Cationic lipids and lipid nanoparticles

Abstract

The present invention relates to cationic lipids and salts thereof (e.g., pharmaceutically acceptable salts), as well as compositions comprising such lipids, which are useful for the delivery of bioactive agents.

Description

Cross-reference to related applications

[0001] This application claims priority to PCT Application No. PCT / CN2023 / 073740, filed on January 29, 2023, and PCT Application No. PCT / CN2023 / 143089, filed on December 29, 2023. The entire contents of the foregoing applications are hereby incorporated by reference. Technical Field

[0002] The present invention relates to cationic lipid compounds and compositions comprising such compounds. The present invention also relates to methods for preparing the compounds and compositions, as well as application methods and uses of the compounds and compositions, for example, for delivering bioactive agents (such as RNA preparations) to cells and tissues. Background Art

[0003] Lipid-containing particles have been used as encapsulation and delivery vehicles for the delivery of therapeutic agents, such as nucleic acids, small molecules, and proteins, to cells and other intracellular compartments. There is a continued need to develop novel lipids to encapsulate therapeutic agents and to improve the safety, efficacy, and targeting of these nanoparticle delivery vehicles. Summary of the Invention

[0004] Amine-containing lipids that can be used to formulate lipid nanoparticle (LNP) compositions are disclosed. Such LNP compositions may have properties that facilitate the delivery of nucleic acid payloads, such as CRISPR / Cas gene-editing components, to cells.

[0005] The present invention provides a compound as shown in formula (I) or a pharmaceutically acceptable salt, isomer or prodrug thereof; in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; X1, X2 and X3 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; and at least one of X1, X2 and X3 is independently selected from -S(O)2-, -OS(O)2O-, -OS(O)2N(R6)-, -N(R7)S(O)2O- or -N(R6)S(O)2N(R7)-; Y is -C- or -N-; L1, L2, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

[0006] The present invention provides another compound as shown in formula (I) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is a C1-C3 alkyl group; R2 is a C1-C3 alkyl group; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, or L5-M1-L6-R8; wherein L5 and L6 are each independently a single bond or a C1-C12 alkylene group; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or heteroalkyl; X1 is independently selected from -OS(O)2O-, -OS(O)2N(R6)-, -N(R7)S(O)2O- or -N(R6)S(O)2N(R7)-; X2 and X3 are each independently selected from a single bond, -C(=O)O-, -OC(=O)- or -OC(=O)O-; Y is -C-; L1 and L2 are each independently a single bond or a C1-C6 alkylene group; L3 and L4 are each independently C1-C18 alkylene or L7-M2-L8; wherein L7 and L8 are each independently a single bond or C1-C10 alkylene; M1 and M2 are each independently selected from a single bond, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6 and R7 are each independently selected from H or C1-C3 alkyl; preferably, R6 and / or R7 are H; R8 is selected from H, C1-C18 alkyl or C2-C18 alkenyl.

[0007] The present invention provides another compound as shown in formula (II) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; X2 and X3 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1, L2, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

[0008] The present invention provides another compound as shown in formula (III) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L1, L2, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

[0009] The present invention provides a compound as shown in formula (III), in: R1 is a C1-C3 alkyl group; R2 is a C1-C3 alkyl group; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, or L5-M1-L6-R8; wherein L5 and L6 are each independently a single bond or a C1-C12 alkylene group; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or heteroalkyl; X2 and X3 are each independently selected from a single bond, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1 and L2 are each independently a single bond or a C1-C6 alkylene group; L3 and L4 are each independently C1-C18 alkylene or L7-M2-L8; wherein L7 and L8 are each independently selected from a single bond or C1-C10 alkylene; M1 and M2 are each independently selected from a single bond, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6 is selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl.

[0010] The present invention provides another compound as shown in formula (IV) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; X2 and X3 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1, L2, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

[0011] The present invention provides another compound as shown in formula (V) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; X2 and X3 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1, L2, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

[0012] The present invention provides another compound as shown in formula (V), in: R1 is a C1-C3 alkyl group; R2 is a C1-C3 alkyl group; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, or L5-M1-L6-R8; wherein L5 and L6 are each independently a single bond or a C1-C12 alkylene group; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or heteroalkyl; X2 and X3 are each independently selected from a single bond, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1 and L2 are each independently a single bond or a C1-C6 alkylene group; L3 and L4 are each independently C1-C18 alkylene or L7-M2-L8; wherein L7 and L8 are each independently selected from a single bond or C1-C10 alkylene; M1 and M2 are each independently selected from a single bond, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6 is selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl.

[0013] The present invention provides another compound of formula (VI) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; X2 and X3 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1, L2, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

[0014] The present invention provides another compound of formula (VII) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; X2 and X3 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1, L2, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

[0015] The present invention provides another compound of formula (VIII) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; X2 and X3 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

[0016] The present invention provides another compound of formula (IX) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; X2 and X3 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

[0017] In some embodiments, R1, L1 or R2, and the nitrogen atom to which they are attached together form a 5-membered, 6-membered, or 7-membered ring.

[0018] In some embodiments, L1 is a linear C2 alkylene group. In some embodiments, L1 is a linear C3 alkylene group. In some embodiments, L1 is a linear C4 alkylene group. In some embodiments, L1 is a C1-C5 alkylene group; alternatively, L1 is selected from C2 alkylene, linear C3 alkylene, or linear C4 alkylene.

[0019] In some embodiments, X1 is -OS(O)2-. In some embodiments, X1 is -S(O)2O-. In some embodiments, X1 is -OS(O)2O-. In some embodiments, X1 is -OS(O)2N(R6)-, wherein R6 is selected from H, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C2-C18 alkenyl, or substituted or unsubstituted C2-C18 alkynyl. In some embodiments, X1 is -OS(O)2N(R6)-, wherein R6 is H. In some embodiments, X1 is -(R7)NS(O)2O-, wherein R7 is selected from H, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C2-C18 alkenyl, or substituted or unsubstituted C2-C18 alkynyl. In some embodiments, X1 is -(R7)NS(O)2O-, wherein R7 is H. In some embodiments, X1 is -N(R6)S(O)2N(R7)-, wherein R6 and R7 are independently selected from H, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C2-C18 alkenyl, or substituted or unsubstituted C2-C18 alkynyl. In some embodiments, X1 is N(R6)S(O)2N(R7)-, wherein R6 is H. In some embodiments, X1 is N(R6)S(O)2N(R7)-, wherein R7 is H. In some embodiments, X1 is N(R6)S(O)2N(R7)-, wherein R6 and R7 are both H. In some embodiments, X1 is -OC(=O)O-.

[0020] In some embodiments, L2 is absent. In some embodiments, L2 is a single bond. In some embodiments, L2 is a single bond or C1-C5 alkylene; in some embodiments, L2 is a linear C1 alkylene. In some embodiments, L2 is a linear C2 alkylene. In some embodiments, L2 is a linear C3 alkylene. In some embodiments, L2 is L7-M2-L8, wherein M2 is -C(=O)O-, L7 and L8 are independently absent or independently selected from C1 alkylene. In some embodiments, L2 is L7-M2-L8, wherein M2 is -C(=O)O-, L7 and L8 are independently absent or independently selected from C2 alkylene. In some embodiments, L2 is L7-M2-L8, wherein M2 is -C(=O)O- or -OC(=O)-, L7 and L8 are independently selected from a single bond, C1 alkylene, or C2 alkylene.

[0021] In some embodiments, Y is -C-.

[0022] In some embodiments, L3 is L7-M2-L8, wherein L7 and L8 are independently selected from linear C1-C7 alkylene, M2 is selected from -(R7)NS(O)2O-, and R7 is H. In some embodiments, L3 is L7-M2-L8, wherein L7 and L8 are independently selected from linear C1-C7 alkylene, M2 is selected from -C(=O)O-, -OC(=O)-, and -OC(=O)O-. In some embodiments, L3 is a single bond or C1-C5 alkylene; alternatively, L3 is linear C1 alkylene, linear C2 alkylene, or linear C3 alkylene. In some embodiments, L3 is L7-M2-L8, wherein L7 and L8 are independently selected from linear C1-C10 alkylene, M2 is selected from -C(=O)O-, -OC(=O)-, or -OC(=O)O-. In some embodiments, L3 is L7-M2-L8, wherein L7 and L8 are independently linear C1-C7 alkylene, and M2 is selected from -C(=O)O- or -OC(=O)-. In some embodiments, L3 is L7-M2-L8, wherein L7 is linear C1-C3 alkylene and L8 is linear C3-C7 alkylene, and M2 is -C(=O)O- or -OC(=O)-.

[0023] In some embodiments, L4 is L7-M2-L8, wherein L7 and L8 are independently selected from linear C1-C7 alkylene, M2 is selected from -(R7)NS(O)2O-, and R7 is H. In some embodiments, L4 is L7-M2-L8, wherein L7 and L8 are independently selected from linear C1-C7 alkylene, M2 is selected from -C(=O)O-, -OC(=O)-, and -OC(=O)O-. In some embodiments, L4 is L7-M2-L8, wherein L7 and L8 are independently selected from linear C1-C7 alkylene, and M2 is OC(=O)-. In some embodiments, L4 is a single bond or C1-C5 alkylene; alternatively, L3 is linear C1 alkylene, linear C2 alkylene, or linear C3 alkylene. In some embodiments, L4 is L7-M2-L8, wherein L7 and L8 are independently linear C1-C10 alkylene, and M2 is selected from -C(=O)O-, -OC(=O)-, or -OC(=O)O-. In some embodiments, L4 is L7-M2-L8, wherein L7 and L8 are independently linear C1-C7 alkylene, and M2 is selected from -C(=O)O- or -OC(=O)-. In some embodiments, L4 is L7-M2-L8, wherein L7 is linear C1-C3 alkylene and L8 is linear C3-C7 alkylene, and M2 is -C(=O)O- or -OC(=O)-.

[0024] In some embodiments, X2 is a single bond. In some embodiments, X2 is -C(=O)O- or -OC(=O)-. In some embodiments, X2 is -(R7)NS(O)2O-, wherein R7 is H. In some embodiments, X3 is independently selected from a single bond. In some embodiments, X3 is C(=O)O- or -OC(=O)-.

[0025] In some embodiments, R3 is C1-C18 alkyl. In some embodiments, R3 is C1-C10 alkyl. In some embodiments, R3 is C2-C18 alkenyl. In some embodiments, R3 is C2-C10 alkenyl. In some embodiments, R3 is C6-C18 alkyl, preferably, R3 is C6-C10 alkyl. In some embodiments, R3 is C6-C18 alkenyl, alternatively, R3 is C10-C18 alkenyl, preferably, R3 is C17 alkenyl. In some embodiments, R3 is Preferably, R3 is In some embodiments, R3 is Preferably, R3 is In some embodiments, R3 is L5-M1-L6-R8; L5 and L6 are independently a single bond or C1-C8 alkylene; M1 is selected from a single bond, -C(=O)O-, or -OC(=O)-; R8 is selected from C8-C18 alkyl or C8-C18 alkenyl. In some embodiments, R3 is L5-M1-L6-R8; L5 and L6 are independently a single bond or C3-C8 alkylene; M1 is -C(=O)O- or -OC(=O)-; R8 is selected from C8-C18 alkyl or C8-C18 alkenyl.

[0026] In some embodiments, R4 and R5 are independently selected from heteroalkyl. In some embodiments, R4 and R5 are independently selected from -OR' or -SR', wherein R' is selected from substituted or unsubstituted C1-C18 alkyl (e.g., C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl), substituted or unsubstituted C2-C18 alkenyl (e.g., C4 alkenyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C10 alkenyl, C11 alkenyl, C12 alkenyl, C13 alkenyl) or substituted or unsubstituted C2-C18 alkynyl (e.g., C4 alkynyl, C5 alkynyl, C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C10 alkynyl, C11 alkynyl, C12 alkynyl, C13 alkynyl). In some embodiments, R4 and R5 are independently selected from -O(CH2)7Me. In some embodiments, R4 and R5 are independently selected from -O(CH2)8Me. In some embodiments, R4 and R5 are independently selected from -S(CH2)8Me. In some embodiments, R4 and R5 are independently selected from -S(CH2)7Me. In some embodiments, R4 and R5 are independently selected from C1-C18 alkyl. In some embodiments, R4 and R5 are independently selected from C1-C8 alkyl. In some embodiments, R4 and R5 are independently selected from branched C1-C18 alkyl. In some embodiments, R4 and R5 are independently H or heteroalkyl; preferably, R4 and R5 are independently -O-alkyl or -S-alkyl. In some embodiments, R4 and R5 are independently H or -O(CH2) 5-11 CH3; Preferably, R4 and R5 are independently H or -O(CH2)7CH3. In some embodiments, R4 and R5 are independently H or -S(CH2) 5-11 CH3; preferably, R4 and R5 are independently H or -S(CH2)7CH3. In some embodiments, R4 and R5 are independently selected from H, C6-C18 alkyl, or C6-C18 alkenyl; alternatively, R4 and R5 are independently linear C8-C18 alkyl. In some embodiments, R4 is H, and R5 is selected from C6-C18 alkyl or C6-C18 alkenyl. In some embodiments, R4 and R5 are independently branched C10-C18 alkyl.

[0027] The present invention also provides a lipid composition comprising: a bioactive agent; and a lipid component, wherein the lipid component comprises: a) an ionizable lipid; b) a neutral lipid; c) a helper lipid; and d) a PEG lipid; wherein the ionizable lipid is a compound of formula (I)-(IX), including any group selection described above.

[0028] In some embodiments, the neutral lipid is an uncharged lipid or a zwitterionic lipid. In some embodiments, the neutral lipid is DSPC or DPME. In some embodiments, the neutral lipid is DSPC. In some embodiments, the helper lipid is cholesterol. In some embodiments, the PEG lipid comprises dimyristoyl glycerol (DMG). In some embodiments, the PEG lipid comprises PEG-2k. In some embodiments, the PEG lipid is PEG-DMG. In some embodiments, the PEG lipid is PEG-2k DMG (DMG-PEG2000). In some embodiments, the PEG lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000.

[0029] In some embodiments, the content of the ionizable lipid is about 30-70 mol% of the lipid component. In some embodiments, the content of the ionizable lipid is about 40-60 mol% of the lipid component. In some embodiments, the content of the ionizable lipid is about 40-50 mol% of the lipid component. In some embodiments, the content of the ionizable lipid is about 45-50 mol% of the lipid component. In some embodiments, the content of the ionizable lipid is about 45 mol%, 46.3 mol%, 48.5 mol% or 50 mol% of the lipid component.

[0030] In some embodiments, the neutral lipids are present at about 3-20 mol% of the lipid component. In some embodiments, the neutral lipids are present at about 5-15 mol% of the lipid component. In some embodiments, the neutral lipids are present at about 8-12 mol% of the lipid component. In some embodiments, the neutral lipids are present at about 9-10 mol% of the lipid component; alternatively, the neutral lipids are present at about 9 mol%, 9.4 mol%, or 10 mol% of the lipid component.

[0031] In some embodiments, the helper lipid is present in an amount of about 30-60 mol% of the lipid component. In some embodiments, the helper lipid is present in an amount of about 35-50 mol% of the lipid component. In some embodiments, the helper lipid is present in an amount of about 38-44.5 mol% of the lipid component; alternatively, the helper lipid is present in an amount of about 38 mol%, 39 mol%, 40 mol%, 40.8 mol%, 42.7 mol%, 43 mol%, 43.5 mol%, 44 mol%, or 44.5 mol% of the lipid component.

[0032] In some embodiments, the PEG lipid is present in an amount of about 0.5-5 mol% of the lipid component. In some embodiments, the PEG lipid is present in an amount of about 1.0-3.5 mol% of the lipid component. In some embodiments, the PEG lipid is present in an amount of about 1.5-3 mol% of the lipid component; alternatively, the PEG lipid is present in an amount of about 1.5 mol%, 1.6 mol%, 1.7 mol%, 2 mol%, 2.5 mol% or 3 mol% of the lipid component.

[0033] In some embodiments, the ionizable lipids are present in an amount of 40-60 mol% of the lipid component; the neutral lipids are present in an amount of 5-15 mol% of the lipid component; the helper lipids are present in an amount of 35-50 mol% of the lipid component; and the PEG lipids are present in an amount of 1.0-3.5 mol% of the lipid component.

[0034] In some embodiments, the ionizable lipids are present at 50 mol% of the lipid component; the neutral lipids are present at 9 mol% of the lipid component; the helper lipids are present at 38 mol% of the lipid component; and the PEG lipids are present at 3 mol% of the lipid component.

[0035] In some embodiments, the ionizable lipids are present at about 46.3 mol% of the lipid component; the neutral lipids are present at about 9.4 mol% of the lipid component; the helper lipids are present at about 42.7 mol% of the lipid component; and the PEG lipids are present at about 1.6 mol% of the lipid component.

[0036] In some embodiments, the ionizable lipids are present at about 48.5 mol% of the lipid component; the neutral lipids are present at about 10 mol% of the lipid component; the helper lipids are present at about 39 mol% of the lipid component; and the PEG lipids are present at about 2.5 mol% of the lipid component.

[0037] In some embodiments, the ionizable lipids are present at about 47.5 mol% of the lipid component; the neutral lipids are present at about 10 mol% of the lipid component; the helper lipids are present at about 40 mol% of the lipid component; and the PEG lipids are present at about 1.5 mol% of the lipid component.

[0038] In some embodiments, the ionizable lipids are present at about 49.5 mol% of the lipid component; the neutral lipids are present at about 10 mol% of the lipid component; the helper lipids are present at about 40 mol% of the lipid component; and the PEG lipids are present at about 1.5 mol% of the lipid component.

[0039] In some embodiments, the ionizable lipids are present at about 45 mol% of the lipid component; the neutral lipids are present at about 9 mol% of the lipid component; the helper lipids are present at about 43 mol% of the lipid component; and the PEG lipids are present at about 3 mol% of the lipid component.

[0040] In some embodiments, the ionizable lipids are present at about 45 mol% of the lipid component; the neutral lipids are present at about 9 mol% of the lipid component; the helper lipids are present at about 43.5 mol% of the lipid component; and the PEG lipids are present at about 2.5 mol% of the lipid component.

[0041] In some embodiments, the ionizable lipids are present at about 45 mol% of the lipid component; the neutral lipids are present at about 9 mol% of the lipid component; the helper lipids are present at about 44 mol% of the lipid component; and the PEG lipids are present at about 2 mol% of the lipid component.

[0042] In some embodiments, the ionizable lipids are present at about 45 mol% of the lipid component; the neutral lipids are present at about 9 mol% of the lipid component; the helper lipids are present at about 44.5 mol% of the lipid component; and the PEG lipids are present at about 1.5 mol% of the lipid component.

[0043] In some embodiments, the ionizable lipids are present at about 46.3 mol% of the lipid component; the neutral lipids are present at about 9.4 mol% of the lipid component; the helper lipids are present at about 41.8 mol% of the lipid component; and the PEG lipids are present at about 2.5 mol% of the lipid component.

[0044] In some embodiments, the composition is an LNP (lipid nanoparticle) composition. In some embodiments, the lipid component comprises a non-cationic lipid and a PEG lipid. In some embodiments, the non-cationic lipid is a neutral lipid. In some embodiments, the composition further comprises a buffer. In some embodiments, the composition further comprises a bioactive agent. In some embodiments, the bioactive agent comprises a polypeptide. In some embodiments, the bioactive agent comprises a nucleic acid. In some embodiments, the nucleic acid component comprises RNA. In some embodiments, the RNA component comprises modified RNA. In some embodiments, the RNA component comprises a sequence encoding an RNA-guided DNA binder (such as a Cas nuclease mRNA). In some embodiments, the nucleic acid component comprises RNA, wherein the RNA component comprises mRNA. In some embodiments, the RNA component comprises Class II Cas nuclease mRNA. In some embodiments, the RNA component comprises mRNA encoding Cas9, Cpf1, c2c2, cas12i, cas12f, cas12b, cas12c, cas12d, cas12e, cas12g, cas12j, or cas12k nuclease. In some embodiments, the RNA component comprises a gRNA nucleic acid. In some embodiments, the RNA component comprises Cas nuclease mRNA and gRNA. In some embodiments, the gRNA nucleic acid is or encodes a dual-guide RNA (dgRNA). In some embodiments, the gRNA nucleic acid is or encodes a single-guide RNA (sgRNA). In some embodiments, the gRNA is a modified gRNA. In some embodiments, the composition further comprises at least one template nucleic acid. In some embodiments, the N / P ratio of the composition is about 3-10. In some embodiments, the N / P ratio of the composition is about 5-8. In some embodiments, the N / P ratio of the composition is about 6.

[0045] The present invention also provides a method for delivering a bioactive agent to a cell, comprising contacting the cell with the above composition.

[0046] In some embodiments, the cell is a hepatocyte, such as a hepatocyte.

[0047] The present invention also provides a gene editing method, comprising contacting cells with the above composition.

[0048] The present invention also provides a DNA cleavage method, comprising contacting cells with the above composition.

[0049] In some embodiments, the above method comprises administering the composition to a human body. In some embodiments, the above method comprises administering the composition to a cell. In some embodiments, the cell is a eukaryotic cell.

[0050] In some embodiments, the methods comprise administering an mRNA formulated in a first LNP composition, and a second LNP composition comprising one or more mRNAs, a gRNA, and a template nucleic acid.

[0051] In some embodiments, the methods comprise administering mRNA and gRNA nucleic acids formulated in a single LNP composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Shown are the editing efficiencies of TTR-targeted cells after administration of different LNP formulations in mice.

[0053] Figure 2 Shown are the editing efficiencies of TTR-targeted cells after in vivo administration of different LNP formulations in PCH (humanized mouse model).

[0054] Figure 3 The editing efficiency of TTR targeting after administration of different LNP formulations in NHP (non-human primate) in vivo is shown.

[0055] Figure 4 Shown are the editing efficiencies of TTR-targeted cells after administration of different LNP formulations of YZL026 in mice.

[0056] Figure 5 Shown are the editing efficiencies of TTR-targeted cells after administration of different LNP formulations of YZL002 in mice.

[0057] Figure 6 Shown are the editing efficiencies of TTR-targeted cells after administration of different LNP formulations of YZL026 in rats.

[0058] Figure 7 Shown are the editing efficiencies of TTR-targeted cells after administration of different LNP formulations of YZL002 in rats. DETAILED DESCRIPTION

[0059] Cationic lipids

[0060] Certain compounds of the present invention may have one or more chiral centers and may therefore exist in optically active forms. Similarly, when a compound contains an alkenyl or olefinic group, cis and trans isomers may exist. The present invention encompasses R- and S-isomers and mixtures thereof, including racemic mixtures and mixtures of cis and trans isomers. Other asymmetric carbon atoms may also be present in substituents (such as alkyl groups). All of these isomers and mixtures thereof are within the scope of the present invention. If a specific stereoisomer is desired, it can be prepared by methods known in the art, such as stereospecific reactions using starting materials containing resolved asymmetric centers, or by generating a mixture of stereoisomers and then resolving them using known methods. For example, a racemic mixture can be reacted with a single enantiomer of another compound, which, because they have different melting points, boiling points, and solubilities, can be separated by conventional methods such as crystallization.

[0061] Prodrugs are derivatives of the compounds of the present invention that contain groups that can be cleaved chemically or metabolically and are converted into compounds of the present invention with in vivo pharmaceutical activity under solvolysis or physiological conditions. Prodrugs include acid derivatives well known to those skilled in the art, such as esters prepared by reacting an acidic compound with a hydroxyl group, or amines prepared by reacting a parent acid compound with an appropriate amine. Simple aliphatic or aromatic esters derived from the acidic groups contained in the compounds of the present invention are preferred prodrugs. In some cases, double ester prodrugs can be prepared, such as (acyloxy) alkyl esters or ((alkoxycarbonyl)oxy) alkyl esters. Particularly preferred prodrug esters include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, morpholineethyl, and N,N-diethylglycinamido esters.

[0062] In some embodiments, compounds of the invention comprise the structure shown below.

[0063] Representative compounds of the general formula:

[0064] Composition

[0065] The lipid composition can be provided in the form of a particle composition. The particle can be, for example, a microsphere (including unilamellar and multilamellar vesicles, such as "liposomes" - lamellar lipid bilayer structures, which are substantially spherical in certain embodiments and, in more specific embodiments, can comprise an aqueous core, e.g., comprising a majority of the RNA molecule), a dispersed phase in an emulsion, a micelle, or the internal phase in a suspension.

[0066] According to an exemplary embodiment, the particles may be composed of cationic lipids (such as the compounds shown in Table 1), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and PEG derivatives (such as DMG-PEG) in different molar ratios.

[0067] In certain embodiments, the composition is an LNP (lipid nanoparticle) composition. In certain embodiments, the lipid component comprises a non-cationic lipid and a PEG lipid. In certain embodiments, the non-cationic lipid is a neutral lipid.

[0068] "Neutral lipids" suitable for use in the lipid compositions of the present invention include a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present invention include, but are not limited to, 5-heptadecanethiol-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), palmitoyllinoleoylphosphatidylcholine (PLPC), 1,2-diachidoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg lecithin (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PLPC), 1,2-diazao ... Phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-diacaproyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-diicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid is selected from distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE).

[0069] "Helper lipids" include steroids, sterols, and alkylresorcinols. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecanylresorcinol, and cholesterol hemisuccinate. In certain embodiments, the helper lipid can be cholesterol or a derivative thereof, such as cholesterol hemisuccinate.

[0070] In some embodiments, the PEG derivatives can be coupled to one or more additional molecules, such as lipids. In any embodiment described herein, the PEG lipid can be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilauroylglycinamide, PEG-dimyristoylglycinamide, PEG-dipalmitoylglycinamide and PEG-distearoylglycinamide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[β]-oxy)formamido-3',6'-dioxaoctyl]formamido-ω-methyl-polyethylene glycol), PEG-DMB (3,4-ditetradecyloxybenzyl-ω-methyl-polyethylene glycol ether), 1,2-dimyristoylglycerol. In some embodiments, the PEG lipid may be PEG2k-DMPE, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE), 1,2-distearoyl-sn-glycero, methoxypolyethylene glycol, polyethylene glycol-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearoyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In certain such embodiments, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In other embodiments, the PEG lipid may be PEG2k-DSPE. In some embodiments, the PEG lipid may be PEG2k-DMA. In other embodiments, the PEG lipid may be PEG2k-C-DMA. In certain embodiments, the PEG lipid may be compound S027 disclosed in WO2016 / 010840. In some embodiments, the PEG lipid may be PEG2k-DSA. In other embodiments, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16. In some embodiments, the PEG lipid may be PEG2k-C18. In preferred embodiments, the PEG lipid comprises a glycerol group. In preferred embodiments, the PEG lipid comprises a dimyristoylglycerol (DMG) group. In preferred embodiments, the PEG lipid comprises PEG-2L. In preferred embodiments, the PEG lipid is PEG-DMG. In preferred embodiments, the PEG lipid is PEG-2k-DMG.In a preferred embodiment, the PEG lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000. In a preferred embodiment, the PEG-2k-DMG is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000. In some embodiments, the PEG lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecanoyl acetamide (ALC-0159).

[0071] In some embodiments, the composition further comprises a bioactive agent. In some embodiments, the bioactive agent comprises a polypeptide, optionally in combination with a nucleic acid. In some embodiments, the bioactive agent comprises a nucleic acid, such as RNA. In some embodiments, the composition comprises a nucleic acid component. In some representative embodiments, the composition comprises a nucleic acid, such as siRNA, miRNA, shRNA, antisense RNA, etc. In some embodiments, the nucleic acid component comprises DNA, which may be referred to as a DNA component. In some embodiments, the nucleic acid component comprises RNA. In some embodiments, the component (such as an RNA component) may comprise mRNA, such as an mRNA encoding an RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder is a Cas nuclease. In certain embodiments, the composition may comprise mRNA encoding Cas9, Cpf1, c2c2, cas12i, cas12f, cas12b, cas12c, cas12d, cas12e, cas12g, cas12j, or cas12k. In certain embodiments, the composition may comprise gRNA. In some compositions comprising mRNA encoding an RNA-guided DNA binder, the composition further comprises a gRNA nucleic acid (such as a gRNA). In some embodiments, the composition comprises an RNA-guided DNA binder and gRNA. In some embodiments, the composition comprises a Cas nuclease mRNA and a gRNA. In some embodiments, the composition comprises a Class II Cas nuclease mRNA and a gRNA. In some embodiments, the composition comprises a small molecule compound, optionally lipophilic.

[0072] "mRNA" refers to a polynucleotide comprising an open reading frame that can be translated into a polypeptide (i.e., a substrate for translation by ribosomes and aminoacyl-tRNA). The mRNA may comprise a phosphate-sugar backbone containing a ribose residue or an analog thereof (e.g., a 2'-methoxyribose residue). In some embodiments, the sugar of the mRNA phosphate-sugar backbone consists essentially of a ribose residue, a 2'-methoxyribose residue, or a combination thereof. Generally speaking, the mRNA does not contain a large amount of thymidine residues (e.g., 0 or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). The mRNA may contain modified uridines at some or all uridine sites.

[0073] As used herein, "Cas nucleases" include Cas cleavage enzymes, Cas nickases, and dCas DNA binders. Cas cleavage enzymes / nickases and dCas DNA binders include Csm or Cmr complexes of type III CRISPR systems, their Cas10, Csm1, or Cmr2 subunits, Cascade complexes of type I CRISPR systems, their Cas3 subunits, and class II Cas nucleases. Herein, "class II Cas nucleases" refer to single-chain polypeptides with RNA-guided DNA binding activity. Class II Cas nucleases include class II Cas cleavage enzymes / nickases (such as H840A, D10A, or N863A variants), which also have RNA-guided DNA cleavage or nickase activity; and class II dCas DNA binders whose cleavage / nickase activity has been inactivated. Class II Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9 (1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9 (1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modified forms thereof. In some embodiments, Cas9 is SaCas9. The Cpf1 protein (Zetsche et al., Cell, 2015, Vol. 163, pp. 1-13) is homologous to Cas9 and contains a RuvC-like nuclease domain. The Cpf1 sequence in the Zetsche paper is incorporated herein by reference in its entirety. See Makarova et al., Nature Reviews Microbiology, vol. 13, 2015, pp. 722-736; Shmakov et al., Molecular Cell, vol. 60, 2015, pp. 385-397.

[0074] "Guide RNA," "gRNA," and simply "guide" are used interchangeably herein to refer to the cognate guide nucleic acid of an RNA-guided DNA binder. The guide RNA may comprise a modified RNA as described herein. The gRNA may be a crRNA (also known as CRISPR RNA) or a combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may exist as a single RNA molecule (single-stranded guide RNA, sgRNA) or as two separate RNA molecules (double-stranded guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to all types. The trRNA may be a native sequence or a trRNA sequence that has been modified or mutated relative to the native sequence.

[0075] In some embodiments, the sgRNA is a "Cas9sgRNA" capable of mediating RNA-guided DNA cleavage by the Cas9 protein. In some embodiments, the sgRNA is a "Cpf1sgRNA" capable of mediating RNA-guided DNA cleavage by the Cpf1 protein. In certain embodiments, the gRNA comprises a crRNA and a tracr RNA sufficient to form an active complex with the Cas9 protein and mediate RNA-guided DNA cleavage. In certain embodiments, the gRNA comprises a crRNA sufficient to form an active complex with the Cpf1 protein and mediate RNA-guided DNA cleavage. See Zetsche 2015.

[0076] Certain embodiments of the present invention also provide nucleic acids encoding gRNAs described herein (e.g., expression cassettes). "Guiding RNA nucleic acid" herein refers to gRNAs (e.g., sgRNAs or dgRNAs) and gRNA expression cassettes (nucleic acids encoding one or more gRNAs).

[0077] In certain embodiments, lipid compositions (such as LNP compositions) include modified nucleic acids, including modified RNA. Modified nucleosides or nucleotides may be present in RNA (e.g., gRNA or mRNA). GRNA or mRNA containing one or more modified nucleosides or nucleotides are referred to as "modified" RNA, for describing the presence of one or more non-natural and / or naturally occurring components or configurations (replacing or supplementing classical A, G, C, and U residues). In certain embodiments, modified RNA is synthesized with non-classical nucleosides or nucleotides (herein referred to as "modification").

[0078] Modified nucleosides and nucleotides may comprise one or more of the following changes: (i) replacement of non-bridging oxygen atoms and / or bridging oxygen atoms in the phosphodiester backbone bond (representative backbone modifications); (ii) changes in the ribose composition (such as replacement of the 2' hydroxyl group of ribose, a representative sugar modification); (iii) replacement of the phosphate group as a whole with a "dephospho" linker (representative backbone modification); (iv) modification or replacement of natural bases (including the use of non-classical bases, a representative base modification); (v) replacement or modification of the ribose-phosphate backbone (representative backbone modification); (vi) modification of the 3' or 5' end of the polynucleotide (such as removal, modification or replacement of the terminal phosphate group, or conjugation of a molecule, cap or linker, such 3' or 5' end cap modification may comprise a sugar and / or backbone modification); (vii) modification or replacement of a sugar (representative sugar modification). Certain embodiments comprise 5' end modifications of the mRNA, gRNA or nucleic acid. Certain embodiments comprise 3' end modifications of the mRNA, gRNA or nucleic acid. Modified RNAs can include both 5' and 3' modifications, and can also include one or more modified residues at non-terminal positions. In certain embodiments, gRNAs include at least one modified residue; in certain embodiments, mRNAs include at least one modified residue.

[0079] In some embodiments, RNA or nucleic acid is chemically or biologically modified to enhance stability. Exemplary modifications include base deletion (by deletion or nucleotide substitution) or base modification (e.g., chemical modification). As used herein, "chemical modification" refers to the introduction of a modification that is different from the chemical structure present in natural RNA or nucleic acid molecules, including covalent modification (e.g., the introduction of modified nucleotides (e.g., nucleotide analogs) or the addition of side chain groups that are not naturally present in such RNA or nucleic acid molecules).

[0080] In some embodiments of backbone modification, the phosphate group of the modified residue can be modified by replacing one or more of its oxygen atoms with different substituents. In addition, the modified residue (e.g., a modified residue in a modified nucleic acid) can comprise replacing the entire unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, backbone modification of the phosphate backbone can produce the following two results: forming an uncharged linker or forming a charged linker with an asymmetric charge distribution.

[0081] Examples of modified phosphate groups include: phosphorothioates, phosphoroselenoates, boranophosphates, boranophosphates, hydrogen phosphonates, phosphoramidates, alkyl / aryl phosphonates, and phosphotriesters. The phosphorus atom in the unmodified phosphate group is achiral, but replacing the non-bridging oxygen atom with the above atoms / groups can produce a chiral phosphorus atom. The chiral phosphorus atom can be in an "R" configuration (referred to herein as Rp) or an "S" configuration (referred to herein as Sp). The backbone modification can also be achieved by replacing the bridging oxygen atom (the oxygen connecting the phosphate and the nucleoside) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), or carbon (bridging methylene phosphonates). The replacement can occur on a single or two bridging oxygen atoms. In certain backbone modifications, the phosphate group can be replaced by a non-phosphorus linking group. In some embodiments, the charged phosphate group can be replaced by a neutral group, including but not limited to: methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformal, formal, oxime, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine, and methyleneoxymethylimino.

[0082] In some embodiments, the compositions or formulations disclosed herein comprise an mRNA encoding an RNA-guided DNA-binding agent (such as a Cas nuclease or a Class II Cas nuclease) open reading frame (ORF). In some embodiments, an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA-binding agent (such as a Cas nuclease or a Class II Cas nuclease) is provided, used, or administered. The mRNA may comprise one or more of the following elements: a 5' end cap structure, a 5' untranslated region (UTR), a 3' UTR, and a polyadenylation tail. The mRNA may comprise a modified open reading frame, for example, for encoding a nuclear localization sequence or encoding a target protein by selecting different codons.

[0083] The mRNA in the disclosed LNP compositions can encode, for example, a secreted hormone, enzyme, receptor, polypeptide, peptide, or other target protein that is normally secreted. In one embodiment of the present invention, the mRNA can optionally have a chemical or biological modification, such as a modification that increases the stability and / or half-life of the mRNA, or a modification that improves or otherwise promotes protein production.

[0084] In addition, suitable modifications include changes to one or more nucleotides in the codon so that the codon encodes the same amino acid but is more stable than the codon in the wild-type mRNA. For example, it has been shown that the stability of RNA is negatively correlated with a higher number of cytidine (Cs) and / or uridine (Us) residues, and RNA without C and U residues has stability to most RNases (Heidenreich et al., Journal of Biological Chemistry, Vol. 269, pp. 2131-8, 1994). In some embodiments, the number of C and / or U residues in the mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by replacing a codon encoding a specific amino acid with another codon encoding the same or related amino acid. The present invention also includes the incorporation of pseudouridine into the mRNA nucleic acid of the present invention. Pseudouridine can be incorporated into the mRNA nucleic acid of the present invention to enhance stability and translation ability and reduce in vivo immunogenicity. See, for example, Kariko, K. et al., Molecular Therapy, Vol. 16, No. 11, pp. 1833-1840, 2008. The substitution and modification of the mRNA of the present invention can be carried out by methods well known to those skilled in the art.

[0085] The term "modification" also includes the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequence of the present invention (e.g., modifications to the 3' and / or 5' ends of an mRNA molecule encoding a functional secreted protein or enzyme). Such modifications include: adding bases to the mRNA sequence (e.g., adding a poly A tail or extending the poly A tail), altering the 3'UTR or 5'UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and introducing elements that can change the structure of the mRNA molecule (e.g., elements that form secondary structures).

[0086] The poly A tail is believed to stabilize natural messenger molecules. Therefore, in one embodiment, a long poly A tail can be added to an mRNA molecule to make the mRNA more stable. The poly A tail can be added by a variety of techniques recognized in the art. For example, a long poly A tail can be added to a synthetic or in vitro transcribed mRNA using poly A polymerase (Yokoe et al., Nature Biotechnology, 1996; 14: 1252-1256). Transcription vectors can also encode long poly A tails. In addition, the poly A tail can be added by direct transcription from a PCR product. In one embodiment, the length of the poly A tail is at least about 90, 200, 300, 400 or at least 500 nucleotides. In another embodiment, the stability of the modified mRNA molecule of the present invention can be controlled by adjusting the length of the poly A tail, thereby regulating protein transcription. For example, because the length of the poly A tail can affect the half-life of the mRNA molecule, the level of resistance of the mRNA to nucleases can be changed by adjusting its length, thereby regulating the duration of protein expression in the cell. In one embodiment, the stabilized mRNA molecule is sufficiently resistant to degradation in vivo (eg, by nucleases) that it can be delivered to target cells without the need for a delivery vehicle.

[0087] In certain embodiments, mRNA can be modified by introducing 3' and / or 5' untranslated region (UTR) sequences that are naturally absent in wild-type mRNA. In another embodiment, 3' and / or 5' flanking sequences naturally present in mRNA flanks and encoding a second unrelated protein can be integrated into the mRNA molecule nucleotide sequence encoding therapeutic or functional protein. For example, 3' or 5' sequences from stable mRNA molecules (such as globin, actin, GAPDH, tubulin, histone or tricarboxylic acid cycle enzymes) can be integrated into 3' and / or 5' regions of justice mRNA nucleic acid molecules to improve the stability of justice mRNA molecules. See, for example, U.S. Patent No. 2003 / 0083272. A more detailed description of mRNA modifications can be found in U.S. Patent No. 2017 / 0210698A1 pages 57-68, the contents of which are incorporated herein by reference.

[0088] The compositions and methods disclosed herein may include template nucleic acids. The template can be used to change or insert a nucleic acid sequence at or near the target site of an RNA-guided DNA binding protein (such as a Class II Cas nuclease). In some embodiments, the method includes introducing a template into a cell. In some embodiments, a single template can be provided. In other embodiments, two or more templates can be provided to edit at two or more target sites. For example, different templates can be provided to edit a single gene or two different genes in a cell.

[0089] In some embodiments, the template can be used for homologous recombination. In some embodiments, homologous recombination may cause the template sequence or a partial sequence thereof to be integrated into the target nucleic acid molecule. In another embodiment, the template can be used for homology-directed repair, a process involving DNA chain invasion of the nucleic acid cleavage site. In some embodiments, homology-directed repair may cause the edited target nucleic acid molecule to include a template sequence. In other embodiments, the template can be used for gene editing mediated by non-homologous end joining. In some embodiments, the template sequence has no similarity to the nucleic acid sequence adjacent to the cleavage site. In some embodiments, the template or a partial sequence thereof can be integrated. In some embodiments, the template comprises flanking inverted terminal repeat (ITR) sequences.

[0090] In some embodiments, lipid nanoparticles (LNPs) are prepared by mixing an aqueous RNA solution with an organic solvent-based lipid solution. Suitable solutions or solvents include, but are not limited to, water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, ether, cyclohexane, tetrahydrofuran, methanol, and isopropanol. For example, the organic solvent can be 100% ethanol. Pharmaceutically acceptable buffers, such as those used for in vivo administration of LNPs, can also be used. In certain embodiments, a buffer is used to maintain the pH of the LNP-containing composition at 6.5 or higher. In certain embodiments, a buffer is used to maintain the pH of the LNP-containing composition at 7.0 or higher. In certain embodiments, the pH of the composition is in the range of 7.2-7.7. In other embodiments, the pH of the composition is in the range of 7.3-7.7 or 7.4-7.6. In other embodiments, the pH of the composition is specifically 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of the composition can be measured using a micro pH probe. In certain embodiments, the composition is added with a cryoprotectant. Cryoprotectants include, but are not limited to, sucrose, trehalose, glycerol, DMSO, and ethylene glycol. In a typical composition, the cryoprotectant content can reach 10%, such as sucrose. In certain embodiments, the composition contains tri-salt sucrose (TSS). In certain embodiments, the cryoprotectant content in the LNP composition is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In certain embodiments, the sucrose content in the LNP composition is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In certain embodiments, the LNP composition contains a buffer. In certain embodiments, the buffer can be selected from phosphate buffered saline (PBS), Tris buffer, citrate buffer or a mixture thereof. In certain embodiments, the buffer contains NaCl. In certain embodiments, the buffer does not contain NaCl. The typical concentration range of NaCl is 20-45mM or 40-50mM. In certain embodiments, the NaCl concentration is 45mM. In certain embodiments, the buffer is a Tris buffer, and the typical concentration range of Tris is 20-60mM or 40-60mM. In certain embodiments, the Tris concentration is 50mM. In certain embodiments, the buffer contains both NaCl and Tris. In certain exemplary embodiments, the lipid nanoparticle (LNP) composition comprises a Tris buffer containing 5% sucrose and 45 mM NaCl. In other exemplary embodiments, the composition comprises about 5% (w / v) sucrose, about 45 mM NaCl, and about 50 mM Tris (pH 7.5). The amounts of salt, buffer, and cryoprotectant can be adjusted to maintain the osmotic pressure of the entire composition.

[0091] In certain embodiments, the particles are nanoparticles. In certain embodiments, the particle (including the encapsulated nucleic acid) and the targeting moiety on the particle surface have a particle size ranging from about 10 nanometers to about 500 nanometers. In certain embodiments, the particle size ranges from about 10 nanometers to about 350 nanometers. In certain embodiments, the particle size ranges from about 50 nanometers to about 250 nanometers. In certain embodiments, the particle size ranges from about 10 nanometers to about 200 nanometers. In certain embodiments, the particle size ranges from about 20 nanometers to about 200 nanometers. In certain embodiments, the particle size ranges from about 50 nanometers to about 200 nanometers. In certain embodiments, the particle size ranges from about 75 nanometers to about 200 nanometers. In certain embodiments, the particle size ranges from about 90 nanometers to about 200 nanometers. In certain embodiments, the particle size ranges from about 100 nanometers to about 200 nanometers. In certain embodiments, the particle size ranges from about 120 nanometers to about 200 nanometers. In certain embodiments, the particle has a size in the range of about 150 nanometers to about 200 nanometers. In certain embodiments, the particle has a size in the range of about 50 nanometers to about 150 nanometers. In certain embodiments, the particle has a size greater than about 10 nanometers. In certain embodiments, the particle has a size greater than about 20 nanometers. In certain embodiments, the particle has a size greater than about 30 nanometers. In certain embodiments, the particle has a size greater than about 40 nanometers. In certain embodiments, the particle has a size greater than about 50 nanometers. In certain embodiments, the particle has a size greater than about 60 nanometers. In certain embodiments, the particle has a size greater than about 70 nanometers. In certain embodiments, the particle has a size greater than about 80 nanometers. In certain embodiments, the particle has a size greater than about 90 nanometers. In certain embodiments, the particle has a size greater than about 100 nanometers. In certain embodiments, the particle has a size greater than about 200 nanometers. In certain embodiments, the particle has a size of no more than about 500 nanometers. In certain embodiments, the particle (including the encapsulated nucleic acid) has a size in the range of about 5 nanometers to about 200 nanometers. In certain embodiments, the particle (including the encapsulated nucleic acid) has a particle size in the range of about 50 nanometers to about 60 nanometers. In certain embodiments, the particle (including the encapsulated nucleic acid) has a particle size in the range of about 55 nanometers to about 58 nanometers. In certain embodiments, the particle size is the hydrodynamic diameter.

[0092] According to certain embodiments, the lipid phase may comprise about 20%-70% (mol percent) of membrane-stabilizing lipids. For example, the membrane-stabilizing lipids may comprise about 40%-60% of the lipid phase. In some embodiments, more than one membrane-stabilizing lipid may be used in the lipid phase. For example, the membrane-stabilizing lipids may comprise cholesterol (about 30%-50% (mol percent) of the lipid phase) and a phospholipid (e.g., DSPC), which may comprise about 5%-15% (mol percent) of the lipid phase.

[0093] According to certain embodiments, the additional PEG derivative (bound to the lipid) may comprise about 0.5%-10% of the lipid phase composition.

[0094] In some embodiments, the lipids can be suspended in an organic solution, such as ethanol. In some embodiments, the nucleic acids can be present in an acetate buffer solution.

[0095] In certain embodiments, nucleic acids can be mixed with a lipid mixture in a microfluidizer to form particles encapsulating / carrying nucleic acids.

[0096] Explanation of terms

[0097] It should be noted that, in this application, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes a plurality of compositions, reference to "a cell" includes a plurality of cells, and so on. Unless otherwise indicated, the use of "or" is inclusive and means "and / or."

[0098] As used herein, the term "contacting" refers to establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means establishing a physical connection between the mammalian cell and the nanoparticle. In the biological field, methods for contacting cells with external entities, whether in vivo or in vitro, are well known. For example, contacting a nanoparticle composition with a mammalian cell in a mammal can be carried out by a variety of administration routes (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and may involve different amounts of the nanoparticle composition. In addition, a nanoparticle composition can be contacted with multiple mammalian cells.

[0099] As used herein, the term "delivering" refers to providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic agent to a subject may involve administering a nanoparticle composition comprising the therapeutic and / or prophylactic agent to the subject (e.g., via intravenous, intramuscular, intradermal, or subcutaneous injection). Administering a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

[0100] As used herein, "N / P ratio" refers to the molar ratio of lipids containing ionized nitrogen atoms (eg, compounds of Formula I) to phosphate groups in RNA in a nanoparticle composition comprising a lipid component and RNA.

[0101] The composition may also include salts of one or more compounds. These salts may be pharmaceutically acceptable salts. As used herein, "pharmaceutically acceptable salts" refers to derivatives obtained by converting an acidic or basic group present in the parent compound into its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral salts or organic acid salts of basic groups (e.g., amines); alkali metal salts or organic salts of acidic groups (e.g., carboxylic acids); and the like. Typical acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphenate, camphensulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, gluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, dodecylsulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valine, and the like. Typical alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts disclosed herein include conventional non-toxic salts of the parent compound formed from a non-toxic inorganic or organic acid. The pharmaceutically acceptable salts disclosed herein can be synthesized from a parent compound containing a basic or acidic group by conventional chemical methods. Typically, these salts can be prepared by reacting the free acid or base form of these compounds with an appropriate amount of base or acid in water or an organic solvent, or in a mixture of the two; typically, non-aqueous media (such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile) are preferred.Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), all of which are incorporated herein by reference in their entirety.

[0102] As used herein, the "polydispersity index" or "PDI" is a ratio used to describe the uniformity of the particle size distribution of a system. A smaller value, such as less than 0.3, indicates a narrower particle size distribution. In certain embodiments, the polydispersity index can be less than 0.1.

[0103] In some embodiments, the polydispersity index (PDI) of lipid nanoparticles (LNPs) described herein can be between about 0.005 and about 0.75. In some embodiments, the PDI of LNP can be between about 0.01 and about 0.5. In some embodiments, the PDI of LNP can be between about 0 and about 0.4. In some embodiments, the PDI of LNP can be between about 0 and about 0.35. In some embodiments, the PDI of LNP can be between about 0 and about 0.3. In some embodiments, the PDI of LNP can be between about 0 and about 0.25. In some embodiments, the PDI of LNP can be between about 0 and about 0.2. In some embodiments, the PDI of LNP can be less than about 0.08, 0.1, 0.15, 0.2 or 0.4.

[0104] In this article, "alkyl" refers to a branched or straight-chain saturated hydrocarbon group containing 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. Alkyl can be cyclic or acyclic. Alkyl can be branched or non-branched (i.e. linear). Alkyl can also be substituted or unsubstituted. For example, alkyl can be substituted by one or more groups, including but not limited to alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halogen, hydroxyl, nitro, silicon, sulfoxide, sulfonate, carboxylate or thiol, as described herein. "low alkyl" refers to an alkyl group containing 1 to 6 (e.g., 1 to 4) carbon atoms.

[0105] As used herein, "alkenyl" refers to an aliphatic group containing at least one carbon-carbon double bond, including "unsubstituted alkenyl" and "substituted alkenyl," the latter of which refers to an alkenyl group in which a hydrogen on one or more carbon atoms in the alkenyl group is replaced by a substituent. These substituents may appear on one or more carbon atoms that are or are not contained in a double bond. In addition, these substituents include all possible substituents discussed for alkyl groups, unless prohibited by stability. For example, an alkenyl group may be substituted with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups. Exemplary alkenyl groups include, but are not limited to, vinyl (-CH=CH2-), allyl (-CH2CH=CH2), cyclopentenyl (-C5H7), and 5-hexenyl (-CH2CH2CH2CH2CH=CH2)

[0106] "Alkylene" refers to a divalent alkyl radical, which can be branched or straight-chain. Any of the monovalent alkyl groups mentioned above can be converted to an alkylene group by abstracting the second hydrogen atom from the alkyl group. Typical alkylene groups include C2-4 alkylene and C2-3 alkylene. Common alkylene groups include, but are not limited to, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, etc. Alkylene groups can also be substituted or unsubstituted. For example, an alkylene group can be substituted with one or more groups, including, but not limited to, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halogen, hydroxyl, nitro, silyl, sulfoxide, sulfonate, carboxylate, or thiol, as described herein.

[0107] "Alkenylene" refers to a divalent, straight or branched, unsaturated, acyclic hydrocarbon group containing at least one carbon-carbon double bond and, in one embodiment, no carbon-carbon triple bond. Any of the monovalent alkenyl groups mentioned above can be converted to an alkenylene group by abstracting the second hydrogen atom from the alkenyl group. Typical alkenylene groups include C2-6 alkenylene groups.

[0108] When "Cx-y" or "Cx-Cy" is used with a chemical group such as an alkyl or alkylene group, it refers to a group containing from x to y carbon atoms. For example, "Cx-y alkyl" (such as C1-8 alkyl or C1-18 alkyl) or "Cx-Cy alkyl" (such as C1-C8 alkyl or C1-C18 alkyl) refers to a substituted or unsubstituted saturated hydrocarbon group containing a chain of from x to y carbon atoms, including straight-chain and branched alkyl and alkylene groups.

[0109] "Heteroalkyl" refers to an alkyl group in which one or more backbone atoms are replaced by atoms other than carbon, for example, oxygen, nitrogen (e.g., -NH-, -N(alkyl)-, or -N(aryl)-), sulfur (e.g., -S-, -S(=O)-, or -S(=O)-), or combinations thereof. In some embodiments, the heteroalkyl group is connected to the remainder of the molecule via a carbon atom of the heteroalkyl group. In some embodiments, the heteroalkyl group is connected to the remainder of the molecule via a heteroatom of the heteroalkyl group. In some embodiments, the heteroalkyl group is a C1-C8 heteroalkyl group. Exemplary heteroalkyl groups include, but are not limited to, -O(CH2)7Me, -O(CH2)8Me, -S(CH2)8Me, -S(CH2)7Me. In certain embodiments, heteroalkyl is selected from -OR' or -SR' (e.g., R4 and R5 are independently selected from -OR' or -SR'), wherein R' is selected from substituted or unsubstituted C1-C18 alkyl (e.g., C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl), substituted or unsubstituted C2-C18 alkenyl (e.g., C4 alkenyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C10 alkenyl, C11 alkenyl, C12 alkenyl, C13 alkenyl), or substituted or unsubstituted C2-C18 alkynyl (e.g., C4 alkynyl, C5 alkynyl, C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C10 alkynyl, C11 alkynyl, C12 alkynyl, C13 alkynyl). "Heteroalkylene" or "heteroalkylene chain" refers to a straight or branched divalent heteroalkyl chain that connects the rest of the molecule to a radical. Unless otherwise specifically stated in this specification, a heteroalkyl or heteroalkylene group can be optionally substituted. Exemplary heteroalkylene groups include, but are not limited to, -OCH2CH2O-, -OCH2CH2OCH2CH2O-, or -OCH2CH2OCH2CH2OCH2CH2O-.

[0110] As used herein, "alkoxy" refers to any alkyl group attached through an oxygen bridge (ie, -OC 1-3 Alkyl, where C 1-3 Alkyl is as defined herein.) Examples of such groups include, but are not limited to, methoxy, ethoxy, and propoxy.

[0111] In this article, "cycloalkyl" refers to a saturated monocyclic, bicyclic or tricyclic hydrocarbon ring with a specified number of carbon atoms. For example, C3.7 cycloalkyl refers to a cycloalkyl ring containing 3 to 7 carbon atoms. Cycloalkyl can be selectively substituted by one or more substituents. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [2.1.1] hexyl, bicyclo [2.2.1] heptyl, adamantyl, etc. In certain embodiments, the atoms constituting the cycloalkyl can be replaced by atoms other than carbon, such as oxygen, nitrogen (e.g., -NH-, -N (alkyl) - or -N (aryl) -), sulfur (e.g., -S-, -S (= O) - or -S (= O) 2 -).

[0112] While the invention is described in conjunction with the described embodiments, it will be understood that these embodiments are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, including equivalents to the specific features that may be included within the scope of the invention, as defined by the appended claims.

[0113] The above general description and detailed description and the following examples are exemplary and illustrative only and are not restrictive. The headings used herein are for organizational purposes only and should not be construed as limiting the desired subject matter in any way. If any referenced document contradicts a term defined in this specification, the present specification shall prevail. Unless otherwise indicated, all ranges given in this application include endpoints.

[0114] Example

[0115] Example 1:

[0116] YZL002

[0117] The entire synthesis process of YZL002

[0118] Synthesis of YZL002-1

[0119] Under a nitrogen atmosphere, SM (5.00 g, 34 mmol, 1.0 eq) and pyridine (2.6 g, 34 mmol, 1.0 eq) were dissolved in anhydrous tetrahydrofuran (THF, 40 ml). This solution was then added dropwise to a solution of SO₂Cl₂ (4.6 g, 34 mmol, 1.0 eq) in tetrahydrofuran (40 ml) at -78°C. After stirring for 2 hours, N,N-diethylpropane-1,3-diamine (9.0 g, 102 mmol, 3.0 eq) was added dropwise. After the addition was complete, the reaction mixture was slowly warmed to room temperature and stirred overnight. After the reaction was complete, the mixture was filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 5:1) to obtain YZL002-1 (3.0 g, yield: 26%, total yield for the two-step reaction) as a yellow oil.

[0120] Synthesis of YZL002-2

[0121] YZL002-1 (3.0 g, 8.87 mmol) was dissolved in tetrahydrofuran (THF, 20 ml) and then 1M hydrochloric acid solution (20 ml) was added at room temperature. The mixture was stirred at room temperature for 2 hours. The pH value of the mixture was then adjusted to 7 with saturated sodium carbonate solution (30 ml). The mixture was concentrated under vacuum and the resulting residue was dissolved in dichloromethane (DCM, 50 ml), filtered and concentrated under vacuum to give the crude product YZL002-2 (3.2 g, yellow oil), which was used directly in the next reaction without purification.

[0122] Synthesis of YZL002-3

[0123] YZL002-2 (3.2 g, crude product, 1.0 eq) was dissolved in dichloromethane (DCM, 10 ml), followed by the addition of INTC (4.2 g, 13 mmol, 2.4 eq), EDCI·HCl (2.27 g, 11.9 mmol, 2.2 eq), DIEA (4.16 g, 32.6 mmol, 6.0 eq) and DMAP (132 mg, 1.0 mmol, 0.2 eq). The mixture was stirred at 35°C for 3 hours. The mixture was then washed with saturated sodium carbonate solution (20 ml) and extracted with dichloromethane (2 x 20 ml). The combined organic phases were washed with brine (20 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane:methanol=10:1) to give the target compound YZL002-3 (1.2 g, yield: 18%) as a yellow oil.

[0124] Synthesis of YZL002

[0125] YZL002-3 (1.2 g, 1.9 mmol, 1.0 eq) was dissolved in dichloromethane (DCM, 10 ml), followed by the addition of linoleic acid (1.5 g, 4.55 mmol, 2.4 eq), EDCI·HCl (848 mg, 4.18 mmol, 2.2 eq), DIEA (1.79 g, 11.4 mmol, 6.0 eq) and DMAP (50 mg, 0.38 mmol, 0.2 eq). The mixture was stirred at 35°C for 3 hours. The mixture was then washed with saturated sodium carbonate solution (20 ml) and extracted with dichloromethane (2 x 20 ml). The combined organic phases were washed with brine (20 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane:methanol=10:1) to give the title compound (320 mg, yield: 18.8%) as a yellow oil.

[0126] LCMS: m / z = 887.3 [M+H] + ;t R = 2.241 minutes;

[0127] 1 H NMR (400MHz, CDCl3): δ5.43-5.27(m,4H),4.48(t,J=5.6Hz,1H),4.21-4.11(m,6H),3.61-3.51(m,2H), 3.45-3.35(m,2H),3.30-3.20(m,2H),2.77(t,J=1.6Hz,1H),2.68-2.59(m,2H),2.55(q,J=6.8Hz,4H), 2.51-2.43(m,1H),2.40(t,J=7.6Hz,2H),2.31(t,J=7.6Hz,2H), 2.05(q,J=6.8Hz,4H), 1.97-1.87(m,2 H), 1.76-1.67 (m, 2H), 1.66-1.50 (m, 6H), 1.36-1.24 (m, 35H), 1.08 (t, J = 7.2Hz, 6H), 0.93-0.84 (m, 9H).

[0128] YZL019

[0129] YZL019 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0130] The structure of YZL019 is as follows:

[0131] LCMS: m / z = 916.0 [M+H] + ;t R =2.544 minutes;

[0132] 1 H NMR (400MHz, CDCl3): δ5.44-5.28(m,4H),4.48(t,J=5.2Hz,1H),4.21-4.10(m,6H),3.62-3.51(m,2H), 3.45-3.35(m,2H),3.30-3.20(m,2H),2.77(t,J=6.8Hz,2H),2.66-2.60(m,2H),2.54(q,J=7.2Hz,4H), 2.51-2.43(m,1H),2.40(t,J=7.6Hz,2H),2.31(t,J=8.0Hz,2H),2.05(q,J=6.8Hz,4H),1.97-1.87(m,2 H),1.76-1.69(m,2H),1.65-1.49(m,8H),1.32-1.24(m,37H),1.08(t,J=7.2Hz,6H),0.89-0.86(m,9H).

[0133] YZL021

[0134] YZL021 can be prepared by a method similar to that used to synthesize YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0135] The structure of YZL021 is as follows:

[0136] LCMS: m / z = 914.0 [M+H] + ;t R = 2.365 minutes;

[0137] 1H NMR (400MHz, CDCl3): δ5.44-5.25(m,4H),4.86(quin,J=6.4Hz,1H),4.23-4.09(m,6H),3.30-3.20(m ,2H),2.80-2.71(m,2H),2.66-2.60(m,2H),2.55(q,J=7.2Hz,4H),2.46(quin,J=5.6Hz,1H),2.39(t, J=7.2Hz,2H),2.37-2.26(m,4H),2.04(q,J=6.8Hz,4H),1.94(quin,J=7.2Hz,2H),1.77-1.69(m,2H) ,1.66-1.56(m,2H),1.55-1.45(m,4H),1.34-1.21(m,39H),1.08(t,J=7.2Hz,6H),0.93-0.83(m,9H).

[0138] YZL022

[0139] YZL022 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0140] The structure of YZL022 is as follows:

[0141] LCMS: m / z = 920.5 [M+H] + ;t R = 2.525 minutes;

[0142] 1 H NMR (400MHz, CDCl3): δ5.46-5.27(m,4H),4.23-4.09(m,6H),3,80(t,J=7.2H z,1H),3.30-3.19(m,2H),2.77(t,J=6.4Hz,2H),2.70-2.51(m,11H),2.51-2 .42(m,1H),2.31(t,J=7.6Hz,2H),2.15-1.98(m,6H),1.80-1.68(m,2H),1.6 6-1.52(m,6H),1.46-1.20(m,35H),1.09(t,J=6.8Hz,6H),0.94-0.82(m,9H).

[0143] YZL025

[0144] YZL025 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0145] The structure of YZL025 is as follows:

[0146] LCMS: m / z=823.4[M+H] + ;t R =0.156 minutes;

[0147] 1 H NMR (400MHz, CDCl3): δ5.43-5.28(m,8H),4.21-4.10(m,6H),3.30-3.21(m,2 H),2.83-2.73(m,4H),2.66-2.60(m,2H),2.54(q,J=7.2Hz,4H),2.50-2.42( m,1H),2.31(t,J=7.6Hz,4H),2.05(q,J=6.8Hz,8H),1.77-1.68(m,2H),1.66 -1.56(m,4H),1.38-1.26(m,29H),1.07(t,J=7.2Hz,6H),0.95-0.84(m,6H).

[0148] YZL026

[0149] YZL026 can be prepared by a method similar to that used to synthesize YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0150] The structure of YZL026 is shown below

[0151] YZL027 can be prepared by a method similar to that used to synthesize YZL026, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0152] LCMS: m / z = 919.5 [M+H] + ;

[0153] 1 H NMR(CDCl3,400MHz)δ4.11-4.06(m,6H),4.02-3.94(m,4H),3.22-3.14(m,2H),2.62 -2.56(m,2H),2.52(q,J=7.2Hz,4H),2.45-2.34(m,1H),2.28-2.18(m,8H),1.72-1.64(m ,2H),1.60-1.48(m,12H),1.27-1.16(m,41H),1.03(t,J=7.2Hz,6H),0.84-0.78(m,6H).

[0154] YZL031

[0155] YZL031 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or reduction of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0156] The structure of YZL031 is as follows:

[0157] LCMS: m / z = 918.5 [M+H] + ;t R = 2.222 minutes;

[0158] 1 H NMR (400MHz, CDCl3) δ4.64 (br s, 1H), 4.21-4.14 (m, 2H), 4.14-4.08 (m, 2H), 4.05 (t, J = 6.8Hz, 4H), 3.18 -3.12(m,2H),3.11-3.05(m,2H),2.64-2.59(m,2H),2.55(q,J=7.2Hz,4H),2.34-2.26(m,10H),1. 76-1.68(m,2H),1.67-1.56(m,13H),1.34-1.25(m,29H),1.07(t,J=6.8Hz,6H),0.92-0.84(m,6H).

[0159] YZL032

[0160] YZL032 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0161] The structure of YZL032 is shown below

[0162] LCMS: m / z = 887.0 [M+H] + ;t R = 2.108 minutes;

[0163] 1 H NMR(CDCl3,400MHz)δ5.70-5.58(m,2H),5.56-5.45(m,2H),4.73(br s,1H),4.62(d,J=6.8Hz,4H),4.23-4.06(m,4H),3.20-3.12(m,2H),3.08(d,J=6.0Hz,2H),2.67-2.62(m,2H),2.59(q,J=7.2Hz,4H),2.36 -2.26(m,8H),2.10(q,J=7.2Hz,4H),1.79-1.71(m,2H),1.68-1.55(m,8H),1.40-.123(m,30H),1.09(m,J=7.2Hz,6H),0.91-0.85(m,6H).

[0164] YZL033

[0165] YZL033 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or reduction of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0166] The structure of YZL033 is as follows:

[0167] LCMS: m / z = 923.6 [M+H] + ;t R =2.453 minutes;

[0168] 1H NMR(CDCl3,400MHz)δ4.48(t,J=5.6Hz,2H),4.16(d,J=6.0Hz,6H),3.61-3 .52(m,4H),3.45-3.36(m,4H),3.29-3.22(m,2H),2.61-2.54(m,2H),2.50 -2.44(m,1H),2.41(t,J=7,6Hz,4H),2.33(s,6H),1.96-1.89(m,4H),1.80 -1.73(m,2H),1.60-1.51(m,8H),1.38-1.22(m,41H),0.90-0.84(m,12H).

[0169] YZL041

[0170] YZL041 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0171] The structure of YZL041 is as follows:

[0172] LCMS: m / z = 936.7 [M+H] + ;t R =8.737 minutes;

[0173] 1 H NMR(CDCl3,400MHz)δ4.48(t,J=5.6Hz,2H),4.24-4.10(m,6H),3.61-3.51(m ,4H),3.46-3.36(m,4H),3.22-3.15(m,2H),2.71-2.65(m,2H),2.61(q,J=7. 2Hz,4H),2.51=2.44(m,1H),2.41(t,J=7.6Hz,4H),1.97-1.88(m,4H),1.61- 1.49(m,8H),1.38-1.23(m,41H),1.07(t,J=7.2Hz,6H),0.92-0.84(m,12H).

[0174] YZL045

[0175] YZL045 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0176] The structure of YZL045 is as follows:

[0177] LCMS: m / z = 936.7 [M+H] + ;t R =8.737 minutes;

[0178] 1 H NMR(400MHz, CDCl3)δ5.56(br s,2H),4.48(t,J=5.2Hz,2H),4.22-4.06(m,4H),3.61-3.51(m,4H),3.45-3.36(m,4H),3.19-3.1 3(m,2H),3.10(d,J=6.4Hz,2H),2.75-2.71(m,2H),2.67(q,J=7.2Hz,4H),2.44-2.37(m,4H),2.35 -2.27(m,1H),1.97-1.88(m,4H),1.60-1.51(m,9H),1.33-1.25(m,39H),1.08(t,J=7.2Hz,6H),0.91-0.86(m,12H).

[0179] YZL046

[0180] YZL046 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0181] The structure of YZL046 is shown below

[0182] LCMS: m / z = 808.6 [M+H] + ;t R = 2.342 minutes;

[0183] 1H NMR (400MHz, CDCl3) δ5.42-5.29(m,8H),4.21-4.15(m,2H),4.14-4.08(m,2H),3.14-4.07(m,3H),2.77(t,J=6.4H z,4H),2.69-2.64(m,2H),2.60(q,J=7.2Hz,4H),2.35-2.25(m,5H),2.04(q,J=6.8Hz,8H),1.66-1.57(m,4H),1.39 -1.25(m,31H),1.05(t,J=7.2Hz,6H),0.93-0.86(m,6H).

[0184] YZL058

[0185] YZL058 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0186] The structure of YZL058 is as follows:

[0187] LCMS: m / z = 822.3 [M+H] + ;t R = 2.229 minutes;

[0188] 1 H NMR(400MHz,CDCl3)δ6.37(br s,1H),5.43-5.28(m,8H),4.22-4.11(m,6H),3.39-3.30(m,1H),3.30 -3.16(m,2H),2.84-2.73(m,5H),2.50(s,3H),2.48-2.40(m,2H),2.3 2(t,J=7.6Hz,4H),2.05(q,J=7.2Hz,8H),2.01-1.94(m,2H),1.92-1.72(m,4H),1.66-1.57(m,4H),1.36-1.26(m,28H),0.92-0.86(m,6H).

[0189] YZL081

[0190] YZL081 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or reduction of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0191] The structure of YZL081 is as follows:

[0192] LCMS: m / z = 935.7 [M+H] + ;t R =9.74 minutes.

[0193] 1 H NMR (400MHz, CDCl3) δ4.91 (br s, 1H), 4.48 (t, J = 5.6Hz, 2H), 4.20 (d, J = 6.0Hz, 2H), 4.18-4.11 (m, 4H), 3.60-3.52 (m, 4H), 3.44-3.35 (m, 4H), 3.26 -3.20(m,2H),2.78 -2.71(m,2H),2.67-2.60(m,4H),2.51-2.44(m,1H),2.41(t,J=7.6Hz,4H),1.98-1.88 (m,4H),1.87-1.78(m,4H),1.60-1.51(m,8H),1.36-1.22(m,40H),0.94-0.84(m,12H).

[0194] YZL082

[0195] YZL082 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0196] The structure of YZL082 is as follows:

[0197] LCMS: m / z = 807.5 [M+H] + ;

[0198] 1H NMR (400MHz, CDCl3) δ5.48–5.15(m,7H),4.20(d,J=5.6Hz,2H),4.16(d,J=6.0Hz,3H),3.27–3.15(m,2H),2.80–2.68(m,5H),2.68–2.57 (m,4H),2.31(t,J=7.6Hz,4H),2.05(q,J=6.4Hz,7H),1.90–1.79(m,4H),1.70–1.54(m,4H),1.43–1.23(m,27H),0.89(t,J=6.8Hz,6H).

[0199] YZL085

[0200] YZL085 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0201] The structure of YZL085 is as follows:

[0202] 1 H NMR (400MHz, CDCl3) δ4.49 (dd, J=7.2, 4.0Hz, 2H), 4.14 (qd, J=11.2, 5.6Hz, 4H), 3.56 (dt,J=9.2,6.8Hz,4H),3.44–3.34(m,4H),3.27–3.16(m,2H),3.10(d,J=6.4Hz,2H),2 .88–2.76(m,2H),2.73(s,3H),2.40(dd,J=9.2,6.0Hz,4H),1.92(dd,J=13.2,7.6Hz,4 H),1.88–1.75(m,3H),1.64–1.45(m,8H),1.40–1.16(m,40H),0.88(t,J=6.8Hz,12H).

[0203] YZL086

[0204] YZL086 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0205] The structure of YZL-086 is as follows:

[0206] LCMS: m / z=806.6[M+H] + ;t R = 2.303 minutes;

[0207] 1 H NMR (400MHz, CDCl3) δ5.43-5.29(m,8H),5.16(brs,1H),4.21-4.08(m,4H),3.19-3.13(m,2H),3.09(d,J=6.4Hz,2H),2.77(t,J=4.0Hz,4H),2.70 -2.65(m,2H),2.62-2.54(m,4H),2.36-2.24(m,5H),2.05(q,J=7.2Hz,8H),1.84-1.74(m,4H),1.67 -1.56(m,4H),1.41-1.22(m,29H),0.94-0.84(m,6H).

[0208] YZL087

[0209] YZL087 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0210] The structure of YZL087 is as follows:

[0211] LCMS: m / z = 902.5 [M+H] + ;t R = 2.223 minutes;

[0212] 1 H NMR (400MHz, CDCl3) δ5.30 (br s, 1H), 4.22-4.08 (m, 4H), 4.05 (t, J = 6.8Hz, 4H), 3.20-3.12 (m, 2H) m 3.09(d,J=6.4Hz,2H),2.70-2.63(m,2H),2.61-2.52(m,4H),2.35-2.24(m,9H) ,1.84-1.74(m,4H),1.67-1.56(m,12H),1.43-1.14(m,41H),0.94-0.83(m,6H).

[0213] YZL089

[0214] YZL089 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0215] The structure of YZL089 is as follows:

[0216] LCMS: m / z = 950.0 [M+H] + ;t R =9.599 minutes;

[0217] 1 H NMR(400MHz, CDCl3)δ4.48(t,J=5.6Hz,2H),4.19-4.12(m,5H),3.61-3.52(m,4H),3.47-3.37(m,4H),3.33-3.20 (m,2H),2.76-2.70(m,2H),2.66-2.57(m,4H),2.52-2.44(m,1H),2.41(t,J=7.6Hz,4H),1.98-1.88(m,4H),1.85 -1.70(m,6H),1.61-1.50(m,8H),1.40-1.20(m,43H),0.91 -0.85(m,12H).

[0218] YZL090

[0219] YZL090 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0220] The structure of YZL090 is as follows:

[0221] 1H NMR (400MHz, CDCl3) δ5.53–5.21(m,8H),4.16(t,J=6.0Hz,5H),3.33–3.22(m,2H),3.01–2.85(m,5H),2.77(t,J=6.4Hz,4H),2 .32(t,J=7.6Hz,4H),2.05(q,J=6.8Hz,8H),1.99–1.85(m,5H),1.70–1.50(m,4H),1.44–1.13(m,28H),0.89(t,J=6.4Hz,6H).

[0222] YZL097

[0223] YZL097 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0224] The structure of YZL097 is as follows:

[0225] LCMS: m / z = 949.5 [M+H] + ;t R =0.173 minutes;

[0226] 1 H NMR (400MHz, CDCl3): δ4.48 (t, J=5.6Hz, 2H), 4.19 (d, J=6.0Hz, 2H), 4.17-4 .14(m,3H),3.64(t,J=6.8Hz,1H),3.60-3.52(m,4H),3.45-3.35(m,4H),3.2 2-3.16(m,2H),2.58-2.51(m,2H),2.50-2.37(m,8H),1.96-1.88(m,4H),1.6 5-1.51(m,13H),1.50-1.44(m,2H),1.37-1.24(m,45H),0.92-0.85(m,12H).

[0227] YZL098

[0228] YZL098 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0229] The structure of YZL098 is as follows:

[0230] LCMS: m / z = 821.5 [M+H] + ;t R =2.297 minutes;

[0231] 1 H NMR (400MHz, CDCl3): δ5.45-5.25(m,8H),4.19(d,J=5.6Hz,2H),4.15(d,J=6.0Hz,4H),3.22-3.13(m,2H),2.77(t,J=6.8Hz,4H),2.54-2.46(m,3 H),2.46-2.37(m,4H),2.31(t,J=7.6Hz,4H),2.05(q,J=7.2Hz,8H),1.66 -1.54(m,9H),1.49-1.42(m,2H),1.37-1.27(m,28H),0.93-0.85(m,6H).

[0232] YZL101

[0233] YZL101 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0234] The structure of YZL101 is as follows:

[0235] LCMS: m / z=520.5[M+H] + ;t R =11.501 minutes;

[0236] 1H NMR(400MHz,CDCl3)δ5.30(br s,1H),4.49(t,J=5.6Hz,1H),4.24-4.07(m,4H),3.62-3.52(m,4H),3.50-3.35(m,4H),2.51-2.45(m,2H),2.40(t,J=7.6Hz ,8H),2.35-2.27(m,1H),1.98-1.87(m,4H),1.61-1.50(m,12H),1.48-1.40(m,2H),1.38-1.22(m,41H),0.95-0.84(m,12H).

[0237] YZL102

[0238] YZL102 can be prepared by a method similar to that for synthesizing YZL002, wherein the selection and amount of raw materials, addition or deletion of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0239] The structure of YZL102 is as follows:

[0240] LCMS: m / z=820.5[M+H] + ;t R = 2.329 minutes;

[0241] 1 H NMR (400MHz, CDCl3) δ5.69–5.20 (m, 8H), 4.15 (qd, J=11.4, 5.6Hz, 4H), 3.28–3. 16(m,2H),3.10(d,J=6.4Hz,2H),2.77(t,J=6.4Hz,4H),2.71–2.62(m,2H),2.5 4(d,J=26.4Hz,4H),2.31(t,J=7.6Hz,6H),2.05(dd,J=13.2,6.4Hz,8H),1.79– 1.55(m,9H),1.48(d,J=19.6Hz,3H),1.41–1.18(m,28H),0.89(t,J=6.4Hz,6H).

[0242] The structure of INTC is as follows:

[0243] Intermediate a:

[0244] To a mixture of 4,4-diethoxybutyronitrile (9.4 g, 60 mmol, 1 eq) and n-octanol (3 eq) was added pyridine tosylate (0.05 eq) at room temperature. The reaction mixture was heated to 105°C and stirred for at least 18 hours without a reflux condenser and open to air. The reaction mixture was then cooled to room temperature and directly purified using silica gel column chromatography (eluting with a gradient of n-hexane / ethyl acetate) to yield 11.4 g (32.3 mmol, 54% yield) of the desired product as a clear oil. Mass spectrum: 372 m / z [M+Na].

[0245] INTE:

[0246] Intermediate a (10.9 g, 31 mmol, 1 eq) was dissolved in ethanol (1 M), followed by the addition of aqueous potassium hydroxide (2.5 M, 2.5 eq) at room temperature. After installing a reflux condenser on the reaction vessel, the reaction mixture was heated to 110°C and stirred for 20-24 hours. The reaction mixture was then cooled to room temperature, acidified to pH 5 with 1N aqueous hydrochloric acid, and extracted with n-hexane. The combined organic extracts were washed with water and brine, dried over anhydrous sodium sulfate or magnesium sulfate, filtered, and concentrated under vacuum to yield 8.22 g (22.1 mmol, 71% yield) of the desired product as a clear oil, which was used without further purification. Mass spectrum: 366 m / z [MH].

[0247] INTE can be prepared by a method similar to that for synthesizing INTF, wherein the selection and amount of raw materials, addition or reduction of conventional steps, and specific reaction conditions can be adjusted by those skilled in the art according to actual conditions.

[0248] The structure of INTE is as follows:

[0249] LCMS: m / z=329.26[M+H] + .

[0250] Under a nitrogen atmosphere, azelaic acid (11.2 g, 59.5 mmol, 1.0 eq) was dissolved in anhydrous tetrahydrofuran (THF, 100 mL), followed by the dropwise addition of oxalyl chloride ((COCl)2, 7.55 g, 59.5 mmol, 1.0 eq) and dimethylformamide (DMF, 1 mL) at 0°C. The mixture was stirred at room temperature for 2 hours. (E)-non-2-en-1-ol (8.45 g, 59.5 mmol, 1.0 eq) was then added dropwise at 0°C. After the addition was complete, the mixture was slowly warmed to room temperature and stirred overnight. The mixture was filtered and concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 40:1) to afford INTF (11.0 g, 59.2% yield, total yield for the two-step reaction) as a yellow oil.

[0251] The structure of INTF is as follows:

[0252] LCMS: m / z=313.23[M+H] + .

[0253] 1 H NMR (400MHz, CDCl3) δ5.68–5.59(m,1H),5.55–5.46(m,1H),4.62(d,J=6.8Hz,2H),2.32(dt,J=15.0, 7.5Hz, 4H), 2.10 (q, J = 7.1Hz, 2H), 1.62 (d, J = 6.2Hz, 4H), 1.38–1.23 (m, 14H), 0.88 (t, J = 6.6Hz, 3H).

[0254] Example 2:

[0255] Materials and methods

[0256] 1. Nanoparticle Preparation

[0257] Lipid nanoparticles (LNPs) were prepared by mixing an aqueous phase containing nucleic acids with an ethanolic phase containing lipids and excipients using a microfluidic chip device as previously described (Chen D, et al. (2012) Rapid discovery of potent siRNA-containing lipid nanoparticles by controlled microfluidic formulation. J Am Chem Soc 134(16):6948–6951). Specifically, the ethanolic phase contained a mixture of an ionizable lipid, 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000). The molar ratio of ionizable lipid / DSPC / cholesterol / DMG-PEG2000 was 50 / 9 / 38 / 3. The ethanolic phase was prepared by dissolving the mixture. The aqueous phase was prepared in 25 mM citrate buffer containing RNA. The resulting LNPs were dialyzed for 2 hours at room temperature using a 30,000 Dalton molecular weight cutoff (MWCO) filter cartridge in a buffer solution containing 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS). The final LNPs were stored at -80°C until further use.

[0258] 2. LNP Characterization

[0259] The particle size and polydispersity of LNPs were measured using dynamic light scattering (DLS). Dynamic light scattering ("DLS") was used to determine the average particle size and polydispersity index ("PDI") of the LNP samples. The average particle size and polydispersity were measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument. Prior to testing, the LNP samples were diluted in 1×PBS at a volume ratio of 1:99. The average hydrodynamic diameter of each sample was measured by reporting the average particle size and PDI. The zeta potential of the LNPs was also measured using a Malvern Zetasizer. Prior to measurement, the samples were diluted in 10 mM NaCl at a volume ratio of 1:19.

[0260] Using fluorescence-based detection methods ( ThermoFisher Scientific) was used to determine the encapsulation efficiency (EE (%)), which was calculated as (total RNA - free RNA) / total RNA. LNP samples were diluted to the appropriate concentration with 1× TE buffer containing 1% Triton-X 100 for total RNA determination, or diluted with 1× TE buffer for free RNA determination. A standard curve was prepared using the starting RNA solution used in the formulation according to the manufacturer's instructions. The dye was added to each standard and sample and incubated at room temperature in the dark for approximately 5 minutes. Samples were read using a Tecan INFINITE 200PRO at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. After subtracting the fluorescence value of the reagent blank from the fluorescence value of each sample, total RNA and free RNA were calculated based on the appropriate standard curve.

[0261] Typically, when preparing LNPs, the encapsulation efficiency is >80%, the particle size is <150 nm, and the PDI is <0.2.

[0262] The values ​​of average particle size, polydispersity and EE% for various LNP compositions are reported in Tables 1-2.

[0263] Table 1: Summary of LNP formulation data for mice

[0264] Table 2: Summary of LNP formulation data for PCH and NHP

[0265] Table 3: sgRNA used in the present invention

[0266] Table 4: Cas9 encoding DNA sequences and their corresponding RNAs

[0267] 3. In vitro LNP delivery

[0268] Primary cynomolgus monkey hepatocytes (TPCS, CCH-100CYS-PQ) were cultured according to the manufacturer's protocol. Cells were seeded at a density of 30,000 cells per well in 96-well plates. Twenty-four hours later, cells were treated with lipid nanoparticles (LNPs) encapsulated with various concentrations of RNA (1.56 nM, 3.125 nM, 6.25 nM, or 12.5 nM). Seventy-two hours after transfection, cells were washed with PBS, and genomic DNA was extracted using Quick Extract DNA Extraction Solution (Epicentre, QE09050) according to the manufacturer's recommended protocol.

[0269] LNPs were prepared using in vitro transcribed Cas9 mRNA (SEQ ID NO. 4, Table 4) and chemically modified sgRNA (targeting NHP TTR) (SEQ ID NO. 2) as described in Example 2. Detailed information on these formulations, including average particle size, polydispersity, and encapsulation efficiency, is provided in Table 2. Figure 2 As shown, in vitro editing effects were observed for each formulation. LNP formulations of YZL06 (LNP017), YZL081 (LNP018), YZL082 (LNP019), YZL085 (LNP020), YZL086 (LNP021), YZL087 (LNP022), YZL089 (LNP023), YZL098 (LNP024), and YZL101 (LNP025) successfully delivered Cas9 mRNA and edited TTR. These data demonstrate that LNPs using novel lipids are effective for delivering CRISPR / Cas9 cargo to cynomolgus monkey hepatocytes and have great potential for in vitro application.

[0270] 4. Animal Experiments

[0271] All mice were purchased from Charles River Labs. Six-week-old female BALB / C mice received an intravenous injection of LNP at a dose of 0.1 mg / kg via the tail vein. All animals were euthanized and necropsied 168 hours after administration. Clinical symptoms, body weight, organ weights, and histopathology were assessed. The liver was minced into small pieces, and genomic DNA was extracted using the TIANamp Genomic DNA kit according to the manufacturer's recommended protocol.

[0272] LNPs were prepared as described in Example 2 using in vitro transcribed Cas9 mRNA (SEQ ID NO. 4) and chemically modified sgRNA (targeting mouse TTR) (SEQ ID NO. 1, Table 3). Detailed information on these formulations, including average particle size, polydispersity, and encapsulation efficiency, is provided in Table 1. Animals in each group were euthanized 7 days after dosing. Figure 1As shown, in vivo editing was observed in the livers of mice that received LNPs targeting TTR. LNP formulations containing lipids YZL097 (LNP001), YZL090 (LNP002), YZL031 (LNP003), YZL046 (LNP004), YZL081 (LNP005), and YZL082 (LNP006) successfully delivered Cas9 mRNA and edited TTR at very low doses, while the control lipid LP01 had almost no activity. These results indicate that LNPs using novel lipids have better in vivo delivery efficiency of CRISPR / Cas9 cargo at very low doses than the commercial lipid LP01.

[0273] LNPs were prepared as described in Example 2 using in vitro transcribed Cas9 mRNA (SEQ ID NO. 4) and chemically modified sgRNA (targeting NHP TTR) (SEQ ID NO. 2). Detailed information on these preparations is shown in Table 2, including average particle size, polydispersity, and encapsulation efficiency. Similarly, LNPs targeting NHP TTR demonstrated a strong and sustained effect in reducing serum TTR protein in cynomolgus monkeys in in vitro non-human primate (NHP) studies (data not shown). In the in vivo screening, cynomolgus monkeys received an intravenous injection of LNPs at a dose of 3 mg / kg. In the in vivo editing, liver tissue was collected 14 and 28 days after administration by ultrasound-guided biopsy. The liver was cut into small pieces, and genomic DNA was extracted using the TIANamp Genomic DNA kit according to the manufacturer's recommended protocol. In addition, blood was collected and serum was separated every 7 days after administration. As Figure 3 As shown, in vivo editing was observed in the livers of mice that received TTR-targeted LNPs (approximately 15%-80% editing efficiency). LNP formulations containing lipids YZL002 (LNP008), YZL019 (LNP009), YZL021 (LNP010), YZL022 (LNP011), YZL026 (LNP013), YZL033 (LNP014), YZL081 (LNP018), YZL082 (LNP019), YZL085 (LNP020), YZL086 (LNP021), YZL087 (LNP022), YZL089 (LNP023), YZL101 (LNP025), YZL027 (LNP026), YZL031 (LNP027), YZL058 (LNP028), and YZL102 (LNP029) successfully delivered Cas9 mRNA and edited TTR. Even though the delivery efficiency of individual samples was promising, exceeding 50%, these results indicate that LNPs using the novel lipids have extremely high CRISPR / Cas9 cargo delivery efficiency in NHPs.

[0274] The mRNA used in this example is an N1-methyl pseudouracil modification of uracil. When the nucleotide sequence of RNA (including those in the sequence listing) is mentioned in the examples, it should be understood that T should be replaced with U. The U in the examples represents N1-methyl pseudouracil.

[0275] 5. Formulation optimization

[0276] All mice were purchased from Charles River Labs. Six-week-old female BALB / C mice received an intravenous injection of LNP at a dose of 0.15 mg / kg via the tail vein. All animals were euthanized and necropsied 168 hours after administration. Clinical symptoms, body weight, organ weights, and histopathology were assessed. The liver was minced into small pieces, and genomic DNA was extracted using the TIANamp Genomic DNA kit according to the manufacturer's recommended protocol.

[0277] LNPs were prepared as described in Example 2 using in vitro transcribed Cas9 mRNA (SEQ ID NO. 4) and chemically modified sgRNA (targeting mouse TTR) (SEQ ID NO. 1). Detailed information on these formulations, including average particle size, polydispersity, and encapsulation efficiency, is provided in Table 5. Animals in each group were euthanized 7 days after dosing. Figure 4 and Figure 5 As shown, in vivo editing was observed in the livers of mice that received TTR-targeted LNPs. These results suggest that different formulations of the same lipid material have an impact on the delivery efficiency of LNPs in mice. Furthermore, each cationic lipid material has its own optimal formulation.

[0278] LNPs were prepared as described in Example 2 using in vitro transcribed Cas9 mRNA (SEQ ID NO. 4) and chemically modified sgRNA (targeting rat TTR) (SEQ ID NO. 5, Table 8). Detailed information on these formulations, including average particle size, polydispersity, and encapsulation efficiency, is provided in Table 6. All rats were purchased from Charles River Labs. Female Sprague-Dawley rats aged 6-8 weeks were used. LNPs were administered via tail vein injection at a dose of 0.3 mg / kg and a dose volume of 7 mL / kg body weight. All animals were euthanized for autopsy 168 hours after administration. Clinical symptoms, body weight, organ weight, and histopathology were evaluated. Figure 6 and Figure 7 As shown, in vivo editing was observed in the liver of rats that received TTR-targeted LNPs. These results suggest that different formulations of the same lipid material have an impact on the delivery efficiency of LNPs in rats.

[0279] The data of LNP preparations for mice and rats are shown in Tables 5 and 6, respectively. The specific composition of LNP is listed in Table 7.

[0280] Table 5: Summary of Mouse LNP Formulation Data

[0281] Table 6: Summary of rat LNP formulation data

[0282] Table 7: Summary of LNP composition

[0283] Table 8: sgRNA used in the present invention

[0284] 6. PCR Amplification

[0285] All samples were amplified by a two-step nested PCR and prepared for sequencing. Specifically, 1 μL of primers (reverse / forward primers at a final concentration of 5 μM) was added to 5 μL of Kapa HiFi 2X Mix (Roche) and 4 μL of template DNA / water. The first PCR reaction was run for 20 cycles. The second PCR was used to add Nextera XT chemistry, index, and i5 / i7 adapter regions, run for 5-10 cycles, and use the product of "PCR 1" as the template. The double-indexed samples were checked on a 2% agarose gel to ensure that the PCR reaction had occurred before being combined and gel purified.

[0286] 7. Deep Sequencing

[0287] PCR samples were purified using AMPure XP magnetic beads. Final library quality control was performed using an Agilent Bioanalyzer 2100. Illumina deep sequencing was performed on an Illumina MiniSeq TM Primer design was based on the Nextera XT adapter sequence.

[0288] 8. Data Analysis

[0289] Sequencing results were processed using a custom Python-based tool to extract raw barcode counts for each tissue. These raw counts were then normalized using an R script before further analysis. Correlation analysis assumed a Gaussian distribution to obtain Pearson correlation coefficients. 2 The values ​​(0-1) were calculated using the squared Pearson correlation coefficient.

Claims

1. A compound as shown in formula (I) or a pharmaceutically acceptable salt, isomer or prodrug thereof; in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; X1, X2 and X3 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; and at least one of X1, X2 and X3 is independently selected from -S(O)2-, -OS(O)2O-, -OS(O)2N(R6)-, -N(R7)S(O)2O- or -N(R6)S(O)2N(R7)-; Y is -C- or -N-; L1, L2, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

2. A compound as shown in formula (I) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is a C1-C3 alkyl group; R2 is a C1-C3 alkyl group; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, or L5-M1-L6-R8; wherein L5 and L6 are each independently a single bond or a C1-C12 alkylene group; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or heteroalkyl; X1 is independently selected from -OS(O)2O-, -OS(O)2N(R6)-, -N(R7)S(O)2O- or -N(R6)S(O)2N(R7)-; X2 and X3 are each independently selected from a single bond, -C(=O)O-, -OC(=O)- or -OC(=O)O-; Y is -C-; L1 and L2 are each independently a single bond or a C1-C6 alkylene group; L3 and L4 are each independently C1-C18 alkylene or L7-M2-L8; wherein L7 and L8 are each independently a single bond or C1-C10 alkylene; M1 and M2 are each independently selected from a single bond, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6 and R7 are each independently selected from H or C1-C3 alkyl; preferably, R6 and / or R7 are H; R8 is selected from H, C1-C18 alkyl or C2-C18 alkenyl.

3. A compound represented by formula (II) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; X2 and X3 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1, L2, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

4. A compound as shown in formula (III) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L1, L2, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

5. A compound represented by formula (III), in: R1 is a C1-C3 alkyl group; R2 is a C1-C3 alkyl group; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, or L5-M1-L6-R8; wherein L5 and L6 are each independently a single bond or a C1-C12 alkylene group; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or heteroalkyl; X2 and X3 are each independently selected from a single bond, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1 and L2 are each independently a single bond or a C1-C6 alkylene group; L3 and L4 are each independently C1-C18 alkylene or L7-M2-L8; wherein L7 and L8 are each independently selected from a single bond or C1-C10 alkylene; M1 and M2 are each independently selected from a single bond, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6 is selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl.

6. A compound as shown in formula (IV) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; X2 and X3 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1, L2, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

7. A compound as shown in formula (V) or a pharmaceutically acceptable salt, isomer or prodrug thereof, in: R1 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R2 is C1-C3 alkyl, which is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, or L5-M1-L6-R8; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl or heteroalkyl; wherein said C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; X2 and X3 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1, L2, L3 and L4 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene, C2-C18 alkynylene or L7-M2-L8, wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; L5, L6, L7 and L8 are each independently selected from a single bond, C1-C18 alkylene, C2-C18 alkenylene or C2-C18 alkynylene; wherein the C1-C18 alkylene, C2-C18 alkenylene and C2-C18 alkynylene are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol; M1 and M2 are each independently selected from a single bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R6)-, -(R7)NS(O)2O-, -N(R6)S(O)2N(R7)-, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6, R7 and R8 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl; wherein the C1-C18 alkyl, C2-C18 alkenyl and C2-C18 alkynyl are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto.

8. A compound as shown in formula (V), in: R1 is a C1-C3 alkyl group; R2 is a C1-C3 alkyl group; Or R1, L1 or R2 and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring; R3 is selected from C1-C18 alkyl, or L5-M1-L6-R8; wherein L5 and L6 are each independently a single bond or a C1-C12 alkylene group; R4 and R5 are each independently selected from H, C1-C18 alkyl, C2-C18 alkenyl or heteroalkyl; X2 and X3 are each independently selected from a single bond, -C(=O)O-, -OC(=O)- or -OC(=O)O-; L1 and L2 are each independently a single bond or a C1-C6 alkylene group; L3 and L4 are each independently C1-C18 alkylene or L7-M2-L8; wherein L7 and L8 are each independently selected from a single bond or C1-C10 alkylene; M1 and M2 are each independently selected from a single bond, -C(=O)O-, -OC(=O)- or -OC(=O)O-; R6 is selected from H, C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl.

9. The compound of any one of claims 1-2, wherein X1 is -OS(O)2N(R6)-, R6 is H or C1-C3 alkyl, preferably, R6 is H.

10. The compound of any one of claims 1-2, wherein X1 is -(R7)NS(O)2O-, R7 is H or C1-C3 alkyl, preferably, R7 is H.

11. The compound of any one of claims 1-2, wherein X1 is -N(R6)S(O)2N(R7)-, R6 and R7 are independently selected from H or C1-C3 alkyl; preferably, R6 and / or R7 are H.

12. The compound of any one of claims 1-11, wherein X2 is a bond.

13. The compound of any one of claims 1-11, wherein X2 is -C(=O)O- or -OC(=O)-.

14. The compound of any one of claims 1-13, wherein X3 is a bond.

15. The compound of any one of claims 1-13, wherein X3 is -C(=O)O- or -OC(=O)-.

16. The compound of any one of claims 1 to 15, wherein R1, L1 or R2, and the nitrogen atom to which they are attached together form a 5-membered, 6-membered or 7-membered ring.

17. The compound of any one of claims 1-16, wherein L1 is C1-C5 alkylene, optionally, L1 is selected from C2 alkylene, linear C3 alkylene or linear C4 alkylene.

18. The compound of any one of claims 1-17, wherein L2 is a bond or a C1-C5 alkylene, optionally, L2 is selected from C1 alkylene, C2 alkylene or linear C3 alkylene.

19. The compound of any one of claims 1 to 17, wherein L2 is L7-M2-L8, M2 is -C(=O)O- or -OC(=O)-, and L7 and L8 are each independently selected from a bond, C1 alkylene, or C2 alkylene.

20. The compound of any one of claims 1-19, wherein L3 is a bond or C1-C5 alkylene, optionally, L3 is linear C1 alkylene, linear C2 alkylene or linear C3 alkylene.

21. The compound of any one of claims 1-19, wherein L3 is L7-M2-L8, L7 and L8 are each a linear C1-C10 alkylene group, and M2 is selected from -C(=O)O-, -OC(=O)-, or -OC(=O)O-.

22. The compound of any one of claims 1-19, wherein L3 is L7-M2-L8, L7 and L8 are each independently a linear C1-C7 alkylene, and M2 is selected from -C(=O)O- or -OC(=O)-.

23. The compound of any one of claims 1-19, wherein L3 is L7-M2-L8, L7 is linear C1-C3 alkylene, L8 is linear C3-C7 alkylene, and M2 is -C(=O)O- or -OC(=O)-.

24. The compound of any one of claims 1-23, wherein L4 is a bond or C1-C5 alkylene, optionally, L3 is linear C1 alkylene, linear C2 alkylene, or linear C3 alkylene.

25. The compound of any one of claims 1-23, wherein L4 is L7-M2-L8, L7 and L8 are each independently a linear C1-C10 alkylene group, and M2 is selected from -C(=O)O-, -OC(=O)-, or -OC(=O)O-.

26. The compound of any one of claims 1-23, wherein L4 is L7-M2-L8, L7 and L8 are each independently a linear C1-C7 alkylene, and M2 is selected from -C(=O)O- or -OC(=O)-.

27. The compound of any one of claims 1-23, wherein L4 is L7-M2-L8, L7 is linear C1-C3 alkylene, L8 is linear C3-C7 alkylene, and M2 is -C(=O)O- or -OC(=O)-.

28. The compound of any one of claims 1-27, wherein R3 is a C6-C18 alkyl group, preferably, R3 is a C6-C10 alkyl group.

29. The compound of any one of claims 1-27, wherein R3 is C6-C18 alkenyl, alternatively, R3 is C10-C18 alkenyl, preferably, R3 is C17 alkenyl.

30. The compound of any one of claims 1-27, wherein R3 is Preferably, R3 is 31. The compound of any one of claims 1 to 27, wherein R3 is Preferably, R3 is 32. A compound as described in any one of claims 1-27, wherein R3 is L5-M1-L6-R8; L5 and L6 are each independently a bond or C1-C8 alkylene; M1 is selected from a bond, -C(=O)O- or -OC(=O)-; R8 is selected from C8-C18 alkyl or C8-C18 alkenyl.

33. A compound as described in any one of claims 1-27, wherein R3 is L5-M1-L6-R8; L5 and L6 are each independently a bond or a C3-C8 alkylene; M1 is -C(=O)O- or -OC(=O)-; R8 is selected from C8-C18 alkyl or C8-C18 alkenyl.

34. The compound of any one of claims 1-33, wherein R4 and R5 are each independently H or heteroalkyl; preferably, R4 and R5 are each independently -O-alkyl or -S-alkyl.

35. The compound of any one of claims 1 to 33, wherein R4 and R5 are each independently H or -O(CH2) 5-11 CH3; preferably, R4 and R5 are each independently H or -O(CH2)7CH3.

36. A compound as described in any one of claims 1-33, wherein said R4 and R5 are each independently H or -S(CH2) 5-11 CH3; preferably, R4 and R5 are each independently H or -S(CH2)7CH3.

37. A compound as described in any one of claims 1-33, wherein the R4 and R5 are each independently selected from H, C6-C18 alkyl or C6-C18 alkenyl; alternatively, R4 and R5 are each independently linear C8-C18 alkyl.

38. The compound of any one of claims 1-33, wherein R4 is H and R5 is selected from C6-C18 alkyl or C6-C18 alkenyl.

39. The compound of any one of claims 1-33, wherein R4 and R5 are each independently a branched C10-C18 alkyl group.

40. The compound of the preceding claim, wherein the compound is selected from the group consisting of:

41. A composition comprising a compound as claimed in any one of claims 1 to 40 and a lipid component.

42. The composition of claim 41, wherein the composition comprises a bioactive agent and a lipid component comprising: a) an ionizable lipid; b) a neutral lipid; c) a helper lipid; and d) a PEG lipid; wherein the ionizable lipid is a compound as described in the preceding claims.

43. The composition of claim 42, wherein the neutral lipid is an uncharged lipid or a zwitterionic lipid, optionally wherein the neutral lipid is DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) or DPME (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine).

44. The composition of claim 42 or 43, wherein the helper lipid is cholesterol.

45. The composition of any one of claims 42-44, wherein the PEG lipid is PEG-DMG (polyethylene glycol-dioleoylglycerol), preferably, the PEG lipid is PEG-2k DMG (DMG-PEG2000).

46. ​​A composition as described in any of claims 42-45, wherein the amount of the ionizable lipids accounts for 40-60 mol% of the lipid component; the amount of the neutral lipids accounts for 5-15 mol% of the lipid component; the amount of the helper lipids accounts for 35-50 mol% of the lipid component; and the amount of the PEG lipids accounts for 1.0-3.5 mol% of the lipid component.

47. The composition of any one of claims 42-46, wherein the amount of ionizable lipid is about 40-50 mol% of the lipid component.

48. The composition of any one of claims 42-47, wherein the amount of ionizable lipid is about 45-50 mol% of the lipid component.

49. The composition of any one of claims 42-48, wherein the amount of neutral lipids is about 8-12 mol% of the lipid component.

50. The composition of any one of claims 42-49, wherein the amount of neutral lipids is about 9-10 mol% of the lipid component, alternatively, the neutral lipids are about 9 mol%, 9.4 mol% or 10 mol% of the lipid component.

51. A composition as described in any of claims 42-50, wherein the amount of the helper lipid accounts for about 38-44.5 mol% of the lipid component, optionally, the helper lipid accounts for about 38 mol%, 39 mol%, 40 mol%, 40.8 mol%, 42.7 mol%, 43 mol%, 43.5 mol%, 44 mol% or 44.5 mol% of the lipid component.

52. The composition of any one of claims 42-51, wherein the amount of PEG lipid comprises about 1.5-3 mol% of the lipid component, alternatively, the PEG lipid comprises about 1.5 mol%, 1.6 mol%, 1.7 mol%, 2 mol%, 2.5 mol% or 3 mol% of the lipid component.

53. The composition of any one of claims 42-52, wherein the bioactive agent comprises a nucleic acid.

54. A composition as described in claim 53, wherein the nucleic acid component comprises RNA; alternatively, the RNA component comprises mRNA and gRNA; preferably, the mRNA is type II Cas nuclease mRNA.

55. A method of delivering a drug to a cell, the method comprising the step of contacting the cell with a composition according to any one of claims 41 to 54.

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