is directly integrated into a hinge domain that separates the scFv domain from the cell membrane domain within the CAR. This system can disassemble or combine different key functions of the CAR (e.g., activation and co-stimulation) within different chains of the receptor complex, thereby mimicking the complexity of the native TCR structure. This integrated system can switch between on / off states of scFv and antigen interaction, controlled by the absence / presence of a stimulus.

[0467] Reversible CAR In some implementations, the CAR can be a reversible CAR system. In such a CAR structure, the LID domain (ligand-induced degradation) is incorporated into the CAR system. CAR can be temporarily downregulated by adding a ligand with the LID domain.

[0468] Inhibitory CAR (iCAR) In some implementations, the CAR can be an inhibitory CAR. An inhibitory CAR (iCAR) refers to a bispecific CAR design where a negative signal is used to enhance tumor specificity and limit normal tissue toxicity. This design incorporates a second CAR having a surface antigen recognition domain combined with the inhibitory signaling domain to limit T cell reactivity, even when activation receptor binding occurs simultaneously. This antigen recognition domain targets normal tissue-specific antigens, allowing T cells to be activated in the presence of the first target protein, but T cell activation is inhibited in the presence of the second protein bound to the iCAR.

[0469] As a non-limiting example, iCARs targeting prostate-specific membrane antigen (PMSA) based on the inhibitory domains of CTLA4 and PD1 have shown the ability to selectively restrict cytokine secretion, cytotoxicity, and proliferation induced by T cell activation.

[0470] Chimeric conversion receptor In some embodiments, the cargo or payload may be, or may encode, a chimeric conversion receptor that can convert negative signals into positive signals. As used herein, the term "chimeric conversion receptor" refers to a fusion protein comprising a first extracellular domain and a second transmembrane and intracellular domain, wherein the first domain contains a negative signaling region and the second domain contains a positive intracellular signal transduction region. In some aspects, the fusion protein is a chimeric conversion receptor comprising an extracellular domain of an inhibitory receptor on a T cell, said extracellular domain being fused to a transmembrane and cytoplasmic domain of a co-stimulatory receptor. This chimeric conversion receptor can convert T cell inhibitory signals into T cell stimulatory signals.

[0471] As a non-limiting example, chimeric conversion receptors may include an extracellular domain of PD-1 fused with the transmembrane and cytoplasmic domains of CD28. In some respects, the extracellular domains of other inhibitory receptors such as CTLA-4, LAG-3, TIM-3, KIR, and BTLA may also be fused with transmembrane and cytoplasmic domains derived from co-stimulatory receptors such as CD28, 4-1BB, CD27, OX40, CD40, GTIR, and ICOS.

[0472] In some embodiments, the chimeric conversion receptor may include a recombinant receptor comprising an extracellular cytokine-binding domain of an inhibitory cytokine receptor (e.g., IL-13 receptor α (IL-13Rα1), IL-10R, and IL-4Rα) fused to an intracellular signaling domain of a stimulatory cytokine receptor, such as IL-2R (IL-2Rα, IL-2Rβ, and IL-2Rγ) and IL-7Rα. An example of such a chimeric cytokine receptor is a recombinant receptor containing an extracellular cytokine-binding domain of IL-4Rα linked to the intracellular signaling domain of IL-7Rα.

[0473] In some embodiments, the chimeric conversion receptor may be a chimeric TGFβ receptor. The chimeric TGFβ receptor may include an extracellular domain derived from a TGFβ receptor and an intracellular domain derived from a non-TGFβ receptor, such as TGFβ receptor 1, TGFβ receptor 2, TGFβ receptor 3, or any other TGFβ receptor or variant thereof. The non-TGFβ receptor intracellular domain may be an intracellular domain or fragment thereof derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CD28, 4-1BB (CD137), OX40 (CD134), cd3ζ, CD40, CD27, or combinations thereof.

[0474] Activation-conditional CAR In some embodiments, the cargo or payload may be, or may encode, an activation-conditional chimeric antigen receptor (CAR) that is expressed only in activated immune cells. CAR expression may be coupled to an activation-conditional control region, which is one or more nucleic acid sequences transcribed and / or expressed under the control of an inducible sequence (e.g., CAR). This activation-conditional control region may be a gene promoter upregulated during immune effector cell activation, such as the IL2 promoter or an NFAT binding site.

[0475] CAR targeting tumor cells with specific proteoglycan markers In some implementations, the cargo or payload may be, or may encode, a CAR targeting a specific type of cancer cell. Human cancer cells and metastases may express unique and otherwise aberrant proteoglycans, such as polysaccharide chains (e.g., chondroitin sulfate (CS), dermatan sulfate (DS or CSB), heparan sulfate (HS), and heparin). Therefore, the CAR can be fused with a binding moiety that recognizes cancer-associated proteoglycans. In one example, the CAR can be fused with a VAR2CSA peptide (VAR2-CAR), which binds with high affinity to a specific type of chondroitin sulfate A (CSA) linked to the proteoglycan. The extracellular ScFv portion of the CAR can be replaced with a VAR2CSA variant containing at least a minimal CSA-binding domain, producing a CAR specific for chondroitin sulfate A (CSA) modification. Optionally, the CAR can be fused with a split protein binding system to produce a spy-CAR, wherein the scFv portion of the CAR is replaced by a part of the split protein binding system (e.g., SpyTag and Spy-catcher), and cancer recognition molecules (e.g., scFv and / or VAR2-CSA) are linked to the CAR via the split protein binding system.

[0476] Nucleic acid The lipid nanoparticles disclosed herein may include a payload region (also referred to as a cargo region) that is a nucleic acid. The term "nucleic acid" in its broadest sense includes any compound and / or substance comprising a nucleotide polymer, which may be referred to as a polynucleotide. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof.

[0477] In some implementations, the payload region contains nucleic acid sequences encoding more than one type of cargo or payload.

[0478] In some implementations, the payload region may be a sequence encoding a nucleic acid sequence or a sequence encoding a specific nucleic acid sequence.

[0479] In some implementations, the payload region can be a non-coding nucleic acid sequence or a coding non-coding nucleic acid sequence.

[0480] In some implementations, the payload region can be a coding nucleic acid sequence and a non-coding nucleic acid sequence, or a coding-coding nucleic acid sequence and a non-coding nucleic acid sequence.

[0481] DNA Deoxyribonucleic acid (DNA) is a molecule that carries all the genetic information of an organism and consists of two intertwined chains forming a shape called a double helix. Each chain has a backbone composed of alternating sugars (deoxyribose) and phosphate groups. Each sugar is attached to one of four bases: adenine (A), cytosine (C), guanine (G), and thymine (T). The two chains are held together by bonds between adenine and thymine or between cytosine and guanine. The base sequence along the backbone acts as the instruction for assembling proteins and RNA molecules.

[0482] In some implementations, the payload region may be encoding DNA or encoding-encoding DNA.

[0483] In some implementations, the payload region can be non-coding DNA or coding non-coding DNA.

[0484] In some implementations, the payload region can be either coding DNA or non-coding DNA, or coding DNA or non-coding DNA.

[0485] In some implementations, the DNA may be modified. Types of modification include, but are not limited to, methylation, acetylation, phosphorylation, ubiquitination, and SUMOylation.

[0486] carrier In some embodiments, the starting construct and / or baseline construct described herein may be a vector, such as a plasmid or viral vector, or may be encoded by such a vector. In some embodiments, the starting construct and / or baseline construct is a viral vector or is encoded by such a vector. The viral vector may be, but is not limited to, herpesvirus (HSV) vectors, retroviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, lentiviral vectors, etc. In some embodiments, the viral vector is an AAV vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is an adenovirus vector.

[0487] Adeno-associated virus (AAV) vector The Parvoviridae family comprises small, non-enveloped icosahedral capsid viruses characterized by a single-stranded DNA genome. Parvoviridae consists of two subfamilies: the Parvovirinae, which infects vertebrates, and the Densovirinae, which infects invertebrates. Due to their relatively simple structure and ease of manipulation using standard molecular biology techniques, this family of viruses can be used as biological tools. The viral genome can be modified to contain the minimum components required for assembling functional recombinant viruses or viral particles loaded with or engineered to express or deliver desired payloads to target cells, tissues, organs, or organisms.

[0488] The parvoviridae family includes the genus Dependovirus, which includes adeno-associated viruses (AAVs) capable of replicating in vertebrate hosts, including but not limited to human, primate, cattle, dog, horse, and sheep species.

[0489] The AAV vector genome is a linear, single-stranded DNA (ssDNA) molecule, approximately 5,000 nucleotides (nt) in length. The AAV vector genome may contain a payload region and at least one inverted terminal repeat (ITR) or ITR region. ITRs are typically located flanking the coding nucleotide sequences of non-structural proteins (encoded by Rep genes) and structural proteins (encoded by capsid or Cap genes). Not wishing to be bound by theory, the AAV vector genome typically contains two ITR sequences. The AAV vector genome contains a characteristic T-shaped hairpin structure, defined by 145 self-complementary nucleotides at the 5' and 3' ends of the ssDNA, forming an energy-stable double-stranded region. The double-stranded hairpin structure has multiple functions, including, but not limited to, acting as a primer for the host viral replication cell's endogenous DNA polymerase complex, serving as the starting point for DNA replication.

[0490] In addition to encoding heterologous payloads, the AAV vector genome may contain, wholly or partially, any naturally occurring and / or recombinant AAV serotype nucleotide sequence or variant. AAV variants may have significantly homologous sequences at both the nucleic acid (genome or capsid) and amino acid (capsid) levels to produce generally physically and functionally equivalent constructs that replicate and assemble through similar mechanisms. Chiorini et al., J. Vir.71: 6823-33 (1997); Srivastava et al., J. Vir.45:555-64 (1983); Chiorini et al., J. Vir.73:1309-1319 (1999); Rutledge et al., J. Vir.72:309-319 (1998); and Wu et al., J. Vir.74:8635-47 (2000), the contents of which are incorporated herein by reference in their full text.

[0491] In some implementations, the AAV vector genome contains at least one control element that enables the replication, transcription, and translation of the coding sequence encoded therein. Not all control elements need to be present at all times, as long as the coding sequence can be replicated, transcribed, and / or translated in a suitable host cell. Non-limiting examples of expression control elements include transcription initiation and / or termination sequences, promoter and / or enhancer sequences, effective RNA processing signals such as splicing and polyadenylation signals, sequences stabilizing cytoplasmic mRNA, sequences that enhance translation efficiency (e.g., Kozak concordant sequences), sequences that enhance protein stability, and / or sequences that enhance protein processing and / or secretion.

[0492] AAV vector genomes can be generated through recombination and can be based on adeno-associated virus (AAV) parent or reference sequences. As used herein, “vector genome” is any molecule or portion that transports, transduces, or otherwise acts as a vector for heterologous molecules such as nucleic acids described herein.

[0493] In addition to single-stranded AAV vector genomes (e.g., ssAAV), this disclosure also provides self-complementary AAV (scAAV) vector genomes. The scAAV vector genome contains DNA strands that anneal together to form double-stranded DNA. By skipping second-strand synthesis, scAAV allows for rapid expression in cells.

[0494] In some implementations, the AAV vector genome is scAAV.

[0495] In some implementations, the AAV vector genome is ssAAV.

[0496] In some implementations, the AAV vector genome may be part of the AAV particle, wherein the serotype of the capsid may be, but is not limited to, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, etc. AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42 -11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.50, AAV2- 5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-11 / rh.53, AAV4-8 / r11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.5 8. AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16.12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu .42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60, AAV161.6 / hu.61, AAV33.12 / hu.17,AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAV-DJ 、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAV rh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-1 / hu.1, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVh u.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、 AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu. .37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.44 8、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8,AAV-PAEC11、AAV-PAEC12、AAV-2-pre-miRNA-101、AAV-8h、AAV-8b、AAV-h、AAV-b、AAV SM 10-2、AAVShuffle 100-1、AAV Shuffle 100-3、AAV Shuffle 100-7、AAV Shuffle 10-2、AAVShuffle 10-6、AAV Shuffle 10-8、AAV Shuffle 100-2、AAV SM 10-1、AAV SM 10-8、AAVSM 100-3、AAV SM 100-10、BNP61 AAV、BNP62 AAV、BNP63 AAV、AAVrh.50、AAVrh.43、AAVrh.62、AAVrh.48、AAVhu.19、AAVhu.11、AAVhu.53、AAV4-8 / rh.64、AAVLG-9 / hu.39、AAV54.5 / hu.2 3、AAV54.2 / hu.22、AAV54.7 / hu.24、AAV54.1 / hu.21、AAV54.4R / hu.27、AAV46.2 / hu.28、AAV46.6 / hu.29、AAV128.1 / hu.43、true type AAV (ttAAV)、UPENN AAV10、Japanese AAV 10 serotypes、AAV CBr-7.1、AAV CBr-7.10、AAV CBr-7.2、AAV CBr-7.3、AAV CBr-7.4、AAV CBr-7.5、AAV CBr-7.7、AAV CBr-7.8、AAV CBr-B7.3、AAV CBr-B7.4、AAV CBr-E1、AAV CBr-E2、AAV CBr-E3、AAV CBr-E4、AAV CBr-E5、AAV CBr-e5、AAV CBr-E6、AAV CBr-E7、AAV CBr-E8、AAV CHt-1、AAV CHt-2、AAV CHt-3、AAV CHt-6.1、AAV CHt-6.10、AAV CHt-6.5、AAV CHt-6.6、AAV CHt-6.7、AAV CHt-6.8、AAV CHt-P1、AAV CHt-P2、AAV CHt-P5、AAVCHt-P6、AAV CHt-P8、AAV CHt-P9、AAV CKd-1、AAV CKd-10、AAV CKd-2、AAV CKd-3、AAVCKd-4、AAV CKd-6、AAV CKd-7、AAV CKd-8、AAV CKd-B1、AAV CKd-B2、AAV CKd-B3、AAV CKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAV CKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAV CLv-1、AAV CLv1-1、AAV Clv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AAV CLv-K3、AAV CLv-K6、AAV CLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M11、AAV CLv-M2、AAV CLv-M5、AAV CLv-M6、AAV CLv-M7、AAV CLv-M8、AAV CLv-M9、AAV CLv-R1、AAV CLv-R2、AAV CLv-R3、AAV CLv-R4、AAV CLv-R5、AAV CLv-R6、AAV CLv-R7、AAV CLv-R8、AAV CLv-R9、AAV CSp-1、AAV CSp-10、AAV CSp-11、AAV CSp-2、AAV CSp-3、AAV CSp-4、AAV CSp-6、AAV CSp-7、AAVCSp-8、AAV CSp-8.10、AAV CSp-8.2、AAV CSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8、AAV CSp-8.9、AAV CSp-9、AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAVF9 / HSC9, PHP.B, PHP.A, G2B-26, G2B-13, TH1.1-32 and / or TH1.1-35 and their variants.

[0497] Inverted terminal repeat (ITR) In some embodiments, the AAV vector genome may contain at least one ITR region and a payload region. In some embodiments, the vector genome has two ITRs. These two ITRs are flanked by the payload region at the 5' and 3' ends. The ITR acts as an origin of replication, containing recognition sites for replication. The ITR contains sequence regions that can be complementary and symmetrically arranged. The ITRs incorporated into the vector genome may contain naturally occurring polynucleotide sequences or recombinant-derived polynucleotide sequences.

[0498] ITRs can originate from the same serotype as the capsid or its derivatives. ITRs can have different serotypes than the capsid. In some embodiments, the AAV particle has more than one ITR. In a non-limiting example, the AAV particle has a vector genome containing two ITRs. In some embodiments, the ITRs have the same serotype as each other. In another embodiment, the ITRs have different serotypes. Non-limiting examples include zero, one, or two ITRs having the same serotype as the capsid. In some embodiments, both ITRs of the AAV particle's vector genome are AAV2 ITRs.

[0499] Each ITR can be independently about 100 to about 150 nucleotides in length. ITRs can be about 100-105 nucleotides long, 106-110 nucleotides long, 111-115 nucleotides long, 116-120 nucleotides long, 121-125 nucleotides long, 126-130 nucleotides long, 131-135 nucleotides long, 136-140 nucleotides long, 141-145 nucleotides long, or 146-150 nucleotides long. In some embodiments, the ITR is 140-142 nucleotides long. Non-limiting examples of ITR lengths are 102, 140, 141, 142, 145 nucleotides long, and those having at least 95% identity with these lengths.

[0500] promoter In some implementations, the payload region of the vector genome contains at least one element that enhances transgene target specificity and expression (see, for example, Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are incorporated herein by reference in their entirety). Non-limiting examples of elements that enhance transgene target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (PolyA) signal sequences and upstream enhancers (USEs), CMV enhancers, and introns.

[0501] In some implementations, it is effective when the promoter drives the expression of the polypeptide encoded in the payload region of the vector genome of the AAV particle.

[0502] In some implementations, a promoter is considered effective when it drives expression in the targeted cells.

[0503] In some implementations, promoter-driven payload expression in target tissue persists for a duration of time. Promoter-driven expression can persist for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, etc. The time periods are: days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more than 10 years. The duration of expression can be 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, or 5-10 years.

[0504] In some implementations, the promoter-driven payload expression persists for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, and 20 years. Year, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65 or more.

[0505] Promoters can be naturally occurring or non-natural. Non-limiting examples of promoters include viral promoters, plant promoters, and mammalian promoters. In some embodiments, the promoter may be a human promoter. In some embodiments, the promoter may be truncated.

[0506] Promoters that drive or promote expression in most tissues include, but are not limited to, human elongation factor 1α subunit (EF1α), cytomegalovirus (CMV) immediate early enhancer and / or promoter, chicken β-actin (CBA) and its derivative CAG, β-glucuronidase (GUSB), or ubiquitin C (UBC). Tissue-specific expression elements can be used to restrict expression to certain cell types, such as, but not limited to, muscle-specific promoters, B-cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, astrocyte promoters, or neural system promoters that can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.

[0507] Non-limiting examples of muscle-specific promoters include the mammalian muscle creatine kinase (MCK) promoter, the mammalian desmin (DES) promoter, the mammalian troponin I (TNNI2) promoter, and the mammalian skeletal α-actin (ASKA) promoter (see, for example, U.S. Patent Publication US20110212529, the contents of which are incorporated herein by reference in their entirety).

[0508] Non-limiting examples of tissue-specific expression elements for neurons include neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synaptic protein (Syn), methyl CpG-binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabolite glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or heavy chain (NFH), β-globin small gene nβ2, proenkephalinogen (PPE), enkephalin (Enk), and excitatory amino acid transporter 2 (EAAT2) promoters. Non-limiting examples of tissue-specific expression elements for astrocytes include glial fibrillary acidic protein (GFAP) and the EAAT2 promoter. Non-limiting examples of tissue-specific expression elements for oligodendrocytes include myelin basic protein (MBP) promoters.

[0509] In some implementations, the promoter can be less than 1 kb. The promoter can have values ​​of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 5 The length of 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more nucleotides. The promoter can have lengths of 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800.

[0510] In some implementations, the promoter can be a combination of two or more components of the same or different starting promoters or parental promoters (e.g., but not limited to CMV and CBA). Each component can have 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 410, 420, 430, 440, 450, Lengths of 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more. Each component can have a length of 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800. In some embodiments, the promoter is a combination of a 382-nucleotide CMV enhancer sequence and a 260-nucleotide CBA promoter sequence.

[0511] In some implementations, the vector genome contains a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include CMV, CBA (including derivatives CAG, CBh,...

Claims

1. A compound of formula (I): (I) Or its pharmaceutically acceptable salt. in A does not exist or is -CH2-; B is -CH2- or -CH2CH2-; Where A does not exist, then B is -CH2CH2-, and when A is -CH2-, then B is -CH2-; X is either -NR1R2 or -O-(C=O)R 1’ ; R1 is H or an optional substituted straight-chain C1-C4 alkyl group; R2 is H, an optionally substituted straight-chain C1-C8 alkyl group, -C(=O)R7, -S(=O)2-R7, or... ; R 1’ It is a straight-chain C1-C8 alkyl group that can be optionally substituted; R 20 It is H or an optional substituted straight-chain C1-C8 alkyl group; R 21 It is an optional substituted straight-chain C1-C8 alkyl or -(C=O)-R 22 , where R 22 It is an optional substituted C1-C8 alkyl group; R3 and R4 are independently H, a straight-chain or branched C4-C60 alkyl, a straight-chain or branched C4-C60 alkenyl, or a straight-chain or branched C4-C60 alkynyl, wherein at least one of R3 or R4 is different from H; R5 and R6 are independently straight-chain or branched C4-C60 alkyl, straight-chain or branched C4-C60 alkenyl, or straight-chain or branched C4-C60 alkynyl. R7 is an optionally substituted straight-chain C1-C8 alkyl, optionally substituted straight-chain C2-C8 alkenyl, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group. When any one of R3, R4, R5, and R6 represents an alkenyl group, the alkenyl group independently contains 1 to 6 C=C bonds, each C=C bond independently having an E or Z configuration; When any one of R3, R4, R5, and R6 represents an alkynyl group, the alkynyl group independently contains 1 to 6 C≡C bonds; When R7 represents an alkenyl group, the alkenyl group contains one or two C=C bonds, each C=C bond independently having an E or Z configuration; When any alkyl and / or alkenyl group is substituted, that group is independently substituted by one or more of the following groups: -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 , -O-(C=O)-CH3, -O-(C=O)-(CH2) p CH3, -O-(C=O)-(CH2) p -R 19 ,-(C=O)-OCH3,-(C=O)-O-(CH2) p CH3、-(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -R 19 -(C=O)NH-(CH2) p -R 19 ,-(C=S)-CH3,-O-CH3,-O-(CH2) p CH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p -R 19 -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, optionally substituted 5- to 10-membered heterocyclic groups, C5-C12 aralkyl, C5-C10 aryl, or optionally substituted 5- to 10-membered heteroaryl groups, wherein p and p' are independently 1 to 5; and wherein R 19 It is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5 to 10 heterocyclic groups or optionally substituted 5 to 10 heteroaryl groups; When any 5- to 10-membered heteroaryl group and / or 5- to 10-membered heterocyclic group is substituted, the group is independently selected from C1-C4 alkyl groups, -(CH2) groups, etc. p’’ The group consisting of N(CH3)2 and oxo (=O) is substituted with one or more substituents, where p'' is 1 to 5; The conditions are that A is absent, B is -CH2CH2-, X is -NR1R2, R1 is -CH3, and each of R3, R4, R5, and R6 has carbons at C9 and C6. 10 A C=C bond between carbon and carbon C 12 and C 13 When R2 is a straight-chain C18 alkyl group with one C=C bond, then R2 is different from -CH3, -CH2CH2OH and -CH2CH2NH2.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) has the structure of formula (I1): (I1) in R1 is H or an optional substituted straight-chain C1-C4 alkyl group; R2 is H, an optionally substituted straight-chain C1-C8 alkyl group, -C(=O)R7, -S(=O)2-R7, or... ; R 20 It is H or an optional substituted straight-chain C1-C8 alkyl group; R 21 It is an optional substituted straight-chain C1-C8 alkyl or -(C=O)-R 22 , where R 22 It is an optional substituted C1-C8 alkyl group; R3 and R4 are independently H, a straight-chain or branched C4-C60 alkyl, a straight-chain or branched C4-C60 alkenyl, or a straight-chain or branched C4-C60 alkynyl, wherein at least one of R3 or R4 is different from H; R5 and R6 are independently straight-chain or branched C4-C60 alkyl, straight-chain or branched C4-C60 alkenyl, or straight-chain or branched C4-C60 alkynyl. R7 is an optionally substituted straight-chain C1-C8 alkyl, optionally substituted straight-chain C2-C8 alkenyl, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group. When any one of R3, R4, R5, and R6 represents an alkenyl group, the alkenyl group independently contains 1 to 6 C=C bonds, each C=C bond independently having an E or Z configuration; When any one of R3, R4, R5, and R6 represents an alkynyl group, the alkynyl group independently contains 1 to 6 C≡C bonds; When R7 represents an alkenyl group, the alkenyl group contains one or two C=C bonds, each C=C bond independently having an E or Z configuration; When any alkyl and / or alkenyl group is substituted, that group is independently substituted by one or more of the following groups: -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 , -O-(C=O)-CH3, -O-(C=O)-(CH2) p CH3, -O-(C=O)-(CH2) p -R 19 ,-(C=O)-OCH3,-(C=O)-O-(CH2) p CH3、-(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -R 19 -(C=O)NH-(CH2) p -R 19 ,-(C=S)-CH3,-O-CH3,-O-(CH2) p CH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p -R 19 -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, optionally substituted 5- to 10-membered heterocyclic groups, C5-C12 aralkyl, C5-C10 aryl, or optionally substituted 5- to 10-membered heteroaryl groups, wherein p and p' are independently 1 to 5; and wherein R 19 It is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5- to 10-membered heterocyclic groups or optionally substituted 5- to 10-membered heteroaryl groups. When any 5- to 10-membered heteroaryl group and / or 5- to 10-membered heterocyclic group is substituted, the group is independently selected from C1-C4 alkyl groups, -(CH2) groups, etc. p’’ One or more substituents of the group consisting of N(CH3)2 and oxo (=O) are substituted, wherein p'' is 1 to 5.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein... R1 is H or an optional substituted straight-chain C1-C4 alkyl group; R2 is H, an optionally substituted straight-chain C1-C4 alkyl group, -C(=O)R7, -S(=O)2-R7, or... ; R 20 It is H or an optional substituted straight-chain C1-C8 alkyl group; R 21 It is an optional substituted straight-chain C1-C8 alkyl or -(C=O)-R 22 , where R 22 It is an optional substituted C1-C8 alkyl group; R3 and R4 are independently H, a straight-chain or branched C4-C30 alkyl, a straight-chain or branched C4-C30 alkenyl, or a straight-chain or branched C4-C30 alkynyl; wherein at least one of R3 or R4 is different from H; R5 and R6 are independently straight-chain or branched C4-C30 alkyl, straight-chain or branched C4-C30 alkenyl, or straight-chain or branched C4-C30 alkynyl. R7 is an optionally substituted straight-chain C1-C8 alkyl, optionally substituted straight-chain C2-C8 alkenyl, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group. When any one of R3, R4, R5, and R6 represents an alkenyl group, the alkenyl group independently contains 1 to 6 C=C bonds, each C=C bond independently having an E or Z configuration; When any one of R3, R4, R5, and R6 represents an alkynyl group, the alkynyl group independently contains 1 to 6 C≡C bonds; When R7 represents an alkenyl group, the alkenyl group contains one or two C=C bonds, each C=C bond independently having an E or Z configuration; When any alkyl and / or alkenyl group is substituted, that group is independently substituted by one or more of the following groups: -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 , -O-(C=O)-CH3, -O-(C=O)-(CH2) p CH3, -O-(C=O)-(CH2) p -R 19 ,-(C=O)-OCH3,-(C=O)-O-(CH2) p CH3、-(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -R 19 -(C=O)NH-(CH2) p -R 19 ,-(C=S)-CH3,-O-CH3,-O-(CH2) p CH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p -R 19 -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, optionally substituted 5- to 10-membered heterocyclic groups, C5-C12 aralkyl, C5-C10 aryl, or optionally substituted 5- to 10-membered heteroaryl groups, wherein p and p' are independently 1 to 5; and wherein R 19 It is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5- to 10-membered heterocyclic groups or optionally substituted 5- to 10-membered heteroaryl groups. When any 5- to 10-membered heteroaryl group and / or 5- to 10-membered heterocyclic group is substituted, the group is independently selected from C1-C4 alkyl groups, -(CH2) groups, etc. p’’ The group consisting of N(CH3)2 and oxo (C=O) is substituted with one or more substituents, where p'' is 1 to 5.

4. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein R1 is H or an optional substituted straight-chain C1-C4 alkyl group; R2 is H, an optionally substituted straight-chain C1-C4 alkyl group, -C(=O)R7, -S(=O)2-R7, or... ; R 20 It is H or an optional substituted straight-chain C1-C8 alkyl group; R 21 It is an optional substituted straight-chain C1-C8 alkyl or -(C=O)-R 22 , where R 22 It is an optional substituted C1-C8 alkyl group; R3 and R4 are independently H, a straight-chain or branched C4-C30 alkyl, a straight-chain or branched C4-C30 alkenyl, or a straight-chain or branched C4-C30 alkynyl; wherein at least one of R3 or R4 is different from H; R5 and R6 are independently straight-chain or branched C4-C30 alkyl, straight-chain or branched C4-C30 alkenyl, or straight-chain or branched C4-C30 alkynyl. R7 is an optionally substituted straight-chain C1-C8 alkyl, optionally substituted straight-chain C2-C8 alkenyl, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group. When any one of R3, R4, R5, and R6 represents an alkenyl group, the alkenyl group independently contains 1 to 6 C=C bonds, each C=C bond independently having an E or Z configuration; When any one of R3, R4, R5, and R6 represents an alkynyl group, the alkynyl group independently contains 1 to 6 C≡C bonds; When R7 represents an alkenyl group, the alkenyl group contains one or two C=C bonds, each C=C bond independently having an E or Z configuration; When any alkyl and / or alkenyl group is substituted, that group is independently substituted by one or more of the following groups: -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 , -O-(C=O)-CH3, -O-(C=O)-(CH2) p CH3, -O-(C=O)-(CH2) p -R 19 ,-(C=O)-OCH3,-(C=O)-O-(CH2) p CH3、-(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -R 19 -(C=O)NH-(CH2) p -R 19 -O-CH3, -O-(CH2) p CH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p -R 19 -(C=O)NH2, optionally substituted 5- to 10-membered heterocyclic groups, C5-C12 aryl, C5-C10 aryl or optionally substituted 5- to 10-membered heteroaryl groups, wherein p and p' are independently 1 to 5; and wherein R 19 It is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5- to 10-membered heterocyclic groups or optionally substituted 5- to 10-membered heteroaryl groups. When any 5- to 10-membered heteroaryl group and / or 5- to 10-membered heterocyclic group is substituted, the group is independently selected from C1-C4 alkyl groups, -(CH2) groups, etc. p’’ One or more substituents of the group consisting of N(CH3)2 and oxo (=O) are substituted, wherein p'' is 1 to 5.

5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein R1 is H or an optional substituted straight-chain C1-C4 alkyl group; R2 is H, an optionally substituted straight-chain C1-C4 alkyl group, -C(=O)R7, -S(=O)2-R7, or... ; R 20 It is H or an optional substituted straight-chain C1-C4 alkyl group; R 21 It is an optional substituted straight-chain C1-C4 alkyl group or -(C=O)-R 22 , where R 22 It is an optional substituted C1-C4 alkyl group; R3 and R4 are independently H, a straight-chain or branched C6-C25 alkyl group, or a straight-chain or branched C6-C25 alkenyl group; wherein at least one of R3 or R4 is different from H; R5 and R6 are independently straight-chain or branched C6-C25 alkyl groups or straight-chain or branched C6-C25 alkenyl groups; R7 is an optionally substituted straight-chain C1-C4 alkyl, optionally substituted straight-chain C2-C4 alkenyl, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group. When any one of R3, R4, R5, and R6 represents an alkenyl group, the alkenyl group independently contains 1 to 4 C=C bonds, each C=C bond independently having an E or Z configuration; When R7 represents an alkenyl group, the alkenyl group contains one or two C=C bonds, each C=C bond independently having an E or Z configuration; When any alkyl and / or alkenyl group is substituted, that group is independently substituted by one or more of the following groups: -OH, -(C=O)-(CH2). p -R 19 -O-(C=O)-(CH2) p -R 19 -(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -N(CH3)2、-(C=O)NH-(CH2) p -R 19 , -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p N(CH3)2, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group, wherein p and p' are independently 1 to 5; and wherein R 19 It is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5- to 10-membered heterocyclic groups or optionally substituted 5- to 10-membered heteroaryl groups. When any 5- to 10-membered heteroaryl group and / or 5- to 10-membered heterocyclic group is substituted, the group is independently selected from C1-C4 alkyl groups, -(CH2) groups, etc. p’’ One or more substituents of the group consisting of N(CH3)2 and oxo (=O) are substituted, wherein p'' is 1 to 5.

6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein R1 is H or an optional substituted C1-C2 alkyl group; R2 is H, an optionally substituted straight-chain C1-C4 alkyl group, -C(=O)R7, -S(=O)2-R7, or... ; R 20 It is a straight-chain C1-C3 alkyl group, either H or optionally substituted with -N(CH3)2; R 21 It is a 5- to 10-membered heterocyclic group that is optionally substituted with -N(CH3)2 or optionally substituted with a straight-chain C1-C3 alkyl group, or -(C=O)-R 22 , where R 22 It is a C1-C3 alkyl group that is optionally substituted with -N(CH3)2; R3 and R4 are independently H, a straight-chain or branched C6-C25 alkyl group, or a straight-chain or branched C6-C25 alkenyl group; wherein at least one of R3 or R4 is different from H; R5 and R6 are independently straight-chain or branched C6-C25 alkyl or straight-chain or branched C6-C25 alkenyl groups; R7 is an optionally substituted straight-chain C1-C4 alkyl group, an optionally substituted straight-chain C2-C4 alkenyl group, an optionally substituted 5- to 10-membered heterocyclic group, or an optionally substituted 5- to 10-membered heteroaryl group, wherein when the 5- to 10-membered heteroaryl group and / or the 5- to 10-membered heterocyclic group are substituted, the group is independently selected from C1-C2 alkyl, -(CH2) ... p’’ The group consisting of N(CH3)2 and oxo (=O) is substituted with one or more substituents, where p'' is 1 to 3; When any one of R3, R4, R5, and R6 represents an alkenyl group, the alkenyl group independently contains 1 to 4 C=C bonds, each C=C bond independently having an E or Z configuration; When R7 represents an alkenyl group, the alkenyl group contains one or two C=C bonds, each C=C bond independently having an E or Z configuration; When any alkyl or alkenyl group in R1, R2, and / or R7 is substituted, that group is independently substituted by one or more of the following groups: -OH, -(C=O)-(CH2). p -R 19 -O-(C=O)-(CH2) p -R 19 -(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -N(CH3)2、-(C=O)NH-(CH2) p -R 19 , -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p N(CH3)2, a 5- to 10-membered heterocyclic group optionally substituted with a C1-C4 alkyl group or a 5- to 10-membered heteroaryl group optionally substituted with a C1-C4 alkyl group, wherein p and p' are independently 1 to 5; and wherein R 19 It is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, a 5- to 10-membered heterocyclic group optionally substituted with a C1-C4 alkyl group, or a 5- to 10-membered heteroaryl group optionally substituted with a C1-C4 alkyl group.

7. The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein R1 is H or an optional substituted C1-C2 alkyl group; R2 is H, an optionally substituted straight-chain C1-C4 alkyl group, -C(=O)R7, -S(=O)2-R7, or... ; R 20 It is a straight-chain C1-C3 alkyl group, either H or optionally substituted with -N(CH3)2; R 21 It is a straight-chain C1-C4 alkyl group optionally substituted with -N(CH3)2 or a 5- to 10-membered heterocyclic group optionally substituted with C1-C2 alkyl, or -(C=O)-R 22 , where R 22 It is a C1-C3 alkyl group that is optionally substituted with -N(CH3)2; R3 and R4 are independently H, a straight-chain or branched C6-C25 alkyl group, or a straight-chain or branched C6-C25 alkenyl group; wherein at least one of R3 or R4 is different from H; R5 and R6 are independently straight-chain or branched C6-C25 alkyl or straight-chain or branched C6-C25 alkenyl groups; R7 is an optionally substituted straight-chain C1-C4 alkyl, optionally substituted straight-chain C2-C4 alkenyl, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group, wherein when the 5- to 10-membered heteroaryl group and / or the 5- to 10-membered heterocyclic group is substituted, the group is independently substituted by one or more substituents selected from the group consisting of C1-C2 alkyl, -(CH2)2N(CH3)2 and oxo (=O); When any one of R3, R4, R5, and R6 represents an alkenyl group, the alkenyl group independently contains 1 to 4 C=C bonds, each C=C bond independently having an E or Z configuration; When R7 represents an alkenyl group, the alkenyl group contains one or two C=C bonds, each C=C bond independently having an E or Z configuration; When any alkyl or alkenyl group in R1, R2, and / or R7 is substituted, that group is independently substituted by one or more of the following groups: -OH, -(C=O)-(CH2). p -R 19 -O-(C=O)-(CH2) p -R 19 -(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -N(CH3)2、-(C=O)NH-(CH2) p -R 19 -N(CH3)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p N(CH3)2, a 5- to 10-membered heterocyclic group optionally substituted with one C1-C2 alkyl group or a 5- to 10-membered heteroaryl group optionally substituted with one C1-C2 alkyl group, wherein p and p' are independently 1 to 5; and wherein R 19 It is -N(CH3)2, a 5- to 10-membered heterocyclic group optionally substituted with one C1-C2 alkyl group, or a 5- to 10-membered heteroaryl group optionally substituted with one C1-C2 alkyl group.

8. The compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, wherein the 5 to 10 heteroaryl groups independently represent furanyl, benzofuranyl, benzimidazolyl, quinolinyl, isoquinolinyl, phenylthio, pyrroleyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazoleyl, pyrazinyl, pyridinyl, pyridinyl, or pyrimidinyl.

9. The compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, wherein the 5 to 10-membered heterocyclic group independently represents a lactam, morpholino, piperazine, or piperidinyl.

10. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, wherein R1 is H or CH3, preferably H.

11. The compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein R2 is H or optionally substituted straight-chain C3-C4 alkyl, -C(=O)R7, -S(=O)2-R7, or .

12. The compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein -NR1R2 has one of the following structures: , , , , , , , , , , , , , , , , , , , , , , , , , , or .

13. The compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, wherein -NR1R2 is .

14. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein any one of R3, R4, R5 and R6 is a C6-C25 alkenyl group, and said alkenyl group independently comprises one, two or three C=C bonds.

15. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein R3 and R4 are straight-chain C6-C16 alkyl groups.

16. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein R3 and R4 are branched C8-C12 alkyl groups.

17. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein R3 and R4 are straight-chain C8-C20 alkenyl groups, wherein each alkenyl group comprises one C=C bond.

18. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein R3 is H and R4 is a straight-chain C6-C25 alkenyl group, wherein the alkenyl group comprises one C=C bond.

19. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein R4 is H and R3 is a straight-chain C6-C25 alkenyl group, wherein the alkenyl group comprises one C=C bond.

20. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 and 15 to 19, wherein R5 and R6 are straight-chain C6-C16 alkyl groups.

21. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 and 15 to 19, wherein R5 and R6 are branched C8-C12 alkyl groups.

22. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 and 15 to 19, wherein R5 and R6 are straight-chain C8-C20 alkenyl groups, wherein each alkenyl group comprises one C=C bond.

23. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 and 15 to 19, wherein R5 and R6 are straight-chain C15-C20 alkenyl groups, wherein each alkenyl group comprises two C=C bonds.

24. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 and 15 to 19, wherein R5 and R6 are branched C22-C25 alkenyl groups, wherein each alkenyl group comprises one C=C bond.

25. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 and 15 to 19, wherein R5 and R6 are branched C13-C17 alkenyl groups, wherein each alkenyl group comprises 3 C=C bonds.

26. The compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein R3, R4, R5, and R6 independently have one of the following structures: , , , , , , , , , , or .

27. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 26, wherein R3 and R4 are both different from H and are the same.

28. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 27, wherein R5 and R6 are identical.

29. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 28, wherein R3 and R4 are both different from H and R3, R4, R5 and R6 are the same.

30. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) has the structure of formula (I2) or formula (I3): (I2) (I3) Among them, R1, R2, R3, R4, R5, R6 and R 1’ As defined in any one of claims 1 to 29.

31. The compound of claim 30, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (I2), wherein R1 is H or C1-C2 alkyl, and R2 is -C(=O)R7; and wherein R3, R4, R5, R6 and R7 are as defined in any one of claims 1 to 29.

32. The compound of claim 30, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (I3), wherein R 1’ It is a straight-chain C1-C4 alkyl group substituted with the following group: -(C=O)-(CH2) p -R 19 -O-(C=O)-(CH2) p -R 19 -(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -N(CH3)2、-(C=O)NH-(CH2) p -R 19 -N(CH3)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p N(CH3)2, optionally substituted 5- to 6-membered heterocyclic group or optionally substituted 5- to 6-membered heteroaryl group, wherein p and p' are independently 1 to 5; and wherein R 19 It is -N(CH3)2, optionally substituted 5- to 6-membered heterocyclic group or optionally substituted 5- to 6-membered heteroaryl group; wherein when the 5- to 6-membered heteroaryl group and / or the 5- to 6-membered heterocyclic group is substituted, the group is independently substituted by a substituent selected from the group consisting of C1-C2 alkyl groups; And R3, R4, R5 and R6 are as defined in any one of claims 1 to 29.

33. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure of formula (I1-1), formula (I1-2), formula (I1-3), formula (I1-4), formula (I1-5), or formula (I1-6): (I1-1)、 (I1-2)、 (I1-3)、 (I1-4)、 (I1-5)、 (I1-6)、 Where R1 is H or a straight-chain C1-C4 alkyl group; Where R 10 and R 11 Independently, it is -H, -(C=O)-CH3, or -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 Or -(C=O)-(CH2) p -(C=O)-R 19 ;and Among them, R3, R4, R5, R6, R7, R 19 R 20 and R 21 As defined in any one of claims 1 to 29, and p is 1 to 5.

34. The compound of claim 33, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (I1-4a): (I1-4a), Wherein R1 is H or a straight-chain C1-C4 alkyl group, preferably R1 is H or -CH3, more preferably R1 is -CH3; and wherein R3, R4, R5 and R6 are as defined in any one of claims 1 to 29.

35. The compound of claim 33, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (I1-4b): (I1-4b), Wherein R1 is H or a straight-chain C1-C4 alkyl group, preferably R1 is H or -CH3, more preferably R1 is -CH3; Where R 12 and R 13 Independently, it is an optional substituted straight-chain C1-C4 alkyl group; wherein when the straight-chain alkyl group is substituted, the group is independently substituted with -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2 or a 5-6 membered heteroaryl group, preferably R. 12 and R 13 Independently, it is a straight-chain C1-C4 alkyl group substituted with -NH2, -NHCH3, -N(CH3)2 or -N(CH2CH3)2, more preferably a straight-chain C1-C4 alkyl group substituted with -N(CH3)2; and wherein R3, R4, R5 and R6 are as defined in any one of claims 1 to 29.

36. The compound of claim 33, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (I1-5a): (I1-5a) Wherein R1 is H or a straight-chain C1-C4 alkyl group, preferably R1 is H or -CH3, more preferably R1 is H; Where q ranges from 1 to 5; Where R 14 and R' 14 Independently represents -H, -CH3, -CH2CH3, or -(CH2). p O-(C=O)CH3, where p is 1 to 5, preferably p is 1 to 3, or R 14 and R' 14 The linkage forms a 6-membered heterocyclic group optionally substituted with -CH3 or -CH2CH3; and wherein R3, R4, R5 and R6 are as defined in any one of claims 1 to 29.

37. The compound of claim 33, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (I1-5b): (I1-5b), Where X1 is -NH- or -O-; Where R 15 It is a straight-chain C1-C4 alkyl group, -(CH2) p- R 19 Or –(CH2) p N(CH3)-((CH2) p’ -N(CH3)2), where p and p' are independently 1 to 5, and R 19 As defined in any one of claims 1 to 9; Preferably, R 15 It is -(CH2)2N(CH3)-((CH2)2-N(CH3)2) or -(CH2) p -R 19 Where p is 1 to 3; More preferably, R 15 It is -(CH2)2N(CH3)-((CH2)2-N(CH3)2), -(CH2) p -N(CH3)2 or -(CH2) p -R 23 Where p is 1 to 3, and R 23 It is a 5- to 6-membered heteroaryl or a 5- to 6-membered heterocyclic group optionally substituted with a C1-C2 alkyl group; Even more preferably, R 15 It is -(CH2)2N(CH3)-((CH2)2-N(CH3)2), -(CH2) p -N(CH3)2 or -(CH2) p -R 23 Where p is 1 to 3, and R 23 It is furanyl, phenylthio, pyrroleyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazoleyl, pyrazinyl, pyridinyl, pyridinyl, pyridinyl, piperidinyl, or piperazinyl, wherein R 23 Optional substitution with C1-C2 alkyl groups; Most preferably, R 15 It is -(CH2)2N(CH3)-((CH2)2-N(CH3)2), -(CH2) p -N(CH3)2 or -(CH2) p -R 23 Where p is 1 to 3, and R 23 It is imidazole or piperidinyl, where R 23 Optionally substituted with C1-C2 alkyl groups; and wherein R3, R4, R5 and R6 are as defined in any one of claims 1 to 29.

38. The compound of claim 33, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (I1-5c): (I1-5c), in R 16 It can be optionally modified by straight-chain C1-C4 alkyl groups, oxo (=O) and / or –(CH2). p -N(CH3)2-substituted 5- to 6-membered heteroaryl or 5- to 6-membered heterocyclic group, wherein p is 1 to 3; preferably R 16 It is optionally substituted with a straight-chain C1-C4 alkyl, oxazolyl, pyrrolidinyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazinyl, pyridinyl, pyridinyl, pyridinyl, piperazinyl, piperidinyl, or pyrimidinyl group, wherein p is 1 to 3; more preferably, R 16 It may be optionally dilated by C1-C2 alkyl, oxidized (=O) and / or –(CH2). p -N(CH3)2-substituted imidazolyl or pyrrolidinyl, wherein p is 1 to 3; and wherein R3, R4, R5 and R6 are as defined in any one of claims 1 to 29.

39. The compound of claim 33, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (I1-5d): (I1-5d), in R 17 It can be optionally surrounded by straight-chain C1-C4 alkyl groups or -(CH2). p -N(CH3)2-substituted 5- to 6-membered heteroaryl groups, wherein p is 1 to 3; preferably, R 17 It is optionally substituted with a C1-C2 alkyl group, namely furanyl, phenylthio, pyrroleyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazoleyl, pyrazinyl, pyridinyl, pyridinyl, or pyrimidinyl; more preferably, R 17 It is an imidazole group; The C=C bonds present in formula (I1-5d) independently have an E or Z configuration; and R3, R4, R5 and R6 are defined as in any one of claims 1 to 29.

40. The compound of claim 33, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure of formula (I1-6a), formula (I1-6b), or formula (I1-6c): (I1-6a) (I1-6b) (I1-6c) in R 31 It is a straight-chain C1-C4 alkyl group optionally substituted with -N(CH3)2; preferably, R 31 It is -(CH2)3-N(CH3)2; R 32 It is -N(CH3)2 or optionally a 5- to 6-membered heterocyclic group substituted with a straight-chain C1-C4 alkyl group and z is 1 to 4; preferably, R 32 It is -N(CH3)2 and z is 2 to 4; more preferably, R 32 It is a piperazine or piperidinyl group substituted with a C1-C2 alkyl group and z is 3; and R3, R4, R5 and R6 are as defined in any one of claims 1 to 29.

41. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 30 to 40, wherein R3 and R4 are identical, and R5 and R6 are identical.

42. A compound, or a pharmaceutically acceptable salt thereof, said compound being selected from the group consisting of: compounds 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320a, 320b, 321a, 321b, 3... 22, 323, 324, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 358, 359, 360, and 362.

43. The compound of claim 42 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: 301, 302, 303, 304, 305, 306, 307, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320a, 320b, 321a, 321b, 322 of Table 1. 323, 324, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 358, 359, 360, and 362.

44. The compound of claim 42 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: 301, 302, 303, 304, 305, 306, 307, 309, 311, 312, 313, 314, 315, 316, 318, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 347, 350, 351, 352, 353, 355, 358, 359, 360 and 362 of Table 1.

45. The compound of claim 42 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: 305, 313, 314, 315, 316, 317, 318, 320a, 320b, 321a, 321b, 323, 324, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 340, 350, 351 and 360 of Table 1.

46. ​​The compound of claim 42 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: 301, 303, 304, 305, 306, 307, 311, 312, 313, 314, 315, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 347, 350, 351, 352, 353, 355, 358 and 359 of Table 1.

47. The compound of claim 42 or 46 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: 305, 306, 307, 313, 314, 315, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 350, 351, 352, 353, 355, 358 and 359 of Table 1.

48. The compound according to any one of claims 1 to 47, wherein the compound or a pharmaceutically acceptable salt thereof is in the form of any enantiomer, any diastereomer, any cis or trans geometric isomer, or any mixture thereof.

49. A lipid nanoparticle comprising at least one compound according to any one of claims 1 to 48 or a pharmaceutically acceptable salt thereof.

50. A lipid nanoparticle comprising: (a) Approximately 40 mol% to approximately 100 mol% of ionizable lipids; (b) 0 to approximately 20 mol% of neutral lipids; (c) 0 to approximately 50 mol% of auxiliary lipids; (d) 0 to 5 mol% polymer-conjugated lipids; and (e) 0 to approximately 10 mol% of hydrophobic components; Wherein mol% is based on the total lipids present in the nanoparticles; and The ionizable lipid is at least one compound of formula (I): (I) Or its pharmaceutically acceptable salt. in A does not exist or is -CH2-; B is -CH2- or -CH2CH2-; Where A does not exist, then B is -CH2CH2-, and when A is -CH2-, then B is -CH2-; X is either -NR1R2 or -O-(C=O)R 1’ ; R1 is H or an optional substituted straight-chain C1-C4 alkyl group; R2 is H, an optionally substituted straight-chain C1-C8 alkyl group, -C(=O)R7, -S(=O)2-R7, or... ; R 1’ It is a straight-chain C1-C8 alkyl group that can be optionally substituted; R 20 It is H or an optional substituted straight-chain C1-C8 alkyl group; R 21 It is an optional substituted straight-chain C1-C8 alkyl or -(C=O)-R 22 , where R 22 It is an optional substituted C1-C8 alkyl group; R3 and R4 are independently H, a straight-chain or branched C4-C60 alkyl, a straight-chain or branched C4-C60 alkenyl, or a straight-chain or branched C4-C60 alkynyl, wherein at least one of R3 or R4 is different from H; R5 and R6 are independently straight-chain or branched C4-C60 alkyl, straight-chain or branched C4-C60 alkenyl, or straight-chain or branched C4-C60 alkynyl. R7 is an optionally substituted straight-chain C1-C8 alkyl, optionally substituted straight-chain C2-C8 alkenyl, optionally substituted 5- to 10-membered heterocyclic group or optionally substituted 5- to 10-membered heteroaryl group. When any one of R3, R4, R5, and R6 represents an alkenyl group, the alkenyl group independently contains 1 to 6 C=C bonds, each C=C bond independently having an E or Z configuration; When any one of R3, R4, R5, and R6 represents an alkynyl group, the alkynyl group independently contains 1 to 6 C≡C bonds; When R7 represents an alkenyl group, the alkenyl group contains one or two C=C bonds, each C=C bond independently having an E or Z configuration; When any alkyl and / or alkenyl group is substituted, that group is independently substituted by one or more of the following groups: -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2). p CH3、-(C=O)-(CH2) p -R 19 , -O-(C=O)-CH3, -O-(C=O)-(CH2) p CH3, -O-(C=O)-(CH2) p -R 19 ,-(C=O)-OCH3,-(C=O)-O-(CH2) p CH3、-(C=O)-O-(CH2) p -R 19 -(C=O)-(CH2) p -(C=O)-R 19 -(C=O)-O-(CH2) p N(CH3)-(CH2) p’ -R 19 -(C=O)NH-(CH2) p -R 19 ,-(C=S)-CH3,-O-CH3,-O-(CH2) p CH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2) p O-(C=O)CH3)2、-N(CH3)-(CH2) p -R 19 -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, optionally substituted 5- to 10-membered heterocyclic groups, C5-C12 aralkyl, C5-C10 aryl, or optionally substituted 5- to 10-membered heteroaryl groups, wherein p and p' are independently 1 to 5; and wherein R 19 It is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, optionally substituted 5 to 10 heterocyclic groups or optionally substituted 5 to 10 heteroaryl groups; When any 5- to 10-membered heteroaryl group and / or 5- to 10-membered heterocyclic group is substituted, the group is independently selected from C1-C4 alkyl groups, -(CH2) groups, etc. p’’ The group consisting of N(CH3)2 and oxo (=O) is substituted with one or more substituents, where p'' is 1 to 5; The conditions are that A is absent, B is -CH2CH2-, X is -NR1R2, R1 is -CH3, and each of R3, R4, R5, and R6 has carbons at C9 and C6. 10 A C=C bond between carbon and carbon C 12 and C 13 When R2 is a straight-chain C18 alkyl group with one C=C bond, then R2 is different from -CH3, -CH2CH2OH and -CH2CH2NH2.

51. The lipid nanoparticles of claim 50, wherein the compound or a pharmaceutically acceptable salt thereof is defined as in any one of claims 1 to 48.

52. The lipid nanoparticles according to claim 50 or 51, comprising: (a) The ionizable lipids, approximately 40 mol% to approximately 85 mol%; (b) about 1 mol% to about 20 mol% of the neutral lipids; (c) 0 to about 50 mol% of the aforementioned auxiliary lipids; (d) 0 to about 5 mol% of the polymer-conjugated lipids; and (e) 0 to about 10 mol% of the hydrophobic component.

53. The lipid nanoparticles according to claim 50 or 51, comprising: (a) The ionizable lipids, approximately 40 mol% to approximately 85 mol%; (b) about 1 mol% to about 20 mol% of the neutral lipids; (c) the aforementioned auxiliary lipids, ranging from about 1 mol% to about 50 mol%; (d) 0 to about 5 mol% of the polymer-conjugated lipids; and (e) 0 to about 10 mol% of the hydrophobic component.

54. The lipid nanoparticles according to claim 50 or 51, comprising: (a) The ionizable lipids, approximately 40 mol% to approximately 85 mol%; (b) about 1 mol% to about 20 mol% of the neutral lipids; (c) the aforementioned auxiliary lipids, ranging from about 1 mol% to about 50 mol%; (d) about 1 mol% to about 5 mol% of the polymer-conjugated lipids; and (e) 0 to about 10 mol% of the hydrophobic component.

55. The lipid nanoparticles according to claim 50 or 51, comprising: (a) The ionizable lipids, approximately 40 mol% to approximately 85 mol%; (b) about 1 mol% to about 20 mol% of the neutral lipids; (c) the aforementioned auxiliary lipids, from about 1 mol% to about 50 mol%; (d) about 1 mol% to about 5 mol% of the polymer-conjugated lipids; and (e) The hydrophobic component of about 0.1 mol% to about 10 mol%.

56. The lipid nanoparticles according to claim 50 or 51, comprising: (a) The ionizable lipids, approximately 40 mol% to approximately 85 mol%; (b) about 5 mol% to about 20 mol% of the neutral lipids; (c) the aforementioned auxiliary lipids, from about 1 mol% to about 50 mol%; (d) about 1 mol% to about 4 mol% of the polymer-conjugated lipids; and (e) The hydrophobic component of about 0.1 mol% to about 10 mol%.

57. The lipid nanoparticles according to claim 50 or 51, comprising: (a) The ionizable lipids, approximately 40 mol% to approximately 85 mol%; (b) about 5 mol% to about 10 mol% of the neutral lipids; (c) the aforementioned auxiliary lipids, ranging from about 1 mol% to about 40 mol%; (d) about 1 mol% to about 4 mol% of the polymer-conjugated lipids; and (e) about 0 to about 5 mol% of the hydrophobic component.

58. The lipid nanoparticles according to claim 50 or 51, comprising: (a) The ionizable lipids, approximately 40 mol% to approximately 85 mol%; (b) about 5 mol% to about 10 mol% of the neutral lipids; (c) the aforementioned auxiliary lipids, ranging from about 1 mol% to about 40 mol%; (d) about 1 mol% to about 4 mol% of the polymer-conjugated lipids; and (e) about 0.1 mol% to about 5 mol% of the hydrophobic component.

59. The lipid nanoparticles according to any one of claims 50 to 58, wherein the neutral lipid comprises at least one phospholipid.

60. The lipid nanoparticles according to any one of claims 50 to 59, wherein the neutral lipid comprises at least one phospholipid selected from the group consisting of: dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearyl-sn-glycerol-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), methionine phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC) ), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-disarachido-sn-glycerol-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dienoenoyl-sn-glycerol-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof.

61. The lipid nanoparticles according to any one of claims 50 to 60, wherein the neutral lipid comprises phospholipids DSPC, DOPC, DMPC, PE, or combinations thereof.

62. The lipid nanoparticles according to any one of claims 50 to 61, wherein the auxiliary lipid comprises sterol, alkyl resorcinol, or a combination thereof.

63. The lipid nanoparticles according to any one of claims 50 to 62, wherein the auxiliary lipid comprises cholesterol, 5-heptadecylresorcinol and cholesterol hemisuccinate, cholesterol, cholesterol ketone, cholesterol ketone, coprosterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, or any combination thereof.

64. The lipid nanoparticles according to any one of claims 50 to 63, wherein the auxiliary lipid comprises cholesterol, 5-heptadecylresorcinol, cholesterol hemisuccinate, or any combination thereof.

65. The lipid nanoparticles according to any one of claims 50 to 64, wherein the assisting lipids comprise at least cholesterol.

66. The lipid nanoparticles according to any one of claims 50 to 65, wherein the polymer-conjugated lipid comprises polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2-oxazoline) (POZ), polyamide (ATTA), cationic polymer, polysarcosine (Psar), polyglutamic acid (PGA), 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol (PEG-DMG), or any combination thereof.

67. The lipid nanoparticles according to any one of claims 50 to 66, wherein the polymer-conjugated lipid comprises at least one PEG-lipid.

68. The lipid nanoparticles according to any one of claims 50 to 67, wherein the polymer-conjugated lipid comprises at least one PEG2k-DMG.

69. The lipid nanoparticles according to any one of claims 50 to 68, wherein the hydrophobic component comprises squalene, cardiolipin, α-tocopherol, solanine A, vitamin A, retinol, β-carotene, or any combination thereof.

70. The lipid nanoparticles according to claim 50 or 51, comprising: (a) about 40 mol% to about 80 mol% of the ionizable lipids; (b) Phospholipids of about 5 mol% to about 15 mol% as said neutral lipids; (c) Sterols as said auxiliary lipids, ranging from about 1 mol% to about 40 mol%; (d) about 1 mol% to about 4 mol% of PEG-lipids as lipids conjugated to the polymer; and (e) about 0 to about 5 mol% of squalene, cardiolipin, α-tocopherol, solanine A, vitamin A, retinol, β-carotene or combinations thereof as said hydrophobic components; Where mol% is based on the total lipids present in the nanoparticles.

71. The lipid nanoparticles of claim 70, wherein the phospholipid is DSPC.

72. The lipid nanoparticles according to claim 70 or 71, wherein the sterol is cholesterol.

73. The lipid nanoparticles according to any one of claims 70 to 72, wherein the PEG-lipid is PEG2k-DMG.

74. The lipid nanoparticles according to any one of claims 50 to 73, wherein the hydrophobic component comprises squalene, cardiolipin, α-tocopherol, or a combination thereof.

75. The lipid nanoparticles according to any one of claims 49 to 74, further comprising at least one cargo.

76. The lipid nanoparticles of claim 75, wherein the cargo comprises at least one of small molecules, antibodies, polynucleotides, or polypeptides.

77. The lipid nanoparticles of claim 75, wherein the cargo comprises at least one nucleic acid, such as mRNA.

78. A pharmaceutical composition comprising lipid nanoparticles according to any one of claims 49 to 77 and a pharmaceutically acceptable excipient.

79. The pharmaceutical composition according to claim 78, which is formulated for intramuscular administration.

80. A method for delivering cargo to a cell, the method comprising contacting the cell with lipid nanoparticles according to any one of claims 49 to 77, wherein the lipid nanoparticles contain the cargo.

81. Use of the lipid nanoparticles according to any one of claims 49 to 77 for delivering cargo to cells, wherein the lipid nanoparticles comprise the cargo.

82. A vaccine comprising lipid nanoparticles according to any one of claims 49 to 74, wherein the lipid nanoparticles comprise at least one cargo; preferably, the cargo comprises at least one of a small molecule, an antibody, a polynucleotide, or a polypeptide; more preferably, the cargo comprises at least one nucleic acid, such as mRNA.

83. A method for vaccinating a subject against an infectious agent, the method comprising: (i) exposing the subject to the vaccine according to claim 82, and (ii) Triggering an immune response.

84. The method of claim 83, wherein the infectious agent is a virus, bacteria, or parasite.

85. The method of claim 83, wherein the infectious agent is Campylobacter jejuni, Clostridium difficile, Entamoeba histolytica, enterotoxin B, norovirus or norovirus, Helicobacter pylori (… Helicobacter pylori ), rotavirus, candida yeast, coronaviruses including SARS-CoV, SARS-CoV-2, and MERS-CoV, enterovirus 71, Epstein-Barr virus, Gram-negative bacteria including Bordetella, and Clostridium tetani ( Clostridium Tetani Gram-positive bacteria, Tulafrancsis ( Francisella Tularensis Streptococcus, Staphylococcus, Salmonella, hepatitis, human cytomegalovirus, human immunodeficiency virus, human papillomavirus, influenza, John Cunningham virus, Mycobacterium, poxvirus, Pseudomonas aeruginosa, respiratory syncytial virus, rubella virus, varicella-zoster virus, Zika virus, Chikungunya virus, dengue virus, rabies virus, Trypanosoma cruzi, Chagas disease, Ebola virus, Plasmodium falciparum, Marburg virus, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, Bacillus anthracis, botulinum toxin, ricin, Nipah virus, Shiga toxin and / or Shiga-like toxin.

86. The use of the vaccine according to claim 83 for vaccinating a subject against an infectious agent.

87. The use according to claim 86, wherein the infectious agent is a virus, bacteria, or parasite.

88. The use according to claim 86, wherein the infectious agent is Campylobacter jejuni, Clostridium difficile, Entamoeba histolytica, enterotoxin B, norovirus or norovirus, Helicobacter pylori, rotavirus, Candida albicans, coronaviruses including SARS-CoV, SARS-CoV-2, and MERS-CoV, enterovirus 71, Epstein-Barr virus, Gram-negative bacteria including Bordetella spp., Gram-positive bacteria including Clostridium tetani, Tulafrancsis, Streptococcus spp., Staphylococcus spp., or Salmonella spp. Hepatitis, human cytomegalovirus, human immunodeficiency virus, human papillomavirus, influenza, John Cunningham virus, mycobacteria, poxvirus, Pseudomonas aeruginosa, respiratory syncytial virus, rubella virus, varicella-zoster virus, Zika virus, chikungunya virus, dengue virus, rabies virus, trypanosomiasis, Chagas disease, Ebola virus, Plasmodium falciparum, Marburg virus, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, anthrax bacillus, botulinum toxin, ricin, Nipah virus, Shiga toxin and / or Shiga-like toxin.

89. A method of treating a subject with cancer, the method comprising administering to the subject lipid nanoparticles according to any one of claims 49 to 74, wherein the lipid nanoparticles comprise anticancer cargoes or cargoes that trigger an immune response against cancer cells.

90. The method of claim 89, wherein the cancer is lung cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, kidney cancer, leukemia, central nervous system cancer, myeloma, or melanoma.

91. The lipid nanoparticles according to any one of claims 49 to 74, for use in treating a subject's cancer, wherein the lipid nanoparticles comprise anticancer cargoes or cargoes that trigger an immune response against cancer cells.

92. The lipid nanoparticles for use according to claim 91, wherein the cancer is lung cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, kidney cancer, leukemia, central nervous system cancer, myeloma, or melanoma.

93. Use of the lipid nanoparticles according to any one of claims 46 to 74 for treating a subject's cancer, wherein the lipid nanoparticles comprise anticancer cargoes or cargoes that trigger an immune response against cancer cells.

94. The use according to claim 93, wherein the cancer is lung cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, kidney cancer, leukemia, central nervous system cancer, myeloma, or melanoma.

95. Use of the lipid nanoparticles according to any one of claims 49 to 74 in the preparation of a medicament for treating cancer in a subject, wherein the lipid nanoparticles comprise anticancer cargoes or cargoes that trigger an immune response against cancer cells.

96. The use according to claim 95, wherein the cancer is lung cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, kidney cancer, leukemia, central nervous system cancer, myeloma, or melanoma.

97. The method of claim 89 or 90, the lipid nanoparticles for use according to claim 91 or 92, or the use according to any one of claims 93 to 96, wherein the cargo comprises at least one of a small molecule, an antibody, a polynucleotide, or a polypeptide.

98. The method of claim 89 or 90, the lipid nanoparticles for use according to claim 91 or 92, or the use according to any one of claims 93 to 96, wherein the cargo comprises at least one nucleic acid, such as mRNA.

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