Branched tail lipid compounds and compositions for intracellular delivery of therapeutic drugs

Abstract

The present invention provides novel compounds for delivering one or more therapeutic and / or prophylactic agents to mammalian cells or organs and / or for generating polypeptides, compositions comprising such compounds, and lipid nanoparticle compositions. [Solution] For example, a compound of formula (A), or its N-oxide, or a salt or isomer thereof is provided. In the formula, R'a represents an R'-branched or R'-cyclic structure. JPEG2026077652000423.jpg38170

Description

[Technical Field]

[0001] Related applications This application claims priority and interest in U.S. Provisional Application No. 62 / 902,927, filed September 19, 2019, the entirety of which is incorporated herein by reference.

[0002] This disclosure provides novel compounds for delivering one or more therapeutic and / or prophylactic agents to mammalian cells or organs and / or generating polypeptides, compositions comprising such compounds, and methods involving lipid nanoparticle compositions. In addition to novel lipids, the lipid nanoparticle compositions of the disclosure may comprise, in specific fractions, one or more cationic and / or ionic aminolipids, phospholipids including polyunsaturated lipids, PEG lipids, structural lipids, and / or therapeutic and / or prophylactic agents. [Background technology]

[0003] The effective targeted delivery of biologically active substances such as small molecule drugs, proteins, and nucleic acids is a representative example of ongoing medical challenges. In particular, the delivery of nucleic acids to cells is difficult due to the relative instability and low cellular permeability of such species. Therefore, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and / or prophylactic agents, such as nucleic acids, to cells.

[0004] Lipid-containing nanoparticle compositions, liposomes, and lipoplexes have been proven effective as transport vehicles for biologically active substances such as small molecule drugs, proteins, and nucleic acids into cells and / or intracellular compartments. Such compositions generally contain one or more "cationic" and / or amino(ionic) lipids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), and / or lipids containing polyethylene glycol (PEG lipids). Examples of cationic and / or ionic lipids include amine-containing lipids that can be readily protonated. Although various such lipid-containing nanoparticle compositions have been shown, improvements in safety, efficacy, and specificity remain lacking. [Overview of the Initiative]

[0005] The present disclosure provides novel compounds, compositions, and methods involving them.

[0006] In some embodiments, the disclosure relates to a compound of formula (1-1):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0007] In some embodiments, the disclosure is formula (2-1): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R' a R' 分岐状 or R' 環式 And, R' 分岐状 teeth, [ka] And R' 環式 teeth, [ka] And, R' b teeth, [ka] And, [ka] This indicates a connection point. R aγ and R bγ Each of them is independent of C 2-12 Alkyl or C 2-12 It is alkenyl, R 2 and R 3 Each of them is independent of C 1-14 Alkyl and C 2-14 Selected from the group consisting of alkenils, R 4 teeth, [ka] And, [ka] R indicates a bonding point. 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Each R' is independent of C 1-12 Alkyl or C 2-12 It is alkenyl, Y a C 3-6 It is a carbon ring, R * " a C 1-15 Alkyl and C 2-15 Selected from the group consisting of alkenils, s is either 2 or 3.

[0008] In some embodiments, the disclosure is formula (A): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R' a R' 分岐状 or R' 環式 And, R' 分岐状 teeth, [ka] And R' 環式 teeth, [ka] And, [ka] This indicates a connection point. R aα H is R aβ , R aγ , and R aδ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aβ , R aγ , and R aδ At least one of them is C 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenils, R 2 and R 3 Each is C 1-14 It is alkyl, R 4 These are -(CH2)2OH, -(CH2)3OH, -(CH2)4OH, -(CH2)5OH, and [ka] Selected from the group consisting of, [ka] This indicates a connection point. R 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Each R 5 These are independently OH, C 1-3 Alkyl, C2-3 selected from the group consisting of alkenyl and H, each R 6 is independently OH, C 1-3 alkyl, C 2-3 alkenyl, or H, R 7 is H, M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-, R' is C 1-12 alkyl or C 2-12 alkenyl, Y a is a carbocyclic ring, 3-6 R R * ” a is C 1-15 alkyl and C 2-15 alkenyl, l is selected from the group consisting of 1, 2, 3, 4, and 5, s is 2 or 3, m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0009] In some embodiments, the disclosure relates to a compound of formula (B):

Chemical formula

Chemical formula

Chemical formula

[0010] In some embodiments, the disclosure is formula (Aa): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R aβ , R aγ , and R aδ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aβ , R aγ , and R aδ At least one of them is C 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenils, R 4It is selected from the group consisting of -(CH2)2OH, -(CH2)3OH, -(CH2)4OH, and -(CH2)5OH. M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-, R' is C 1-12 Alkyl or C 2-12 It is Alkenil.

[0011] In some embodiments, the disclosure is formula (Ab): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R aβ , R aγ , and R aδ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aβ , R aγ , and R aδ At least one of them is C 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenils, R 4 It is selected from the group consisting of -(CH2)2OH, -(CH2)3OH, -(CH2)4OH, and -(CH2)5OH. R' is C 1-12 Alkyl or C 2-12 It is Alkenil. [Modes for carrying out the invention]

[0012] The disclosure relates to novel lipids and lipid nanoparticles containing novel lipids (e.g., empty LNPs or filled LNPs). The disclosure also provides methods for delivering therapeutic and / or prophylactic agents to mammalian cells, methods for specifically delivering therapeutic and / or prophylactic agents to mammalian organs, methods for generating a polypeptide of interest in mammalian cells, methods for improving the level of protein produced in mammalian cells compared to LNPs containing other lipids, and methods for treating diseases or disorders in mammals requiring treatment of the disease or disorder. For example, a method for generating a polypeptide of interest in cells may involve contacting a mammalian cell with a nanoparticle containing mRNA, thereby allowing the mRNA to be translated to produce the polypeptide of interest. A method for delivering therapeutic and / or prophylactic agents to mammalian cells or organs may involve administering a nanoparticle composition containing the therapeutic and / or prophylactic agent to a target, the administration of which involves contacting the cell or organ with the composition, thereby delivering the therapeutic and / or prophylactic agent to the cell or organ. Such delivery methods may be in vitro or in vivo.

[0013] This disclosure provides lipids comprising a central amine moiety and at least one biodegradable group. The lipids described herein may be advantageously used in lipid nanoparticles (e.g., empty LNPs or filled LNPs) for the delivery of therapeutic and / or prophylactic agents to mammalian cells or organs. For example, the lipids described herein are little to no immunogenic. For example, lipid compounds of formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc) have lower immunogenicity compared to reference lipids (e.g., MC3, KC2, or DLinDMA). For example, formulations comprising the lipids and therapeutic or prophylactic agents disclosed herein have an increased therapeutic index compared to corresponding formulations comprising a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.

[0014] In some embodiments, the disclosure is formula (A-1): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R' a R' 分岐状 or R' 環式 And, R' 分岐状 teeth, [ka] And R' 環式 teeth, [ka] And, [ka] This indicates a connection point. R aα , R aγ , and R aδ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aβ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 2 and R 3 Each is C 1-14 It is alkyl, R 4 is -(CH2)2OH or [ka] And, [ka] This indicates a connection point. R 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. R 5 and R 6 These are H, R 7 H is, M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-, R' is C 1-12 Alkyl or C 2-12 It is alkenyl, Y a C 3-6 It is a carbon ring, R * " a C 1-15 Alkyl and C 2-15 Selected from the group consisting of alkenils, l is selected from the group consisting of 1, 2, 3, 4, and 5. s is 2 or 3, m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0015] In some embodiments, the disclosure is formula (A-2): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R' a R' 分岐状 or R' 環式 And, R' 分岐状 teeth, [ka] And R' 環式 teeth, [ka] And, [ka] This indicates a connection point. R aα , R aβ , and R aδ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aγ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 2 and R 3 Each is C 1-14 It is alkyl, R 4 is -(CH2)2OH or [ka] And, [ka] This indicates a connection point. R 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. R 5 and R 6 These are H, R 7 H is, M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-, R' is C 1-12 Alkyl or C 2-12 It is alkenyl, Y a C3-6 It is a carbon ring, R * " a C 1-15 Alkyl and C 2-15 Selected from the group consisting of alkenils, l is selected from the group consisting of 1, 2, 3, 4, and 5. s is 2 or 3, m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0016] In some embodiments, the disclosure is formula (A-3): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R' a R' 分岐状 or R' 環式 And, R' 分岐状 teeth, [ka] And R' 環式 teeth, [ka] And, [ka] This indicates a connection point. R aα , R aγ , and R aβ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aδ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 2 and R 3Each is C 1-14 It is alkyl, R 4 is -(CH2)2OH or [ka] And, [ka] This indicates a connection point. R 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. R 5 and R 6 These are H, R 7 H is, M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-, R' is C 1-12 Alkyl or C 2-12 It is alkenyl, Y a C 3-6 It is a carbon ring, R * " a C 1-15 Alkyl and C 2-15 Selected from the group consisting of alkenils, l is selected from the group consisting of 1, 2, 3, 4, and 5. s is 2 or 3, m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0017] In some embodiments, the disclosed compound has one of the following structures. [ka]

[0018] In some embodiments, the disclosure is formula (B-1): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R' a R' 分岐状 or R' 環式 And, R' 分岐状 teeth, [ka] And R' 環式 teeth, [ka] And, [ka] This indicates a connection point. R aα , R aγ , and R aδ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aβ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R bα , R bγ , and R bδ These are H and C, which are independent of each other. 2-30 Alkyl and C 5-20 Selected from the group consisting of alkenyls, R bβ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 4 teeth, [ka] And, [ka] This indicates a connection point. R 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. R 5 and R 6 These are H, M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-, R' is C 1-12 Alkyl or C 2-12 It is alkenyl, Y a C 3-6 It is a carbon ring, R * " a C 1-15 Alkyl and C 2-15 Selected from the group consisting of alkenils, l is selected from the group consisting of 1, 2, 3, 4, and 5. s is 2 or 3, m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0019] In some embodiments, the disclosure is formula (B-2): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R' a R' 分岐状 or R' 環式 And, R' 分岐状 teeth, [ka] And R' 環式 teeth, [ka] And, [ka] This indicates a connection point. R aα , R aβ , and R aδ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aγ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R bα , R bβ , and R bδ These are H and C, which are independent of each other. 2-30 Alkyl and C 5-20 Selected from the group consisting of alkenyls, R bγ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 4 is -(CH2)2OH or [ka] And, [ka] This indicates a connection point. R 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. R 5 and R 6 These are H, M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-, R' is C 1-12 Alkyl or C 2-12 It is alkenyl, Y a C 3-6 It is a carbon ring, R * " a C 1-15 Alkyl and C 2-15 Selected from the group consisting of alkenils, l is selected from the group consisting of 1, 2, 3, 4, and 5. s is 2 or 3, m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0020] In some embodiments, the disclosure is formula (B-3): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R' a R' 分岐状 or R' 環式 And, R' 分岐状 teeth, [ka] And R' 環式 teeth, [ka] And, [ka] This indicates a connection point. R aα , R aβ , and R aγ These are H and C, which are independent of each other. 2-12Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aδ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R bα , R bβ , and R bγ These are H and C, which are independent of each other. 2-30 Alkyl and C 5-20 Selected from the group consisting of alkenyls, R bδ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 4 is -(CH2)2OH or [ka] And, [ka] This indicates a connection point. R 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. R 5 and R 6 These are H, M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-, R' is C 1-12 Alkyl or C 2-12 It is alkenyl, Y a C 3-6 It is a carbon ring, R * " a C 1-15 Alkyl and C 2-15 Selected from the group consisting of alkenils, l is selected from the group consisting of 1, 2, 3, 4, and 5. s is 2 or 3, m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0021] In some embodiments, the disclosure is formula (A-a1): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R aγ and R aδ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aβ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 4 is -(CH2)2OH or [ka] And, [ka] This indicates a connection point. R 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-, R' is C 1-12 Alkyl or C 2-12 It is Alkenil.

[0022] In some embodiments, the disclosure is formula (A-a2): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R aβ and R aδ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aγ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 4 is -(CH2)2OH or [ka] And, [ka] This indicates a connection point. R 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-, R' is C 1-12 Alkyl or C 2-12 It is Alkenil.

[0023] In some embodiments, the disclosure is formula (A-a3): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R aβ and R aγ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aδThese are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 4 is -(CH2)2OH or [ka] And, [ka] This indicates a connection point. R 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-, R' is C 1-12 Alkyl or C 2-12 It is Alkenil.

[0024] In some embodiments, the disclosure is formula (A-b1): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R aγ and R aδ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aβ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 4 is -(CH2)2OH or [ka] And, [ka] This indicates a connection point. R 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. R' is C 1-12 Alkyl or C 2-12 It is Alkenil.

[0025] In some embodiments, the disclosure is formula (A-b2): [ka] A compound thereof, or its N-oxide, or its salt or isomer, In the formula, R aβ and R aδ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aγ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 4 is -(CH2)2OH or [ka] And, [ka] R indicates a bonding point. 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. R' is C 1-12 Alkyl or C2-12 It is Alkenil.

[0026] In some embodiments, the disclosure is formula (A-b3): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R aβ and R aγ These are H and C, which are independent of each other. 2-12 Alkyl and C 2-12 Selected from the group consisting of alkenyls, R aδ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 4 is -(CH2)2OH or [ka] And, [ka] This indicates a connection point. R 10 N(R)² is such that each R is independently C 1-6 Alkyl, C 2-3 n2 is selected from the group consisting of alkenyl and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. R' is C 1-12 Alkyl or C 2-12 It is Alkenil.

[0027] In some embodiments, the disclosure is formula (Ac): [ka] With respect to the compound, or its N-oxide, or its salt or isomer, In the formula, R aγThese are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 4 is -(CH2)2OH or [ka] And, [ka] indicates a bond point, and R' is C 1-12 It is alkyl.

[0028] In some embodiments, the disclosure is formula (Bc): [ka] With respect to a compound, or its N-oxide, or its salt or isomer, in the formula, R aγ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R bγ These are C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. R 4 is -(CH2)2OH or [ka] And, [ka] indicates a bond point, and R' is C 1-12 It is alkyl.

[0029] In some embodiments, the disclosure is formula (Ia): [ka] Regarding the compound, in the formula, R 2and R 3 Each of them is independent of C 1-14 Alkyl and C 2-14 Selected from the group consisting of alkenyls, R aγ and R bγ Each of them is independent of C 2-6 It is alkyl.

[0030] Any compound of formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), and (Bc) contains, where applicable, one or more of the following characteristics:

[0031] In some embodiments, R 4 teeth, [ka] And n2 is 2. In some embodiments, R 4 teeth, [ka] And n2 is 3. In some embodiments, R 4 teeth, [ka] Therefore, n2 is 4.

[0032] In some embodiments, R 10 is -NH2. In some embodiments, R 10 is -NH(C 1-6 It is alkyl. In some embodiments, R 10 is -N(C 1-6 It is alkyl)2. In some embodiments, R 10 is -NH(CH3). In some embodiments, R10 This is -N(CH3)2.

[0033] In some embodiments, R 4 is -(CH2)2OH, -(CH2)3OH, or -(CH2)4OH, and M and M' are each -C(O)O-. In some embodiments, R 4 is -(CH2)2OH, and M and M' are each -C(O)O-. In some embodiments, R 4 is -(CH2)3OH, and M and M' are each -C(O)O-. In some embodiments, R 4 M is -(CH2)4OH, and M and M' are each -C(O)O-.

[0034] In some embodiments, R 4 teeth, [ka] That is the case.

[0035] In some embodiments, R 4 teeth, [ka] And M and M' are -C(O)O-, respectively. In some embodiments, R 4 teeth, [ka] And M and M' are each -OC(O)-. In some embodiments, R 4 teeth, [ka] And M is -OC(O)- and M' is -C(O)O-. In some embodiments, R 4 teeth, [ka] Therefore, M is -C(O)O- and M' is -OC(O)-.

[0036] In some embodiments, l is 1, 2, 3, or 4. In some embodiments, l is 5. In some embodiments, m is 5, 6, 7, 8, or 9. In some embodiments, m is 5. In some embodiments, m is 7.

[0037] In some embodiments, l is 5 and m is 5. In some embodiments, l is 5 and m is 7. In some embodiments, l is 5 and m is 5, 6, 7, 8, or 9. In some embodiments, m is 5 and l is 1, 2, 3, or 4. In some embodiments, m is 7 and l is 1, 2, 3, or 4.

[0038] In some embodiments, R 5 , R 6 、 and R 7 H is each of the following, and m is 5, 6, 7, 8, or 9. In some embodiments, R 5 , R 6 、 and R 7 H is the same as m is 5. In some embodiments, R 5 , R 6 、 and R 7 H is the value of each, and m is 7.

[0039] In some embodiments, R 2 and R 3 Each of them is independent of C 1-14 Alkyl or C 2-14 It is an alkenyl. In some embodiments, R 2 and R 3 Each of them is independent of C 3-14 Alkyl or C 3-14 It is an alkenyl. In some embodiments, R 2and R 3 Independently, C 5-14 Alkyl or C 5-14 It is Alkenil.

[0040] In some embodiments, R 2 and R 3 Each of them is independent of C 1-14 It is alkyl. In some embodiments, R 2 and R 3 Each of them is independent of C 3-14 It is alkyl. In some embodiments, R 2 and R 3 Each of them is independent of C 7-9 It is alkyl.

[0041] In some embodiments, R 2 and R 3 Each of these is a C7 alkyl group. In some embodiments, R 2 and R 3 Each of these is a C8 alkyl group. In some embodiments, R 2 and R 3 These are each C9 alkyl groups.

[0042] In some embodiments, R' a R' 分岐状 And R aα , R aγ , and R aδ H and R aβ is a C2-C6 alkyl group. In some embodiments, R' a R' 分岐状 And R aα , R aβ , and R aδ H and R aγ is a C2-C6 alkyl group. In some embodiments, R' a R' 分岐状 And R aα , R aβ , and R aγ H and R aδ It is a C2-C6 alkyl group.

[0043] In some embodiments, R bα , R bγ , and R bδ H and R bβ is a C2-C6 alkyl group. In some embodiments, R bα , R bβ , and R bδ H and R bγ is a C2-C6 alkyl group. In some embodiments, R bα , R bβ , and R bγ H and R bδ It is a C2-C6 alkyl group.

[0044] In some embodiments, R aα , R aγ , and R aδ H and R aβ It is a C2-C6 alkyl group, and R bα , R bγ , and R bδ H and R bβ is a C2-C6 alkyl group. In some embodiments, R aα , R aγ , and R aδ H and R aβ It is a C2-C6 alkyl group, and R bα , R bβ , and R bδ H and R bγ is a C2-C6 alkyl group. In some embodiments, R aα , R aγ , and R aδ H and R aβ It is a C2-C6 alkyl group, and R bα , R bβ , and R bγ H and R bδ It is a C2-C6 alkyl group.

[0045] In some embodiments, R aα , R aβ , and R aδ H and R aγIt is a C2-C6 alkyl group, and R bα , R bγ , and R bδ H and R bβ is a C2-C6 alkyl group. In some embodiments, R aα , R aβ , and R aδ H and R aγ It is a C2-C6 alkyl group, and R bα , R bβ , and R bδ H and R bγ is a C2-C6 alkyl group. In some embodiments, R aα , R aβ , and R aδ H and R aγ It is a C2-C6 alkyl group, and R bα , R bβ , and R bγ H and R bδ It is a C2-C6 alkyl group.

[0046] In some embodiments, R aα , R aβ , and R aγ H and R aδ It is a C2-C6 alkyl group, and R bα , R bγ , and R bδ H and R bβ is a C2-C6 alkyl group. In some embodiments, R aα , R aβ , and R aγ H and R aδ It is a C2-C6 alkyl group, and R bα , R bβ , and R bδ H and R bγ is a C2-C6 alkyl group. In some embodiments, R aα , R aβ , and R aγ H and R aδ It is a C2-C6 alkyl group, and R bα , R bβ , and R bγH and R bδ It is a C2-C6 alkyl group.

[0047] In some embodiments, R' is C 1-12 Alkyl or C 2-12 It is an alkenyl. In some embodiments, R' is a C2 alkyl. In some embodiments, R' is a C3 or C4 alkyl. In some embodiments, R' is a C3 alkyl. In some embodiments, R' is a C4 alkyl. In some embodiments, R' is a C5 alkyl.

[0048] In some embodiments, R' is a C4 alkyl or C4 alkenyl. In some embodiments, R' is a C5 alkyl or C5 alkenyl. In some embodiments, R' is a C6 alkyl or C6 alkenyl. In some embodiments, R' is a C7 alkyl or C7 alkenyl. In some embodiments, R' is a C8 alkyl or C8 alkenyl. In some embodiments, R' is a C9 alkyl or C9 alkenyl. In some embodiments, R' is a C 10 Alkyl or C 10 It is an alkenyl. In some embodiments, R' is C 11 Alkyl or C 11 It is Alkenil.

[0049] In some embodiments, R' a R' 分岐状 And R aα , R aγ , and R aδ H and R aβ R' is a C2-C6 alkyl group, and R' is a C3-C5 alkyl group. In some embodiments, R' a R' 分岐状 And R aα , R aγ , and R aδ H and R aβ R' is a C2-C6 alkyl group, and R' is a C3 alkyl group. In some embodiments, R'a R' 分岐状 And R aα , R aγ , and R aδ H and R aβ R' is a C2-C6 alkyl group, and R' is a C4 alkyl group.

[0050] In some embodiments, R' a R' 分岐状 And R aα , R aβ , and R aδ H and R aγ R' is a C2-C6 alkyl group, and R' is a C3-C5 alkyl group. In some embodiments, R' a R' 分岐状 And R aα , R aβ , and R aδ H and R aγ R' is a C2-C6 alkyl group, and R' is a C3 alkyl group. In some embodiments, R' a R' 分岐状 And R aα , R aβ , and R aδ H and R bγ R' is a C2-C6 alkyl group, and R' is a C4 alkyl group.

[0051] In some embodiments, R' a R' 分岐状 And R aα , R aβ , and R aγ H and R aδ R' is a C2-C6 alkyl group, and R' is a C3-C5 alkyl group. In some embodiments, R' a R' 分岐状 And R aα , R aβ , and R aγ H and R aδ R' is a C2-C6 alkyl group, and R' is a C3 alkyl group. In some embodiments, R' a R' 分岐状 And Raα , R aβ , and R aγ H and R aδ R' is a C2-C6 alkyl group, and R' is a C4 alkyl group.

[0052] In some embodiments, R * " a It is either C2-alkyl or C3-alkyl.

[0053] In some embodiments, s is 2. In some embodiments, s is 3.

[0054] In some embodiments, s is 2 and R * " a It is either a C2 alkyl or a C3 alkyl group.

[0055] In some embodiments, Y a R * " a teeth, [ka] In some embodiments, Y a R * " a teeth, [ka] That is the case.

[0056] In some embodiments, s is 2 and Y a R * " a teeth, [ka] And R * " a is a C2 alkyl or C3 alkyl group. In some embodiments, s is 2 and Y a R * "a teeth, [ka] And R * " a It is either a C2 alkyl or a C3 alkyl group.

[0057] In some embodiments, any of the compounds from the formulas described herein are suitable for preparing nanoparticle compositions for intramuscular administration.

[0058] In some embodiments, the compounds of formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc) are selected from the compounds in Table 1 and their N-oxides, salts, or isomers. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0059] The central amine moiety of lipids according to formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc) can be protonated at physiological pH. Therefore, lipids can have a positive or partially positive charge at physiological pH. Such lipids are sometimes referred to as cationic or ionic (amino) lipids. Lipids may also be zwitterionic, i.e., neutral molecules that have both positive and negative charges.

[0060] definition As used herein, the terms "alkyl" or "alkyl group" mean linear or branched saturated hydrocarbons comprising one or more optionally substituted carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms). 1-14 The term "alkyl" refers to a linear or branched saturated hydrocarbon containing 1 to 14 carbon atoms, which is optionally substituted. Unless otherwise specified, alkyl groups as used herein refer to both unsubstituted and substituted alkyl groups.

[0061] As used herein, the terms “alkenyl” or “alkenyl group” mean a linear or branched hydrocarbon comprising two or more optionally substituted carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms) and at least one double bond. 2-14 The term "alkenyl" refers to an optionally substituted linear or branched hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may contain 1, 2, 3, 4, or more carbon-carbon double bonds. For example, C 18 Alkenyls can contain one or more double bonds. C containing two double bonds18 The alkenyl group may be a linoleyl group. Unless otherwise specified, the alkenyl group as described herein refers to both unsubstituted and substituted alkenyl groups.

[0062] As used herein, the terms “alkynyl” or “alkynyl group” mean a linear or branched hydrocarbon comprising two or more optionally substituted carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms) and at least one carbon-carbon triple bond. 2-14 The term "alkynyl" refers to an optionally substituted linear or branched hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group may contain 1, 2, 3, 4, or more carbon-carbon triple bonds. For example, C 18 Alkynnyls may contain one or more carbon-carbon triple bonds. Unless otherwise specified, the alkynyl groups described herein refer to both unsubstituted and substituted alkynyl groups.

[0063] As used herein, the terms “carbocyclic” or “carbocyclic group” mean an optionally substituted monocyclic or polycyclic system containing one or more rings of carbon atoms. The rings may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-membered rings. 3-6The term "carbocyclic ring" refers to a single ring containing 3 to 6 carbon atoms. A carbocyclic ring may contain one or more carbon-carbon double or triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl or aryl groups). Examples of carbocyclic rings include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups. As used herein, the term "cycloalkyl" refers to a non-aromatic carbocyclic ring, which may or may not contain double or triple bonds. Unless otherwise specified, carbocyclic rings as described herein refer to both unsubstituted carbocyclic groups and substituted carbocyclic groups, i.e., optionally substituted carbocyclic rings. In some embodiments, the carbocyclic ring is C 3-8 It is a cycloalkyl. In some embodiments, the carbon ring is C 3-6 It is a cycloalkyl. In some embodiments, the carbon ring is C 6-10 It is Ariel.

[0064] "Aryl" includes "compound" or polycyclic systems having at least one aromatic ring that does not contain heteroatoms in its ring structure. Examples include phenyl, benzyl, and 1,2,3,4-tetrahydronaphthalenyl. In some embodiments, "aryl" is an aromatic C 6-10 It is a carbon ring (for example, "aryl" is C 6-10 (It is Ariel.)

[0065] As used herein, the terms “heterocycle” or “heterocyclic group” mean an optionally substituted monocyclic or polycyclic system comprising one or more rings, where at least one ring comprises at least one heteroatom. The heteroatom may be, for example, a nitrogen, oxygen, or sulfur atom. The rings may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14-membered rings. The heterocycle may contain one or more double or triple bonds and may be non-aromatic or aromatic (e.g., heterocycloalkyl or heteroaryl groups). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups. As used herein, the term "heterocycloalkyl" means a non-aromatic heterocycle, which may or may not contain double or triple bonds. Unless otherwise specified, heterocycles as described herein refer to both unsubstituted heterocyclic groups and substituted heterocyclic groups, i.e., optionally substituted heterocycles. In some embodiments, the heterocycle is a 4- to 12-membered heterocycloalkyl. In some embodiments, the heterocycle is a 5 or 6-membered heteroaryl.

[0066] A "heteroaryl" group is an aryl group as defined above, except that it has 1 to 4 heteroatoms in its ring structure, and may also be called an "aryl heterocyclic" or "heteroaromatic." As used herein, the term "heteroaryl" is intended to include a carbon atom and one or more heteroatoms independently selected from the group consisting of nitrogen, oxygen, sulfur, and boron, e.g., one, or 1-2, or 1-3, or 1-4, or 1-5, or 1-6 heteroatoms, or a stable 5, 6, or 7-membered monocyclic or 7, 8, 9, 10, 11, or 12-membered bicyclic aromatic heterocyclic ring consisting of, e.g., 1, 2, 3, 4, 5, or 6 heteroatoms. The nitrogen atom may be substituted or unsubstituted (i.e., N, or R is H or other substituent as defined, NR). The nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., N → O and S(O) p (and p=1 or 2). Note that the total number of S and O atoms in the aromatic heterocycle does not exceed 1.

[0067] Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0068] Furthermore, the terms "aryl" and "heteroaryl" include polycyclic aryl and heteroaryl groups, such as tricyclic and bicyclic groups, such as naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzimidazole, benzothiophene, quinoline, isoquinoline, naphtholidine, indole, benzofuran, purine, benzofuran, deazapurine, and indoridine.

[0069] As used herein, “biodegradable group” is a group that can promote faster lipid metabolism in mammalian entities. Biodegradable groups may be selected from the group consisting of, but are not limited to, -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, aryl groups, and heteroaryl groups. As used herein, “aryl group” is an optionally substituted carbocyclic group containing one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups. As used herein, “heteroaryl group” is an optionally substituted heterocyclic group containing one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups can be optionally substituted. For example, M and M' can be selected from an unrestricted group consisting of optionally substituted phenyl, oxazole, and thiazole groups. In the formulas herein, M and M' can be independently selected from the list of biodegradable groups above. Unless otherwise specified, aryl or heteroaryl groups as described herein refer to both unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.

[0070] Alkyl, alkenyl, and cyclyl (e.g., carbocyryl and heterocyclyl) groups may be optionally substituted unless otherwise specified. Any substituent is not limited to halogen atoms (e.g., chloride, bromide, fluoride, or iodide groups), carboxylic acids (e.g., -C(O)OH), alcohols (e.g., hydroxyl, -OH), esters (e.g., -C(O)OR or -OC(O)R), aldehydes (e.g., -C(O)H), carbonyls (e.g., -C(O)R, alternatively represented by C=O), acyl halides (e.g., -C(O)X, where X is a halide selected from bromide, fluoride, chloride, and iodide), carbonates (e.g., -OC(O)OR), alkoxys (e.g., -OR), acetals (e.g., -C(OR)2R'', where each OR is an alkoxy group that may be the same or different and R''" is an alkyl or alkenyl group), phosphates (e.g., P(O)4 3- ), thiols (e.g., -SH), sulfoxides (e.g., -S(O)R), sulfinic acids (e.g., -S(O)OH), sulfonic acids (e.g., -S(O)2OH), thials (e.g., -C(S)H), sulfates (e.g., S(O)4) 2-), sulfonyl (e.g., -S(O)2-), amide (e.g., -C(O)NR2 or -N(R)C(O)R), azide (e.g., -N3), nitro (e.g., -NO2), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(O)R), amino (e.g., -NR2, -NRH, or -NH2), carbamoyl (e.g., -OC(O)NR2, The substituents can be selected from the group consisting of -OC(O)NRH or -OC(O)NH2), sulfonamides (e.g., -S(O)2NR2, -S(O)2NRH, -S(O)2NH2, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)S(O)2H, or -N(H)S(O)2H), alkyl groups, alkenyl groups, and cyclyl groups (e.g., carbocyryl or heterocyclyl). In all of the foregoing, R is an alkyl or alkenyl group as defined herein. In some embodiments, the substituent itself may be further substituted with, for example, 1, 2, 3, 4, 5, or 6 substituents as defined herein. For example, C 1-6 The alkyl group may be further substituted with one, two, three, four, five, or six substituents described herein.

[0071] Other compounds of the disclosure can be obtained by converting nitrogen-containing compounds to N-oxides by treating them with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (mCPBA) and / or hydrogen peroxide). Thus, all nitrogen-containing compounds shown and claimed are, where permitted by valency and structure, the shown compounds and their N-oxide derivatives (N → O or N + -O -It is deemed to include both (which may be designated as) the nitrogen compounds of the disclosed compounds. Furthermore, in other cases, the nitrogen in the disclosed compounds may be converted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds may be prepared by oxidation of the parent amine with an oxidizing agent such as m-CPBA. All nitrogen-containing compounds shown and claimed are also deemed to encompass both the shown compounds and their N-hydroxy (i.e., N-OH) and N-alkoxy (i.e., N-OR) derivatives, where permitted by valency and structure, R is a substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3-14 membered carbon ring, or 3-14 membered heterocycle.

[0072] Approximately, about: As used herein, the terms “approximately” and “about” refer to values ​​similar to the listed reference values ​​when applied to one or more target values. In certain embodiments, unless otherwise stated or evident from the context (except where such numbers exceed 100% of possible values), the terms “approximately” or “about” refer to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) the listed reference values. For example, when used in the context of the amount of a given compound in a lipid component of a nanoparticle composition, “about” may mean ±10% of the listed values. For example, a nanoparticle composition containing a lipid component having about 40% of a given compound may contain 30–50% of the compound.

[0073] As used herein, the term “compound” means including all isomers and isotopes of the structure shown. “Isotope” refers to an atom having the same number of atoms but a different mass number, resulting from a different number of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. Furthermore, the compounds, salts, or complexes of this disclosure can be prepared in combination with a solvent or water molecule by conventional methods to form solvates and hydrates.

[0074] As used herein, the term “contact” means to establish a physical connection between two or more entities. For example, contacting mammalian cells with a nanoparticle composition means that the mammalian cells and nanoparticles are constructed to share a physical connection. Methods for contacting cells with external entities both in vivo and ex vivo are well known in the field of biology. For example, contacting a nanoparticle composition with mammalian cells placed within a mammal can be carried out by various administration routes (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and may involve varying amounts of lipid nanoparticles (e.g., empty LNPs or filled LNPs). Furthermore, two or more mammalian cells may be contacted with a nanoparticle composition.

[0075] As used herein, the term “deliver” means to provide an entity to a place of interest. For example, delivering a therapeutic and / or prophylactic agent to a place of interest may involve administering a nanoparticle composition containing the therapeutic and / or prophylactic agent to a place of interest (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route). Administering a nanoparticle composition to a mammal or mammalian cells may involve bringing one or more cells into contact with the nanoparticle composition.

[0076] As used herein, the term “enhanced delivery” means that the therapeutic and / or prophylactic agent delivered by nanoparticles to a target cell of interest (e.g., mammalian liver) is delivered at a greater rate (e.g., at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times) compared to the level of delivery of the therapeutic and / or prophylactic agent by control nanoparticles to the target cell of interest (e.g., MC3, KC2, or DLinDMA). The level of delivery of nanoparticles to a particular tissue may be measured by comparing the amount of protein produced in the tissue to the weight of the tissue, comparing the amount of the therapeutic and / or prophylactic agent in the tissue to the weight of the tissue, comparing the amount of protein produced in the tissue to the total amount of protein in the tissue, or comparing the amount of the therapeutic and / or prophylactic agent in the tissue to the total amount of the therapeutic and / or prophylactic agent in the tissue. It will be understood that the improved delivery of nanoparticles to target cells does not need to be determined in the target being treated, but can be determined in a surrogate, such as an animal model (e.g., a rat model). In certain embodiments, nanoparticle compositions comprising compounds by formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc) have substantially the same level of delivery improvement regardless of the route of administration. For example, certain compounds disclosed herein exhibit similar delivery improvement when used to deliver therapeutic and / or prophylactic agents intravenously or intramuscularly. In other embodiments, certain compounds disclosed herein exhibit a higher level of delivery enhancement when used to deliver therapeutic and / or prophylactic agents intramuscularly rather than intravenously.

[0077] As used herein, the terms “specific delivery,” “deliver specifically,” or “specifically deliver” mean that the therapeutic and / or prophylactic agent by nanoparticles is delivered more (e.g., at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times) to the target tissue of interest (e.g., mammalian liver) compared to extra-target cells (e.g., mammalian spleen). The level of nanoparticle delivery to a particular tissue may be measured by comparing the amount of protein produced in the tissue to the weight of the tissue, comparing the amount of therapeutic and / or prophylactic agent in the tissue to the weight of the tissue, comparing the amount of protein produced in the tissue to the total amount of protein in the tissue, or comparing the amount of therapeutic and / or prophylactic agent in the tissue to the total amount of therapeutic and / or prophylactic agent in the tissue. For example, if a therapeutic and / or prophylactic agent is delivered to the kidney at a rate of 1.5, 2, 3, 5, 10, 15, or 20 times or more per gram of tissue compared to delivery to the liver or spleen following systemic administration of the therapeutic and / or prophylactic agent, then in renal vascular targeting, the therapeutic and / or prophylactic agent is specifically delivered to the mammalian kidney compared to the liver and spleen. It will be understood that the ability of nanoparticles to be specifically delivered to target tissue does not need to be determined in the subject being treated, but can be determined in surrogates such as animal models (e.g., rat models).

[0078] As used herein, “encapsulation efficiency” refers to the amount of therapeutic and / or prophylactic agent that becomes part of the nanoparticle composition relative to the initial total amount of therapeutic and / or prophylactic agent used in the preparation of the nanoparticle composition. For example, if 97 mg of therapeutic and / or prophylactic agent out of a total of 100 mg initially provided to the composition is encapsulated in the nanoparticle composition, the encapsulation efficiency may be 97%. As used herein, “encapsulation” may refer to complete, substantial, or partial sealing, containment, encirclement, or packing.

[0079] As used herein, “encapsulated,” “encapsulated,” “filled,” and “associated” may refer to complete, substantial, or partial sealing, containment, encirclement, or packing. As used herein, “encapsulation” or “association” may refer to the process of encapsulating individual nucleic acid molecules within a nanoparticle and / or the process of establishing a physiological and chemical relationship between the individual nucleic acid molecules and the nanoparticle. As used herein, “empty nanoparticles” may refer to nanoparticles that substantially do not contain therapeutic or prophylactic agents. As used herein, “empty nanoparticles” or “empty lipid nanoparticles” may refer to nanoparticles that substantially do not contain nucleic acids. As used herein, “empty nanoparticles” or “empty lipid nanoparticles” may refer to nanoparticles that substantially do not contain nucleotides or polypeptides. As used herein, “empty nanoparticles” or “empty lipid nanoparticles” may refer to nanoparticles that consist substantially only of lipid components. As used herein, “filled nanoparticles” or “filled lipid nanoparticles” (also referred to as “complete nanoparticles” or “complete lipid nanoparticles”) may refer to nanoparticles that contain the components of empty nanoparticles plus therapeutic or prophylactic agents. As used herein, “filled nanoparticles” or “filled lipid nanoparticles” (also referred to as “complete nanoparticles” or “complete lipid nanoparticles”) may refer to nanoparticles comprising the components of empty nanoparticles and nucleotides or polypeptides. As used herein, “filled nanoparticles” or “filled lipid nanoparticles” (also referred to as “complete nanoparticles” or “complete lipid nanoparticles”) may refer to nanoparticles comprising the components of empty nanoparticles and nucleic acids.

[0080] As used herein, “expression” of a nucleic acid sequence refers to the translation of mRNA into a polypeptide or protein, and / or post-translational modification of a polypeptide or protein.

[0081] As used herein, the term "in vitro" refers to events occurring in an artificial environment, such as in a test tube or reaction vessel, during cell culture, or in a petri dish, rather than within living organisms (e.g., animals, plants, or microorganisms).

[0082] As used herein, the term "in vivo" refers to events occurring within a living organism (for example, an animal, plant, or microorganism, or their cells or tissues).

[0083] As used herein, the term “ex vivo” refers to an event occurring outside of a living organism (e.g., an animal, plant, or microorganism, or its cells or tissues). Ex vivo events can occur in environments that have been minimally altered from their natural (e.g., in vivo) environment.

[0084] As used herein, the term “isomer” means any geometric isomer, tautomer, amphoteric, stereoisomer, enantiomer, or diastereomer of a compound. A compound may contain one or more chiral centers and / or double bonds and therefore may exist as a stereoisomer, such as a double bond isomer (i.e., a geometric E / Z isomer) or a diastereomer (e.g., an enantiomer (i.e., (+) or (-)), or a cis / trans isomer). This disclosure encompasses any and all isomers of the compounds described herein, including stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure), as well as mixtures of enantiomers and mixtures of stereoisomers, such as racemates. Mixtures of enantiomers and mixtures of stereoisomers of a compound, and means for separating them into their enantiomer or stereoisomer components, are well known.

[0085] A "tautomer" is one of two or more structural isomers that exist in equilibrium and can be readily converted from one isomer to another. This conversion involves a formal transfer of hydrogen atoms, with the switching of adjacent conjugated double bonds. Tautomers exist as a mixture of sets of tautomers in solution. In a solution where tautomerization is possible, a chemical equilibrium of tautomers will be reached. The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH. The concept of tautomers that can be interconverted by tautomerization is called tautomerism.

[0086] Of the various types of tautomerism possible, two are commonly observed. Keto-enol tautomerism involves the simultaneous shift of electrons and hydrogen atoms. Ring chain tautomerism occurs as a result of an aldehyde group (-CHO) of a sugar chain molecule reacting with one of the hydroxyl groups (-OH) of the same molecule, resulting in the cyclic (ring-shaped) form exhibited by glucose.

[0087] Common tautomerism pairs include ketone-enol, amide-nitrile, lactam-lactim, amide-imido acid tautomerisms, imine-enamine, and enamine-enamine tautomerisms of heterocyclic rings (e.g., nucleic acid bases such as guanine, thymine, and cytosine). An example of tautomerism in disubstituted guanidines is shown below. [ka]

[0088] It should be understood that the disclosed compounds may be represented as different tautomers. Furthermore, if a compound has tautomer forms, it should be understood that all tautomer forms are intended to be included within the scope of the disclosure, and that the naming of the compound does not exclude any tautomer forms.

[0089] As used herein, “lipid component” refers to a component of a nanoparticle composition containing one or more lipids. For example, lipid components may include one or more cationic / ionic lipids, PEGylated lipids, structural lipids, or other lipids such as phospholipids.

[0090] As used herein, “linker” refers to a part that connects two parts, for example, a link between two nucleosides of a cap species. The linker may include, but is not limited to, one or more groups comprising a phosphate group (e.g., phosphate, boranophosphate, thiophosphate, selenophosphate, and phosphonate), an alkyl group, an amidate, or glycerol. For example, two nucleosides of a cap analog may be linked at their 5' positions by a triphosphate base or by a chain comprising two phosphate moieties and a boranophosphonate moiety.

[0091] As used herein, “method of administration” includes intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering the composition to a target. The method of administration may be selected to target delivery to a specific area or system of the body (e.g., specific delivery).

[0092] As used herein, “modified” means unnatural. For example, RNA can be modified RNA; that is, RNA may contain one or more nucleic acid bases, nucleosides, nucleotides, or linkers that are present in unnatural conditions. A “modified” species may also be referred to herein as an “altered” species. Species can be modified or altered chemically, structurally, or functionally. For example, a modified nucleic acid base species may contain one or more substitutions that are not present in nature.

[0093] As used herein, "N:P ratio" refers, for example, to the molar ratio of ionic nitrogen atoms in lipids (within the physiological pH range) to phosphate groups in RNA in a nanoparticle composition containing lipid components and RNA.

[0094] As used herein, “nanoparticle composition” is a composition comprising one or more lipids. A nanoparticle composition is typically about a few micrometers in size or less and may contain a lipid bilayer. A nanoparticle composition may include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition may be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0095] As used herein, “naturally occurring” means occurring naturally without artificial intervention.

[0096] As used herein, “patient” means a person who can or may need treatment, needs treatment, is receiving treatment, is receiving treatment, or is being treated by a specialist trained for a particular disease or condition.

[0097] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid containing a polyethylene glycol component.

[0098] The term "pharmaceutically acceptable" is used herein to mean a compound, material, composition, and / or dosage form that is suitable for use in contact with human and animal tissues, within reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0099] The expression "pharmaceutically acceptable excipients," as used herein, refers to any component other than the compounds specified herein (e.g., a vehicle capable of suspending, complexing, or dissolving the active compound) that is substantially non-toxic and non-inflammatory to the patient. Examples of excipients include anti-adhesion agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), softeners, emulsifiers, fillers (diluents), film-forming agents or coatings, fragrances, flavorings, flow accelerators (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending agents or dispersants, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, gelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and other types disclosed herein.

[0100] While the structural formulas of compounds in this specification may, for convenience, represent a specific isomer, this disclosure includes all isomers, including geometric isomers, optical isomers based on chiral carbons, stereoisomers, and tautomers, and it should be understood that not all isomers may have the same level of activity. In addition, compounds represented by formulas may exist in polymorphism. It should be noted that any crystalline form, mixture of crystalline forms, or their anhydrous or hydrated forms are within the scope of this disclosure.

[0101] The terms "crystalline polymorphism," "polymorphism," or "crystalline form" refer to crystalline structures in which a compound (or its salt or solvate) can crystallize in different crystalline packing arrangements, all having the same elemental composition. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, density hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can result in the dominance of one crystalline form. Crystallographic polymorphisms of a compound can be prepared by crystallization under different conditions.

[0102] The composition may also contain salts of one or more compounds. The salts may be pharmaceutically acceptable salts. As used herein, “pharmaceutically acceptable salt” means a derivative of a compound disclosed in which the parent compound has been modified by converting an existing acidic or base moiety to its salt form (for example, 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 residues such as amines, and alkali salts or organic salts of acidic residues such as carboxylic acids. Typical acid addition salts include acetate, adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfonate, ethanesulfonate, fumarate, glucoheptone, glycerophosphate, hemisulfate, heptone, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxy- Examples include ethanesulfonates, lactobionates, lactates, laurates, sulfates, malates, maleates, malons, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, toluenesulfonates, undecanoic acid, and valerates. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. The pharmaceutically acceptable salts of this disclosure include, for example, conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized by conventional chemical methods from a parent compound containing a basic or acidic moiety.Generally, such salts can be prepared by reacting the free acid or base form of these compounds with an appropriate amount of stoichiometric base or acid in water, an organic solvent, or a mixture of the two, and generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0103] As used herein, “phospholipid” is a lipid comprising a phosphate moiety and one or more carbon chains, such as an unsaturated fatty acid chain. A phospholipid may contain one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturated bonds). Certain phospholipids may facilitate fusion to membranes. For example, cationic phospholipids may interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or intracellular membrane). By fusing phospholipids to a membrane, one or more elements of a lipid-containing composition may pass through the membrane, enabling, for example, the delivery of one or more elements to a cell.

[0104] As used herein, the "polydispersity index" or "PDI" is a ratio that describes the homogeneity of the particle size distribution in a given system. For example, a small value less than 0.3 indicates a narrow particle size distribution.

[0105] As used herein, the terms “polypeptide” or “polypeptide of interest” typically refer to polymers of amino acid residues joined by peptide bonds, which can be produced naturally (e.g., by isolation or purification) or synthetically. The terms “polypeptide,” “peptide,” and “protein” are used herein synonymously to refer to polymers of amino acids of any length. Polymers may include modified amino acids. The term also encompasses amino acid polymers that are naturally occurring or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with labeling components. Also included in the definition are polypeptides containing one or more analogues of amino acids (including, for example, non-natural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include encoded polypeptide products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologues, paralogs, fragments, and other equivalents, variants, and analogues. Polypeptides may be monomers or multimolecular complexes such as dimers, trimers, or tetramers. They may also include single-chain or polychain polypeptides. Most commonly, disulfide bonds are found in polychain polypeptides. The term polypeptide may also be applied to amino acid polymers, in which one or more amino acid residues are artificial chemical analogues of corresponding naturally occurring amino acids. In some embodiments, the “peptide” may be 50 amino acid lengths or less, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid lengths.

[0106] As used herein, “RNA” refers to ribonucleic acid, which may be natural or unnatural. For example, RNA may contain one or more nucleic acid bases, nucleosides, nucleotides, or modifications such as linkers and / or components that are present in nature. RNA may contain cap structures, chain termination nucleosides, stem-loops, poly(A) sequences, and / or polyadenylation signals. RNA may have nucleotide sequences that encode the polypeptide of interest.

[0107] As used herein, “DNA” refers to deoxyribonucleic acid that may exist naturally or unnaturally. For example, DNA may be a synthetic molecule, such as a synthetic DNA molecule produced in vitro. In some embodiments, the DNA molecule is a recombinant molecule. As used herein, “recombinant DNA molecule” refers to a DNA molecule that does not exist as a naturally occurring product but is produced using molecular biological techniques.

[0108] As used herein, “single unit dose” refers to the dose of any therapeutic agent administered in one dose / single dose / single route / single contact point, i.e., in a single administration event.

[0109] As used herein, “divided dose” means dividing a single unit dose or total daily dose into two or more doses.

[0110] As used herein, “total daily dose” refers to the amount given or prescribed over a 24-hour period. This may be administered as a single unit dose.

[0111] As used herein, “size” or “average size” in the context of lipid nanoparticles (e.g., empty LNPs or filled LNPs) refers to the average diameter of the nanoparticle composition.

[0112] As used herein, the terms “subject” or “patient” refer to any organism to which the compositions disclosed may be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0113] As used herein, “target cells” refers to any one or more cells of interest. Cells may be found in vitro, in vivo, in situ, or in the tissues or organs of an organism. Organisms may be animals, preferably mammals, more preferably humans, and most preferably patients.

[0114] As used herein, “target tissue” refers to any one or more types of target tissues to which delivery of a therapeutic and / or prophylactic agent will produce the desired biological and / or pharmacological effect. Examples of target tissues include certain tissues, organs, and their systems or groups. In specific applications, target tissues may be the kidneys, lungs, spleen, vascular endothelium within blood vessels (e.g., coronary or femoral arteries), or tumor tissue (e.g., via intratumoral injection). “Non-target tissue” refers to any one or more types of tissues to which the expression of the encoded protein will not produce the desired biological and / or pharmacological effect. In specific applications, non-target tissues may include the liver and spleen.

[0115] The terms “therapeutic” or “preventive” refer to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or preventive effect, and / or induces a desired biological and / or pharmacological effect. Therapeutic agents are also referred to as “activators” or “activators.” Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0116] As used herein, the term “therapeutic dose” means the amount of a delivered agent (e.g., nucleic acids, drugs, compositions, therapeutic agents, diagnostic agents, prophylactic agents, etc.) that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat the infection, disease, disorder, and / or condition, improve, diagnose, prevent, and / or delay the onset of their symptoms.

[0117] As used herein, “transfection” refers to the introduction of a species (e.g., RNA) into a cell. Transfection may be performed, for example, in vitro, ex vivo, or in vivo.

[0118] As used herein, the term “to treat” means to partially or completely alleviate, improve, improve, mitigate, delay the onset, inhibit the progression, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition. For example, “to treat” cancer may mean to inhibit the survival, growth, and / or spread of the tumor. Treatment may be administered to subjects who show no signs of the disease, disorder, and / or condition, and / or subjects who show only early signs of the disease, disorder, and / or condition, for the purpose of reducing the risk of developing pathologies associated with the disease, disorder, and / or condition.

[0119] As used herein, "zeta potential" refers, for example, to the electrical kinetic potential of lipids in a particulate composition.

[0120] Nanoparticle composition The disclosure also features lipid nanoparticles comprising compounds of formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc) as described herein.

[0121] In some embodiments, the maximum dimensions of the nanoparticle composition are less than or equal to 1 μm (e.g., 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or less) as measured by, for example, dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. Examples of nanoparticle compositions include lipid nanoparticles (LNPs, e.g., empty LNPs or filled LNPs), liposomes, lipid vesicles, and lipoplexes. In some embodiments, the nanoparticle composition is a vesicle containing one or more lipid bilayers. In certain embodiments, the nanoparticle composition contains two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers may be functionalized and / or crosslinked with each other. The lipid bilayers may contain one or more ligands, proteins, or channels.

[0122] The nanoparticle composition contains a lipid component comprising at least one compound according to formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc). For example, the lipid component of the nanoparticle composition may contain one or more compounds from Table 1. The nanoparticle composition may also contain various other components. For example, the lipid component of the nanoparticle composition may include one or more other lipids in addition to lipids according to formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc).

[0123] Cationic / ionic lipids Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may contain one or more cationic and / or ionic lipids (e.g., lipids that may have a positive or partial positive charge at physiological pH) in addition to lipids according to formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc).Cationic and / or ionic lipids include 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethaneamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethaneamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), and 1,2-dilinoleyloxy-N,N-dimethicone. Diaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), octatriacontan-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyl Xy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z Cationic lipids can be selected from a non-limiting group consisting of (12Z)-octadeca-9,12-diene-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)) and (2S)-2-({8-[(3β)-cholest-5-ene-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)). In addition to these, cationic lipids may also be lipids containing a cyclic amine group.

[0124] structural lipids Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may contain one or more structural lipids. Structural lipids may be selected from the group consisting of, but are not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brazicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or combinations thereof. In some embodiments, the structural lipid is [ka] That is the case.

[0125] Phospholipids Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may contain one or more phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids may be assembled into one or more lipid bilayers. Generally, a phospholipid may contain a phospholipid moiety and one or more fatty acid moieties. For example, a phospholipid can be expressed by formula (IV): [ka] It can be a lipid due to, in the formula, R p R represents the phospholipid portion. A and R Brepresents a fatty acid moiety that may be the same or different, or may not have unsaturated fatty acids. The phospholipid moiety may be selected from an unrestricted group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from an unrestricted group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-natural species, including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes, are also considered. For example, phospholipids can be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under suitable reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition upon exposure to an azide. Such reactions may be useful for functionalizing the lipid bilayer of lipid nanoparticles (e.g., empty or filled LNPs) to facilitate transmembrane or cell recognition, or for conjugating lipid nanoparticles (e.g., empty or filled LNPs) with useful components such as targeting or imaging moieties (e.g., dyes).

[0126] Phospholipids useful in the composition and method include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dipalmitoyl-sn-glycero-3 -Phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 dietherPC), 1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-difytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphotidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine The lipid nanoparticles can be selected from a non-limiting group consisting of (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidyethanolamine (phosphatidyethanolamine) (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof. In some embodiments, the lipid nanoparticles (e.g., empty LNPs or filled LNPs) include DSPC. In certain embodiments, the lipid nanoparticles (e.g., empty LNPs or filled LNPs) include DOPE. In some embodiments, lipid nanoparticles (e.g., empty or filled LNPs) include both DSPC and DOPE.

[0127] PEG lipids Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may contain one or more PEG or PEG-modified lipids. Such species may be alternatively referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids may be selected from a non-limiting group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DEG), PEG-modified dialkylglycerol, and mixtures thereof. For example, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids.

[0128] In certain embodiments, the PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0129] In certain embodiments, the PEG lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-distelglycerol (PEG-DSG), PEG-dipalmetrail, PEG-dioleil, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). For example, in some embodiments, the PEG lipid is PEG-DMG.

[0130] In a particular embodiment, the PEG lipid is expressed by formula (PL-I): [ka] The compound, It is either or a salt thereof, in the formula, R 3PL1 is -OR OPL1 And, R OPL1 is hydrogen, optionally substituted alkyl, or oxygen protecting group, r PL1 is an integer between 1 and 100 (including 1 and 100), L 1 This is C, which is optionally substituted. 1-10 It is an alkylene, and C is optionally substituted. 1-10 At least one methylene group of the alkylene is independently and optionally substituted with carbocyclylene, optionally substituted with heterocyclylene, optionally substituted with arylene, optionally substituted with heteroarylene, O, N(R) NPL1 ), S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R NPL1 ), NR NPL1 C(O)O, or NR NPL1 C(O)N(R NPL1 ) is replaced by D is a portion obtained by click chemistry, or a portion that can be cleaved under physiological conditions. m PL1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, A is the formula: [ka] It is, L 2 Each case is independently, combined, or optionally substituted C 1-6 It is an alkylene, and C is optionally substituted. 1-6 One methylene unit of an alkylene can be optionally O, N(R) NPL1 ), S, C(O), C(O)N(R NPL1), NR NPL1 C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R NPL1 ), NR NPL1 C(O)O, or NR NPL1 C(O)N(R NPL1 ) is replaced by R 2SL Each case is independently and arbitrarily substituted C 1-30 Alkyl, optionally substituted C 1-30 Alkenyl, or optionally substituted C 1-30 It is an alkynyl, and optionally R 2SL One or more methylene units are independently, optionally substituted with carbocyclylene, optionally substituted with heterocyclylene, optionally substituted with arylene, optionally substituted with heteroarylene, N(R NPL1 ), O, S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), NR NPL1 C(O)N(R NPL1 ), C(O)O, OC(O), OC(O)O, OC(O)N(R NPL1 ), NR NPL1 C(O)O, C(O)S, SC(O), C(=NR NPL1 ), C(=NR NPL1 )N(R NPL1 ), NR NPL1 C(=NR NPL1 ), NR NPL1 C(=NR NPL1 )N(R NPL1 ), C(S), C(S)N(R NPL1 ), NR NPL1 C(S), NR NPL1 C(S)N(R NPL1 ), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R NPL1 )S(O), S(O)N(R NPL1 ), N(R NPL1 )S(O)N(R NPL1 ), OS(O)N(R NPL1 ), N(R NPL1 )S(O)O, S(O)2, N(R NPL1)S(O)2, S(O)2N(R NPL1 ), N(R NPL1 )S(O)2N(R NPL1 ), OS(O)2N(R NPL1 ), or N(R NPL1 ) Replaced with S(O)2O, R NPL1 Each of these cases independently consists of a hydrogen atom, an optionally substituted alkyl group, or a nitrogen protecting group. Ring B is an arbitrarily substituted carbocyclyl, an arbitrarily substituted heterocyclyl, an arbitrarily substituted aryl, or an arbitrarily substituted heteroaryl. p SL It is either 1 or 2.

[0131] In a particular embodiment, the PEG lipid is of formula (PL-I-OH): [ka] It is a compound of or a salt thereof.

[0132] In a particular embodiment, the PEG lipid is of formula (PL-II-OH): [ka] A compound of, or a salt or isomer thereof, in the formula, R 3PEG is -OR O And, R O is hydrogen, C 1-6 Alkyl or oxygen protecting group, r PEG These are integers from 1 to 100. R 5PEG C 10-40 Alkyl, C 10-40 Alkenyl, or C 10-40 It is an alkynyl, and optionally R 5PEG One or more methylene groups are independently C 3-10 Carbocyclylene, 4-10 member heterocyclylene, C6-10 Arylene, 4-10 member heteroarylene, -N(R) NPEG )-, -O-, -S-, -C(O)-, -C(O)N(R NPEG )-, -NR NPEG C(O)-, -NR NPEG C(O)N(R NPEG )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R NPEG )-, -NR NPEG C(O)O-, -C(O)S-, -SC(O)-, -C(=NR NPEG )-, -C(=NR NPEG )N(R NPEG )-, -NR NPEG C(=NR NPEG )-, -NR NPEG C(=NR NPEG )N(R NPEG )-, -C(S)-, -C(S)N(R NPEG )-, -NR NPEG C(S)-, -NR NPEG C(S)N(R NPEG )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R NPEG )S(O)-, -S(O)N(R NPEG )-,-N(R NPEG )S(O)N(R NPEG )-,-OS(O)N(R NPEG )-,-N(R NPEG )S(O)O-, -S(O)2-, -N(R NPEG )S(O)2-, -S(O)2N(R NPEG )-,-N(R NPEG )S(O)2N(R NPEG )-,-OS(O)2N(R NPEG )-, or -N(R NPEG ) Replaced with S(O)2O-, R NPEG Each of these cases is independent of hydrogen, C 1-6 It is an alkyl group or a nitrogen protecting group.

[0133] In certain embodiments, for the PEG lipid of formula (PL-II-OH), r is an integer between 40 and 50. For example, r is selected from the group consisting of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50. For example, r is 45.

[0134] In a particular embodiment, the PEG lipid of formula (PL-II-OH) is R 5 C 17 It is alkyl.

[0135] In a particular embodiment, the PEG lipid is expressed by formula (PL-II): [ka] It is a compound of which, in the formula, r PEG This is an integer between 1 and 100.

[0136] In certain embodiments, the PEG lipid is a compound of formula (PEG-1): [ka] That is the case.

[0137] In a particular embodiment, the PEG lipid is expressed by formula (PL-III): [ka] A compound of, or a salt or isomer thereof, in which, s PL1 This is an integer between 1 and 100.

[0138] In a particular embodiment, the PEG lipid is given by the following formula: [ka] It is a compound of [the compound].

[0139] In certain embodiments, the formulas (PL-I), (PL-I-OH), (PL-II), (PL-II-OH), (PL-III), and PEG are used. 2k By incorporating one of the lipids, either DMG or PEG-1, into a nanoparticle formulation, the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulation can be improved. For example, by incorporating one of the lipids, either (PL-II-OH), (PL-IIa-OH), (PL-II), or PEG-1, into a nanoparticle formulation, the accelerated blood clearance (ABC) effect can be reduced.

[0140] Adjuvant In some embodiments, lipid nanoparticles (e.g., empty LNPs or filled LNPs) comprising one or more lipids described herein may further comprise one or more adjuvants, e.g., glucopyranosyllipid adjuvants (GLAs), CpG oligodeoxynucleotides (e.g., class A or B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0141] Treatment drugs Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may comprise one or more therapeutic and / or prophylactic agents. Disclosures include methods for delivering therapeutic and / or prophylactic agents to mammalian cells or organs, for generating a polypeptide of interest in mammalian cells, and for treating a disease or disorder in a mammal requiring treatment of the disease or disorder, comprising administering lipid nanoparticles (e.g., empty LNPs or filled LNPs) containing the therapeutic and / or prophylactic agent to a mammal and / or contacting mammalian cells with lipid nanoparticles (e.g., empty LNPs or filled LNPs) containing the therapeutic and / or prophylactic agent.

[0142] Therapeutic and / or prophylactic agents contain biologically active substances and are alternatively referred to as “active agents.” Once delivered to a cell or organ, a therapeutic and / or prophylactic agent may be a substance that brings about a desirable change in the cell, organ, or other body tissue or system. Such a species may be useful in treating one or more diseases, disorders, or conditions. In some embodiments, therapeutic and / or prophylactic agents are small molecule drugs useful in treating specific diseases, disorders, or conditions.

[0143] In some embodiments, therapeutic and / or prophylactic agents are vaccines, compounds (e.g., polynucleotides or nucleic acid molecules encoding proteins or polypeptides or peptides, or proteins or polypeptides or proteins) that induce an immune response, and / or other therapeutic and / or prophylactic agents. Vaccines include compounds and preparations capable of providing immunity against one or more conditions associated with an infection, and may include mRNA encoding antigens and / or epitopes derived from the infection. Vaccines also include compounds and preparations that induce an immune response against cancer cells, and may include mRNA encoding antigens, epitopes, and / or neoepitopes derived from tumor cells. In some embodiments, vaccines and / or compounds capable of inducing an immune response are administered intramuscularly via the compositions of the disclosure.

[0144] In other embodiments, the therapeutic and / or prophylactic agent is a protein, for example, a protein required to enhance or replace a naturally occurring protein of interest. Such a protein or polypeptide may be naturally occurring or may be modified, for example, to increase its half-life, using methods known in the art. Exemplary proteins are intracellular, transmembrane, or secretory proteins.

[0145] Polynucleotides and nucleic acids In some embodiments, therapeutic agents are drugs that enhance (i.e., increase, stimulate, or upregulate) protein expression. Non-limiting examples of the types of therapeutic agents that can be used to enhance protein expression include RNA, mRNA, dsRNA, CRISPR / Cas9 technology, ssDNA, and DNA (e.g., expression vectors). Drugs that upregulate protein expression may upregulate the expression of naturally occurring or unnaturally occurring proteins (e.g., chimeric proteins modified to improve half-life, or those involving desired amino acid changes). Exemplary proteins include intracellular, transmembrane, or secreted proteins, peptides, or polypeptides.

[0146] In some embodiments, the therapeutic agent is a DNA therapeutic agent. The DNA molecule may be double-stranded DNA, single-stranded DNA (ssDNA), or partially double-stranded DNA, i.e., a molecule having a double-stranded portion and a single-stranded portion. In some cases, the DNA molecule may be triple-stranded or partially triple-stranded, i.e., having a triple-stranded portion and a double-stranded portion. The DNA molecule may be a circular DNA molecule or a linear DNA molecule.

[0147] DNA therapeutics can be DNA molecules capable of transcribing genes into cells, for example, DNA molecules that encode and express transcripts. In other embodiments, the DNA molecule is a synthetic molecule, such as a synthetic DNA molecule produced in vitro. In some embodiments, the DNA molecule is a recombinant molecule. Non-limiting exemplary DNA therapeutics include plasmid expression vectors and viral expression vectors.

[0148] The DNA therapeutics described herein, such as DNA vectors, may have a variety of different characteristics. The DNA therapeutics described herein, such as DNA vectors, may include non-coding DNA sequences. For example, the DNA sequence may include at least one regulatory element of a gene, such as a promoter, enhancer, termination element, polyadenylation signal element, splicing signal element, etc. In some embodiments, the non-coding DNA sequence is an intron. In some embodiments, the non-coding DNA sequence is a transposon. In some embodiments, the DNA sequence described herein may have a non-coding DNA sequence that is manipulably bound to a gene that is transcriptionally active. In other embodiments, the DNA sequence described herein may have a non-coding DNA sequence that is not bound to a gene, i.e., the non-coding DNA does not regulate a gene on the DNA sequence.

[0149] In some embodiments, in the LNPs filled with disclosure, one or more therapeutic and / or prophylactic agents are nucleic acids. In some embodiments, one or more therapeutic and / or prophylactic agents are selected from the group consisting of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).

[0150] For example, in some embodiments, when the therapeutic and / or prophylactic agent is DNA, the DNA is selected from the group consisting of double-stranded DNA, single-stranded DNA (ssDNA), partially double-stranded DNA, triple-stranded DNA, and partially triple-stranded DNA. In some embodiments, the DNA is selected from the group consisting of circular DNA, linear DNA, and mixtures thereof.

[0151] In some embodiments, in the LNP filled with disclosure, one or more therapeutic and / or prophylactic agents are selected from the group consisting of plasmid expression vectors, viral expression vectors, and mixtures thereof.

[0152] For example, in some embodiments, when the therapeutic and / or prophylactic agent is RNA, the RNA is selected from the group consisting of single-stranded RNA, double-stranded RNA (dsRNA), partially double-stranded RNA, and mixtures thereof. In some embodiments, the RNA is selected from the group consisting of circular RNA, linear RNA, and mixtures thereof.

[0153] For example, in some embodiments, when the therapeutic and / or prophylactic agent is RNA, the RNA is selected from the group consisting of short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecules, microRNA (miRNA), antagomyl, antisense RNA, ribozymes, dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), loc nucleic acid (LNA), and CRISPR / Cas9 technology, as well as mixtures thereof.

[0154] For example, in some embodiments, when the therapeutic and / or prophylactic agent is RNA, the RNA is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0155] In some embodiments, one or more therapeutic and / or prophylactic agents are mRNA. In some embodiments, one or more therapeutic and / or prophylactic agents are modified mRNA (mmRNA).

[0156] In some embodiments, one or more therapeutic and / or prophylactic agents are mRNAs incorporating microRNA binding sites (miR binding sites). Furthermore, in some embodiments, the mRNA comprises one or more of the following: a stem-loop, a strand termination nucleoside, a poly(A) sequence, a polyadenylation signal, and / or a 5' cap structure.

[0157] mRNA can be naturally occurring or unnatural mRNA. mRNA may contain one or more modified nucleic acid bases, nucleosides, or nucleotides as described below, in which case it may be referred to as “modified mRNA” or “mmRNA”. As used herein, “nucleoside” is defined as a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred herein as “nucleic acid base”). As used herein, “nucleotide” is defined as a nucleoside containing a phosphate group.

[0158] mRNA may contain a 5′ untranslated region (5′-UTR), a 3′ untranslated region (3′-UTR), and / or a coding region (e.g., an open reading frame). mRNA may contain any suitable number of base pairs, including tens (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100), hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900), or thousands (e.g., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000) base pairs. Any number (e.g., all, some, or none) of nucleic acid bases, nucleosides, or nucleotides may be analogues of canonical species, substitutions, modifications, or other unnaturally occurring ones. In certain embodiments, all types of a particular nucleic acid base may be modified. In some embodiments, all uracil or uridine is modified. When all nucleic acid bases, nucleosides, or nucleotides, for example, all uracil or uridine are modified, mRNA may be referred to, for example, as "fully modified" uracil or uridine.

[0159] In some embodiments, the mRNA described herein may include a 5' cap structure, a chain termination nucleotide, optionally a Kozak sequence (also known as a Kozak consensus sequence), a stem-loop, a poly(A) sequence, and / or a polyadenylation signal.

[0160] A 5' cap structure or cap species is a compound containing two nucleoside moieties joined by a linker, and can be selected from naturally occurring caps, unnaturally occurring caps or cap analogs, or anti-reversal cap analogs (ARCA). A CAP species may contain one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap may contain a guanine nucleotide and a guanine (G) nucleotide methylated at position 7, joined by a triphosphate bond at position 5', such as m7G(5′)ppp(5′)G, commonly written as m7GpppG. A cap species may also be an anti-reversal cap analog. A non-restrictive enumeration of possible cap types includes m7GpppG, m7Gpppm7G, m73′dGpppG, m27,O3′GpppG, m27,O3′GppppG, m27,O2′GppppG, m7Gpppm7G, m73′dGpppG, m27,O3′GpppG, m27,O3′GppppG, and m27,O2′GppppG.

[0161] mRNA may contain, or in addition to, a chain termination nucleoside. For example, a chain termination nucleoside may be a nucleoside deoxygenated at the 2' and / or 3' positions of the sugar group. Such species may include 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, and 2',3'-dideoxynucleosides such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, and 2',3'-dideoxythymine. In some embodiments, the incorporation of a chain termination nucleotide into mRNA, for example at the 3' end, may result in mRNA stabilization.

[0162] mRNA may, in addition to or instead of, contain stem loops, such as histone stem loops. Stem loops may contain two, three, four, five, six, seven, eight, or more nucleotide base pairs. For example, a stem loop may contain four, five, six, seven, or eight nucleotide base pairs. Stem loops may be located in any region of mRNA. For example, a stem loop may be located before or after an untranslated region (5' or 3' untranslated region), a coding region, a polyA sequence, or a tail. In some embodiments, stem loops may influence one or more functions of mRNA, such as translation initiation, translation efficiency, and / or transcription termination.

[0163] mRNA may, instead or in addition, contain a poly(A) sequence and / or a polyadenylation signal. The poly(A) sequence may consist entirely or largely of adenine nucleotides or their analogs or derivatives. The poly(A) sequence may also contain stabilizing nucleotides or analogs. For example, the poly(A) sequence may contain deoxythymidine, e.g., inverted (or reverse-bound) deoxythymidine (dT), as a stabilizing nucleotide or analog. Details regarding the use of inverted dT and other stabilizing poly(A) sequence modifications can be found, for example, in WO2017 / 049275A2, the contents of which are incorporated herein by reference. The poly(A) sequence may be a tail located adjacent to the 3' untranslated region of mRNA. In some embodiments, the poly(A) sequence may affect the nuclear export, translation, and / or stability of mRNA.

[0164] mRNA may, in addition to or instead, include a microRNA binding site. MicroRNA binding sites (or miR binding sites) can be used to regulate mRNA expression in various tissue or cell types. In exemplary embodiments, the miR binding site is manipulated to the 3'UTR sequence of mRNA to regulate, for example, enhance, the degradation of mRNA in cells or tissues expressing a homologous miR. Such regulation is useful for regulating or controlling "out-of-target" expression of ir mRNA, i.e., in vivo expression in unwanted cells or tissues. Further details regarding the use of miR binding sites can be found, for example, in WO2017 / 062513A2, the contents of which are incorporated herein by reference.

[0165] In some embodiments, the mRNA is a bicistronic mRNA comprising a first coding region and a second coding region, and having an intervening sequence containing an internal ribosome entry site (IRES) sequence that enables internal translation initiation between the first and second coding regions, or having an intervening sequence that encodes a self-cleaving peptide such as a 2A peptide. IRES sequences and 2A peptides are typically used to enhance the expression of multiple proteins derived from the same vector. For example, various IRES sequences, including encephalomyositis virus IRESs, are known, available, and can be used in the art.

[0166] In some embodiments, the mRNA disclosed comprises one or more modified nucleic acid bases, nucleosides, or nucleotides (referred to as “modified mRNA” or “mmRNA”). In some embodiments, the modified mRNA may have useful properties compared to the reference unmodified mRNA, including improved stability, intracellular retention, improved translation, and / or virtually no induction of the innate immune response in the cell into which the mRNA is introduced. Thus, the use of modified mRNA may improve the efficiency of protein synthesis, intracellular retention of the nucleic acid, and reduced immunogenicity.

[0167] In some embodiments, the mRNA contains one or more (e.g., 1, 2, 3, or 4) different modified nucleic acid bases, nucleosides, or nucleotides. In some embodiments, the mRNA contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified nucleic acid bases, nucleosides, or nucleotides. In some embodiments, the modified mRNA may reduce the degradation of the cell into which the mRNA is introduced compared to the corresponding unmodified mRNA.

[0168] In some embodiments, the modified nucleic acid base is modified uracil. Exemplary nucleic acid bases and nucleosides having modified uracil include pseudouridine (ψ), pyridine-4-onyribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5 - Methoxyuridine (mo5U), Uridine 5-oxyacetic acid (cmo5U), Uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyluridine (cm5U), 1-carboxymethyl-pseudolidine, 5-carboxyhydroxymethyluridine (chm5U), 5-carboxyhydroxymethyluridine methyl ester (mchm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-aminomethyl-2-thiouridine Auridine (nm5s2U), 5-methylaminomethyluridine (mnm5U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-carbamoylmethyluridine (ncm5U), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-propynyluridine, 1-propynylpsuduridine, 5-taurinomethyluridine (τm 5U), 1-taurinomethyl-pseuduridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseuduridine, 5-methyl-uridine (m5U, i.e., having the nucleic acid base deoxythymine), 1-methyl-pseuduridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseuduridine (m1s4ψ), 4-thio-1-methyl-pseuduridine, 3-methyl-pseuduridine (m3ψ), 2-thio-1-methyl-pseuduridine,1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine (D), dihydropseuduridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseuduridine, 2-methoxy-uridine, 2-methoxy-4-thiouridine, 4-methoxy-pseuduridine, 4-meth Xy-2-thio-pseuduridine, N1-methyl-pseuduridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseuduridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2 '-O-methyluridine (Um), 5,2'-O-dimethyluridine (m5Um), 2'-O-methyl-pseudridine (ψm), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine This includes din (cmnm5Um), 3,2'-O-dimethyluridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2'-F-ala-uridine, 2'-F-uridine, 2'-OH-ala-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine).

[0169] In some embodiments, the modified nucleic acid base is modified cytosine. Exemplary nucleic acid bases and nucleosides having modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methylcytidine (m3C), N4-acetylcytidine (ac4C), 5-formylcytidine (f5C), N4-methylcytidine (m4C), 5-methylcytidine (m5C), 5-halocytidine (e.g., 5-iodocytidine), and 5-hydrocytidine. Xymethylcytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolocytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine (s2C), 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine Zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thiocytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl This includes thyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-ala-cytidine, 2'-F-cytidine, and 2'-OH-ala-cytidine.

[0170] In some embodiments, the modified nucleic acid base is a modified adenine.Exemplary nucleic acid bases and nucleosides having modified adenine include α-thio-adenosine, 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine Nopurine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonine N6-carbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenosine, 2-methoxy-adenosine, α-methyl This includes OH-adenosine, 2'-O-methyladenosine (Am), N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m62Am), 1,2'-O-dimethyladenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methylpurine, 1-thioadenosine, 8-azidoadenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0171] In some embodiments, the modified nucleic acid base is modified guanine. Exemplary nucleic acid bases and nucleosides having modified guanosine include α-thio-guanosine, inosine (I), 1-methyl-inosine (m1I), waiosine (imG), methylwaiosine (mimG), 4-demethylwaiosine (imG-14), isowyosine (imG2), wibutosine (yW), peroxywibutosine (o2yW), hydroxywibutosine (OhyW), and low-modified hydroxywibutosine (OhyW) *), 7-deaza-guanosine, quasin (Q), epoxyquasin (oQ), galactosylquasin (galQ), mannosylquasin (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), alkaeosin (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine , 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2 ,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thioguanosine, N2,N2-dimethyl-6-thioguanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), It contains 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ala-guanosine, and 2'-F-guanosine.

[0172] In some embodiments, the disclosed mRNA comprises one or more combinations of the aforementioned modified nucleic acid bases (for example, a combination of two, three, or four of the aforementioned modified nucleic acid bases).

[0173] In some embodiments, the modified nucleic acid bases are pseudouridine (ψ), N1-methylpseudridine (m1ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, or 2'-O-methyluridine. In some embodiments, the mRNA of the disclosure comprises one or more combinations of the aforementioned modified nucleic acid bases (e.g., combinations of two, three, or four of the aforementioned modified nucleic acid bases). In some embodiments, the modified nucleic acid base is N1-methylpseudridine (m1ψ), and the mRNA of disclosure is completely modified with N1-methylpseudridine (m1ψ). In some embodiments, N1-methylpseudridine (m1ψ) corresponds to 75-100% of the uracil in the mRNA. In some embodiments, N1-methylpseudridine (m1ψ) corresponds to 100% of the uracil in the mRNA.

[0174] In some embodiments, the modified nucleic acid base is modified cytosine. Exemplary nucleic acid bases and nucleosides having modified cytosine include N4-acetylcytidine (ac4C), 5-methylcytidine (m5C), 5-halocytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine (hm5C), 1-methylpseudoisocytidine, 2-thiocytidine (s2C), and 2-thio-5-methylcytidine. In some embodiments, the mRNA of the disclosure includes one or more combinations of the aforementioned modified nucleic acid bases (e.g., combinations of two, three, or four of the aforementioned modified nucleic acid bases).

[0175] In some embodiments, the modified nucleic acid base is a modified adenine. Exemplary nucleic acid bases and nucleosides having a modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenosine (m2A), and N6-methyl-adenosine (m6A). In some embodiments, the mRNA of the disclosure includes one or more combinations of the aforementioned modified nucleic acid bases (e.g., combinations of two, three, or four of the aforementioned modified nucleic acid bases).

[0176] In some embodiments, the modified nucleic acid base is a modified guanine. Exemplary nucleic acid bases and nucleosides having modified guanines include inosine (I), 1-methyl-inosine (m1I), waiosine (imG), methylwaiosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, and 7-methyl-8-oxo-guanosine. In some embodiments, the mRNA of the disclosure includes one or more combinations of the aforementioned modified nucleic acid bases (e.g., combinations of two, three, or four of the aforementioned modified nucleic acid bases).

[0177] In some embodiments, the modified nucleic acid bases are 1-methyl-pseudridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine, or α-thio-adenosine. In some embodiments, the mRNA of the disclosure comprises one or more combinations of the aforementioned modified nucleic acid bases (e.g., combinations of two, three, or four of the aforementioned modified nucleic acid bases).

[0178] In some embodiments, the mRNA contains pseudouridine (ψ). In some embodiments, the mRNA contains pseudouridine (ψ) and 5-methylcytidine (m5C). In some embodiments, the mRNA contains 1-methylpseudridine (m1ψ). In some embodiments, the mRNA contains 1-methylpseudridine (m1ψ) and 5-methylcytidine (m5C). In some embodiments, the mRNA contains 2-thiouridine (s2U). In some embodiments, the mRNA contains 2-thiouridine and 5-methylcytidine (m5C). In some embodiments, the mRNA contains 5-methoxyuridine (mo5U). In some embodiments, the mRNA contains 5-methoxyuridine (mo5U) and 5-methylcytidine (m5C). In some embodiments, the mRNA contains 2'-O-methyluridine. In some embodiments, the mRNA comprises 2'-O-methyluridine and 5-methylcytidine (m5C). In some embodiments, the mRNA comprises N6-methyladenosine (m6A). In some embodiments, the mRNA comprises N6-methyladenosine (m6A) and 5-methylcytidine (m5C).

[0179] In certain embodiments, the mRNA of the disclosure is uniformly modified for a specific modification (i.e., completely modified, modified throughout the entire sequence). For example, the mRNA may be uniformly modified with N1-methylpseudridine (m1ψ) or 5-methylcytidine (m5C), meaning that all uridine or all cytosine nucleosides in the mRNA sequence are replaced with N1-methylpseudridine (m1ψ) or 5-methylcytidine (m5C). Similarly, the mRNA of the disclosure for any type of nucleoside residue present in the sequence may be uniformly modified by substitution with modifying residues such as those described above.

[0180] In some embodiments, the mRNA disclosed may have modifications to its coding region (e.g., the open reading frame encoding the polypeptide). In other embodiments, the mRNA may have modifications to regions other than the coding region. For example, in some embodiments, a 5'-UTR and / or 3'-UTR are provided, either or both of which may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the coding region.

[0181] The disclosed mMRNAs may include combinations of modifications to the binding between sugars, nucleic acid bases, and / or nucleosides. These combinations may include any one or more of the modifications described herein.

[0182] When single modifications are listed, the listed nucleoside or nucleotide represents 100% of its modified A, U, G, or C nucleotide or nucleoside. When percentages are listed, these represent the percentage of that particular A, U, G, or C triphosphate nucleic acid base out of the total amount of A, U, G, or C triphosphates present. For example, the combination: 25% 5-aminoallyl-CTP + 75% CTP / 25% 5-methoxy-UTP + 75% UTP refers to a polynucleotide where 25% of the cytosine triphosphates are 5-aminoallyl-CTP, 75% of the cytosine is CTP, 25% of the uracil is 5-methoxy-UTP, and 75% of the uracil is UTP. If modified UTPs are not listed, naturally occurring ATP, UTP, GTP, and / or CTP are used for 100% of the nucleotide sites found in the polynucleotide. In this example, all GTP and ATP nucleotides remain unmodified.

[0183] The mRNA or regions of the disclosed herein can be codon-optimized. Methods for codon optimization are known in the art and may be useful for a variety of purposes, namely matching codon frequencies in a host organism to ensure proper folding; biasing G / C content to increase mRNA stability or reduce secondary structures; minimizing tandem repeat codons or base sequences that may impair gene construction or expression; customizing transcriptional and translational regulatory regions; inserting or removing protein transport sequences; removing / adding post-translational modification sites (e.g., glycosylation sites) in encoded proteins; adding, removing, or shuffling protein domains; inserting or deleting restriction sites; modifying ribosome binding sites and mRNA degradation sites; adjusting translation rates to allow various domains of a protein to fold properly; or reducing or eliminating problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include services and / or proprietary methods from GeneArt (Life Technologies) and DNA2.0 (Menlo Park, CA). In some embodiments, mRNA sequences are optimized using optimization algorithms, for example, to optimize expression in mammalian cells or to improve mRNA stability.

[0184] In certain embodiments, the Disclosure includes polynucleotides having sequence identity with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of any of the polynucleotide sequences described herein.

[0185] The mRNAs of this disclosure may be produced by means available in the art, including, but not limited to, in vitro transcription (IVT) and synthesis methods. Enzymatic (IVT), solid-phase, liquid-phase, combined synthesis methods, micro-region synthesis, and ligation methods may be utilized. In some embodiments, the mRNAs are produced using IVT enzymatic synthesis methods. Accordingly, this disclosure also includes polynucleotides, such as DNA, constructs, and vectors, which may be used to transcribe the mRNAs described herein in vitro.

[0186] Unnaturally modified nucleic acid bases can be introduced into polynucleotides, such as mRNA, during or after synthesis. In certain embodiments, the modifications may be located on nucleoside bonds, purine or pyrimidine bases, or sugars. In certain embodiments, the modifications may be introduced at the ends of the polynucleotide chain or at any other location within the polynucleotide chain by chemosynthesis or by polymerase enzymes.

[0187] Polynucleotides or regions thereof can be conjugated with different functional parts, such as targets or delivery agents, fluorescent labels, liquids, or nanoparticles, using either enzymatic or chemical ligation methods.

[0188] Therapeutic drugs to reduce protein expression In some embodiments, therapeutic agents are those that reduce (i.e., reduce, inhibit, or downregulate) protein expression. Non-limiting examples of the types of therapeutic agents that may be used to reduce protein expression include microRNA binding sites (miR binding sites), microRNAs (miRNAs), antagomils, small (short) interfering RNAs (siRNAs) (including shortmer and dicer substrate RNAs), RNA interference (RNAi) molecules, antisense RNAs, ribozymes, small hairpin RNAs (shRNAs), lock nucleic acids (LNAs), and mRNAs incorporating CRISPR / Cas9 technology.

[0189] Peptide / polypeptide therapeutics In some embodiments, the therapeutic agent is a peptide therapeutic agent. In some embodiments, the therapeutic agent is a polypeptide therapeutic agent.

[0190] In some embodiments, the peptide or polypeptide is of natural origin, for example, isolated from a natural source. In other embodiments, the peptide or polypeptide is a synthetic molecule, for example, a synthetic peptide or polypeptide produced in vitro. In some embodiments, the peptide or polypeptide is a recombinant molecule. In some embodiments, the peptide or polypeptide is a chimeric molecule. In some embodiments, the peptide or polypeptide is a fusion molecule. In some embodiments, the peptide or polypeptide therapeutic agent of the composition is a naturally occurring peptide or polypeptide. In some embodiments, the peptide or polypeptide therapeutic agent of the composition is a modified version of a naturally occurring peptide or polypeptide (for example, containing less than 3, less than 5, less than 10, less than 15, less than 20, or less than 25 amino substitutions, deletions, or additions compared to its wild-type naturally occurring peptide or polypeptide counterpart).

[0191] In some embodiments, in the LNPs filled with disclosure, one or more therapeutic and / or prophylactic agents are polynucleotides or polypeptides.

[0192] Other ingredients Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may contain one or more components in addition to those described in the preceding section. For example, lipid nanoparticles (e.g., empty LNPs or filled LNPs) may contain one or more small hydrophobic molecules such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0193] Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may also contain one or more permeability-enhancing molecules, carbohydrates, polymers, surface modifiers, or other components. Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0194] Polymers may be included and / or used to encapsulate or partially encapsulate nanoparticle compositions. The polymers may be biodegradable and / or biocompatible. The polymers may be selected from, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polyethylacrylates, polyacrylonitriles, and polyarylates. For example, polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-glycolic acid) (PLGA), poly(L-lactic acid-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,Polyalkylenes such as L-lactide, polyalkylcyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyanhydride, poly(orthoester), poly(esteramide), polyamide, poly(ester ether), polycarbonate, polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxide (PEO), polyalkylene refractates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ether, polyvinyl esters such as poly(vinyl acetate), polyvinyl halide such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxane, polystyrene (PS), polyurethane, derivatized cellulose such as alkylcellulose, hydroxyalkylcellulose, cellulose ether, cellulose ester, nitrocellulose, hydroxypropylcellulose, carboxymethylcellulose Lurose, poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), and copolymers thereof. Examples include polymers of acrylic acid such as mixtures, polydioxanones and their copolymers, polyhydroxyalkanoates, polypropylene fumarates, polyoxymethylene, poloxamers, poloxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactidoco-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycols.

[0195] Examples of surface modifiers include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelan, papain, clerodendrum, bromexin, carbocysteine, eprazinon, mesna, ambroxol, sobrelol, domiodol, letostein, stepronin, thioproline, gellin, thymosine, β4, dorunase, alpha, neltenexin, and erdosteine), and DNases (e.g., rhDNase). Surface modifiers may be placed within and / or on the surface of lipid nanoparticles (e.g., empty LNPs or filled LNPs) (e.g., by coating, adsorption, covalent bonding, or other processes).

[0196] Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may also contain one or more functionalized lipids. For example, lipids may be functionalized with alkyne groups that can undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. In particular, lipid bilayers may be functionalized in this manner with one or more groups useful for promoting transmembrane delivery, cell recognition, or imaging. The surface of lipid nanoparticles (e.g., empty LNPs or filled LNPs) may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful for target cell delivery, imaging, and transmembrane delivery are well known in the art.

[0197] In addition to these components, lipid nanoparticles (e.g., empty LNPs or filled LNPs) may contain any substance useful in a pharmaceutical composition. For example, lipid nanoparticles (e.g., empty LNPs or filled LNPs) may contain, but are not limited to, one or more pharmaceutically acceptable excipients or adjuncts such as solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, fluids, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and other kinds. They may also contain excipients such as waxes, butters, colorants, coatings, fragrances, and aromatics.

[0198] Examples of diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, corn starch, powdered sugar, and / or combinations thereof. Granulating agents and dispersants may be selected from a non-limiting list consisting of potato starch, corn starch, tapioca starch, sodium glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregeratinized starch (starch 1500), microcrystalline starch, water-insoluble starch, carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium laudylic acid sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0199] The surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylcellulose, cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose, methylcellulose), and polyoxyethylcellulose. Lensorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitiate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]), polyoxyethylene esters (e.g., polyoxyethylene esters) Lenmonostearate [MYRJ(registered trademark) 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL(registered trademark), sucrose fatty acid ester, polyethylene glycol fatty acid ester (e.g., CREMOPHOR(registered trademark)), polyoxyethylene ether (e.g., polyoxyethylene lauryl ether [BRIJ(registered trademark) 30]), poly(vinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate,Potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC® F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium doxate, and / or combinations thereof may be included.

[0200] The binder may be starch (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), natural and synthetic gums (e.g., acacia, sodium alginate, Irish moss extract, panwar gum, ghatti gum, isapol husk mucus, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate (VEEGUM®, and larch arapinogalactan), alginate, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylate, wax, water, and combinations thereof, or any suitable binder.

[0201] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, α-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl formate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, and / or thimerosal. Examples of antifungal agents include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenol compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, dystoxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE®, KATHON®, and / or EUXYL®.

[0202] Examples of buffers include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphate, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, aminosulfonic acid buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof. The lubricant may be selected from a non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0203] Examples of oils include almond, apricot kernel, avocado, babassu, bergamot, blackcurrant seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grapeseed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, and Litsea cubeba. Examples of ingredients include, but are not limited to, cubeba, macadamia nuts, mallow, mango seeds, meadowfoam seeds, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savory, sea buckthorn, sesame, shea butter, silicone, soy, sunflower, tea tree, thistle, camellia, vetiver, walnut, and wheat germ oil, as well as butyl stearate, tricaprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, simethicone, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0204] formulation Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may comprise a lipid component and one or more additional components, such as therapeutic and / or prophylactic agents. Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may be designed for one or more specific applications or targets. The elements of lipid nanoparticles (e.g., empty LNPs or filled LNPs) may be selected based on a specific application or target and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more elements. Similarly, a particular formulation of a nanoparticle composition may be selected for a specific application or target, for example, depending on the efficacy and toxicity of a particular combination of elements.

[0205] The lipid components of the nanoparticle composition may include, for example, lipids according to formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), phospholipids (unsaturated lipids, e.g., DOPE or DSPC), PEG lipids, and structural lipids. The elements of the lipid components may be provided in specific fractions.

[0206] In some embodiments, the lipid component of the nanoparticle composition includes lipids according to formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), phospholipids, PEG lipids, and structural lipids. In a particular embodiment, the lipid component of the nanoparticle composition comprises about 30 mol% to about 60 mol% of a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), about 0 mol% to about 30 mol% of phospholipids, about 18.5 mol% to about 48.5 mol% of structural lipids, and about 0 mol% to about 10 mol% of PEG lipids, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises about 35 mol% to about 55 mol% of compounds of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), about 5 mol% to about 25 mol% of phospholipids, about 30 mol% to about 40 mol% of structural lipids, and about 0 mol% to about 10 mol% of PEG lipids. In certain embodiments, the lipid component comprises about 50 mol% of the compound, about 10 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In other specific embodiments, the lipid component comprises about 40 mol% of the compound, about 20 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In some embodiments, the phospholipids may be DOPE or DSPC. In other embodiments, the PEG lipids may be PEG-1 or PEG 2k- It may be DMG, and / or structural lipids may be cholesterol.

[0207] In some embodiments, empty lipid nanoparticles (empty LNPs) include compounds of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), phospholipids, structural lipids, and PEG lipids.

[0208] In some embodiments, the filled lipid nanoparticles (filled LNPs) comprise a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents.

[0209] In some embodiments, empty or filled LNPs contain about 40% to about 60% of a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc).

[0210] In some embodiments, empty or filled LNPs contain phospholipids in amounts ranging from about 0% to about 20%. For example, in some embodiments, empty or filled LNPs contain DSPCs in amounts ranging from about 0% to about 20%.

[0211] In some embodiments, empty or filled LNPs contain structural lipids in an amount of about 30% to about 50%. For example, in some embodiments, empty or filled LNPs contain cholesterol in an amount of about 30% to about 50%.

[0212] In some embodiments, empty or filled LNPs contain PEG lipids in amounts of about 0% to about 5%. For example, in some embodiments, empty or filled LNPs contain PEG-1 or PEG 2k -Contains approximately 0% to 5% DMG.

[0213] In some embodiments, an empty or filled LNP contains about 40 mol% to about 60 mol% of a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), about 0 mol% to about 20 mol% of phospholipids, about 30 mol% to about 50 mol% of structural lipids, and about 0 mol% to about 5 mol% of PEG lipids.

[0214] In some embodiments, an empty or filled LNP contains about 40 mol% to about 60 mol% of a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), about 0 mol% to about 20 mol% of DSPC, about 30 mol% to about 50 mol% of cholesterol, and about 0 mol% to about 5 mol% of PEG. 2k-Includes DMG. In some embodiments, empty or filled LNPs contain about 40 mol% to about 60 mol% of the compounds in Table 1, about 0 mol% to about 20 mol% of DSPC, about 30 mol% to about 50 mol% of cholesterol, and about 0 mol% to about 5 mol% of PEG. 2k -DMG and, including

[0215] In some embodiments, an empty or filled LNP contains about 40 mol% to about 60 mol% of a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), about 0 mol% to about 20 mol% of DSPC, about 30 mol% to about 50 mol% of cholesterol, and about 0 mol% to about 5 mol% of PEG-1. In some embodiments, an empty or filled LNP contains about 40 mol% to about 60 mol% of the compounds in Table 1, about 0 mol% to about 20 mol% of DSPC, about 30 mol% to about 50 mol% of cholesterol, and about 0 mol% to about 5 mol% of PEG-1.

[0216] In some embodiments, an empty or filled LNP comprises a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), along with a phospholipid, a structural lipid, and a PEG lipid, where the phospholipid is DSPC and the structural lipid is cholesterol. In some embodiments, an empty or filled LNP comprises a compound of Table 1, along with a phospholipid, a structural lipid, and a PEG lipid, where the phospholipid is DSPC and the structural lipid is cholesterol.

[0217] In some embodiments, an empty or filled LNP comprises a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), a phospholipid, a structural lipid, and a PEG lipid, wherein the structural lipid is cholesterol and the PEG lipid is PEG 2k -DMG. In some embodiments, an empty or filled LNP comprises the compounds of Table 1, a phospholipid, a structural lipid, and a PEG lipid, where the structural lipid is cholesterol and the PEG lipid is PEG 2k -DMG

[0218] In some embodiments, an empty or filled LNP comprises a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), along with a phospholipid, a structural lipid, and a PEG lipid, where the structural lipid is cholesterol and the PEG lipid is PEG-1. In some embodiments, an empty or filled LNP comprises a compound of Table 1, along with a phospholipid, a structural lipid, and a PEG lipid, where the structural lipid is cholesterol and the PEG lipid is PEG-1.

[0219] In some embodiments, an empty or filled LNP comprises a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), a phospholipid, a structural lipid, and a PEG lipid, wherein the phospholipid is DSPC and the PEG lipid is PEG2k -DMG. In some embodiments, an empty or filled LNP comprises the compounds of Table 1, a phospholipid, a structural lipid, and a PEG lipid, where the structural lipid is DSPC and the PEG lipid is PEG 2k -DMG

[0220] In some embodiments, an empty or filled LNP comprises a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), along with a phospholipid, a structural lipid, and a PEG lipid, where the phospholipid is DSPC and the PEG lipid is PEG-1. In some embodiments, an empty or filled LNP comprises a compound of Table 1, along with a phospholipid, a structural lipid, and a PEG lipid, where the phospholipid is DSPC and the PEG lipid is PEG-1.

[0221] In some embodiments, an empty or filled LNP comprises a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), a phospholipid, a structural lipid, and a PEG lipid, wherein the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG 2k -DMG. In some embodiments, an empty or filled LNP comprises the compounds of Table 1, a phospholipid, a structural lipid, and a PEG lipid, where the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG 2k -DMG

[0222] In some embodiments, an empty LNP or a filled LNP comprises a compound of formula (Ac), a phospholipid, a structural lipid, and a PEG lipid, where the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG 2k -DMG. In some embodiments, an empty or filled LNP comprises the compounds of Table 1, a phospholipid, a structural lipid, and a PEG lipid, where the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG 2k -DMG

[0223] In some embodiments, an empty or filled LNP comprises a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), a phospholipid, a structural lipid, and a PEG lipid, where the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG-1.

[0224] In some embodiments, an empty or filled LNP comprises a compound of formula (Ac), a phospholipid, a structural lipid, and a PEG lipid, where the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG-1. In some embodiments, an empty or filled LNP comprises a compound of Table 1, a phospholipid, a structural lipid, and a PEG lipid, where the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG-1.

[0225] Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may be designed for one or more specific applications or targets. For example, a nanoparticle composition may be designed to deliver therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems or groups thereof within the mammalian body. The physicochemical properties of lipid nanoparticles (e.g., empty LNPs or filled LNPs) may be modified to increase selectivity for specific bodily targets. For example, particle size may be adjusted based on the pore size of different organs. The therapeutic and / or prophylactic agents contained in a nanoparticle composition may also be selected based on a desired delivery target or multiple targets. For example, a therapeutic and / or prophylactic agent may be selected for a specific indication, condition, disease, or disorder, and / or for delivery to specific cells, tissues, organs, or systems or groups thereof (e.g., local or specific delivery). In certain embodiments, a nanoparticle composition may contain mRNA encoding a polypeptide of interest that can be translated in cells to produce the polypeptide of interest. Such a composition may be designed to be specifically delivered to a specific organ. In some embodiments, a composition may be designed to be specifically delivered to the mammalian liver.

[0226] The amount of therapeutic and / or prophylactic agents in a nanoparticle composition may depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition, as well as the properties of the therapeutic and / or prophylactic agents. For example, the amount of RNA useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and / or prophylactic agents and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some embodiments, the weight / weight ratio of lipid components to therapeutic and / or prophylactic agents in a nanoparticle composition may be approximately 5:1 to approximately 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the weight / weight ratio of lipid components to therapeutic and / or prophylactic agents may range from about 10:1 to about 40:1. In certain embodiments, the weight / weight ratio is about 20:1.

[0227] The amount of therapeutic and / or prophylactic agents in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0228] In some embodiments, the nanoparticle composition comprises one or more RNAs, one or more RNAs, and lipids, the amounts of which may be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. Generally, a lower N:P ratio is preferred. One or more RNAs, lipids, and their amounts may be selected to have an N:P ratio of about 2:1 to about 30:1, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio may be about 2:1 to about 8:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio could be approximately 5.0:1, 5.5:1, 5.67:1, 6.0:1, 6.5:1, or 7.0:1. For example, the N:P ratio could be approximately 5.67:1.

[0229] physical properties The characteristics of lipid nanoparticles (e.g., empty LNPs or filled LNPs) may depend on their composition. For example, lipid nanoparticles containing cholesterol as a structural lipid (e.g., empty LNPs or filled LNPs) may have different characteristics than lipid nanoparticles containing different structural lipids (e.g., empty LNPs or filled LNPs). Similarly, the characteristics of lipid nanoparticles (e.g., empty LNPs or filled LNPs) may depend on the absolute or relative amounts of their components. For example, lipid nanoparticles containing a higher mole fraction of phospholipids (e.g., empty LNPs or filled LNPs) may have different characteristics than lipid nanoparticles containing a lower mole fraction of phospholipids (e.g., empty LNPs or filled LNPs). The characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0230] Lipid nanoparticles (e.g., empty LNPs or filled LNPs) can be characterized by various methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the nanoparticle composition. Zeta potential may be measured using dynamic light scattering or potentiometric methods (e.g., potentiometric titration). Particle size may also be determined using dynamic light scattering. Furthermore, several properties of the nanoparticle composition, such as particle size, polydispersity index, and zeta potential, may be measured using instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK).

[0231] The average size of lipid nanoparticles (e.g., empty or filled LNPs) can range from tens of nanometers to hundreds of nanometers, as measured, for example, by dynamic light scattering (DLS). For example, the average size may range from approximately 40 nm to approximately 150 nm, for example, approximately 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of lipid nanoparticles (e.g., empty LNPs or filled LNPs) may be approximately 50 nm to 100 nm, approximately 50 nm to 90 nm, approximately 50 nm to 80 nm, approximately 50 nm to 70 nm, approximately 50 nm to 60 nm, approximately 60 nm to 100 nm, approximately 60 nm to 90 nm, approximately 60 nm to 80 nm, approximately 60 nm to 70 nm, approximately 70 nm to 150 nm, approximately 70 nm to 130 nm, approximately 70 nm to 100 nm, approximately 70 nm to 90 nm, approximately 70 nm to 80 nm, approximately 80 nm to 150 nm, approximately 80 nm to 130 nm, approximately 80 nm to 100 nm, approximately 80 nm to 90 nm, approximately 90 nm to 150 nm, approximately 90 nm to 130 nm, or approximately 90 nm to 100 nm. In certain embodiments, the average size of lipid nanoparticles (e.g., empty LNPs or filled LNPs) may be about 70 nm to about 130 nm, or about 70 nm to about 100 nm. In certain embodiments, the average size may be about 80 nm. In other embodiments, the average size may be about 100 nm. In other embodiments, the average size may be about 120 nm.

[0232] Lipid nanoparticles (e.g., empty LNPs or filled LNPs) can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of lipid nanoparticles (e.g., empty LNPs or filled LNPs). A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may have polydispersity indices ranging from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of lipid nanoparticles (e.g., empty LNPs or filled LNPs) can be about 0.10 to about 0.20.

[0233] The zeta potential of lipid nanoparticles (e.g., empty LNPs or filled LNPs) can be used to indicate the electrical potential of a composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Lipid nanoparticles (e.g., empty LNPs or filled LNPs) with relatively low positive or negative charges are generally preferred, as higher charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of lipid nanoparticles (e.g., empty LNPs or filled LNPs) is approximately -10mV to approximately +20mV, approximately -10mV to approximately +15mV, approximately -10mV to approximately +10mV, approximately -10mV to approximately +5mV, approximately -10mV to approximately 0mV, approximately -10mV to approximately -5mV, approximately -5mV to approximately +20mV, approximately The ranges could be -5mV to approximately +15mV, approximately -5mV to approximately +10mV, approximately -5mV to approximately +5mV, approximately -5mV to approximately 0mV, approximately 0mV to approximately +20mV, approximately 0mV to approximately +15mV, approximately 0mV to approximately +10mV, approximately 0mV to approximately +5mV, approximately +5mV to approximately +20mV, approximately +5mV to approximately +15mV, or approximately +5mV to approximately +10mV.

[0234] The efficiency of encapsulation of therapeutic and / or prophylactic agents describes the amount of therapeutic and / or prophylactic agent encapsulated or associated with lipid nanoparticles (e.g., empty or filled LNPs) after preparation, compared to the initial amount provided. The encapsulation efficiency is preferably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing lipid nanoparticles (e.g., empty or filled LNPs) before and after the degradation of the lipid nanoparticles (e.g., empty or filled LNPs) with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in the solution. In the lipid nanoparticles described herein (e.g., empty LNPs or filled LNPs), the encapsulation efficiency of therapeutic and / or prophylactic agents may be at least 50%, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency of therapeutic and / or prophylactic agents is between 80% and 100%.

[0235] Pharmaceutical composition Lipid nanoparticles (e.g., empty LNPs or filled LNPs) can be formulated whole or partially as a pharmaceutical composition. A pharmaceutical composition may comprise one or more lipid nanoparticles (e.g., empty LNPs or filled LNPs). In one embodiment, the pharmaceutical composition comprises a population of lipid nanoparticles (e.g., empty LNPs or filled LNPs). For example, a pharmaceutical composition may comprise one or more lipid nanoparticles (e.g., empty LNPs or filled LNPs) comprising one or more different therapeutic and / or prophylactic agents. A pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients or accessories, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and drugs can be found, for example, in Remington's The Science and Practice of Pharmacy, 21 st It is available in Edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and accessories may be used in any pharmaceutical composition unless any conventional excipient or accessory may be incompatible with one or more components of the nanoparticle composition. An excipient or accessory may be incompatible with the components of nanoparticles (e.g., empty LNPs or filled LNPs) if its combination with the components may result in any undesirable biological or other adverse effects.

[0236] In some embodiments, one or more excipients or accessories may constitute more than 50% of the total mass or volume of the pharmaceutical composition, including the nanoparticle composition. For example, one or more excipients or accessories may constitute 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical regulations. In some embodiments, pharmaceutically acceptable excipients are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipients are approved for human and veterinary use. In some embodiments, the excipients are approved by the U.S. Food and Drug Administration. In some embodiments, the excipients are pharmaceutical grade. In some embodiments, the excipients meet the standards of the United States Pharmacopeia (USP), European Pharmacopeia (EP), British Pharmacopeia, and / or International Pharmacopoeia.

[0237] The relative amounts of one or more lipid nanoparticles (e.g., empty or filled LNPs), one or more pharmaceutically acceptable excipients, and / or any additional components in a pharmaceutical composition according to this disclosure will vary depending on the uniqueness, size, and / or condition of the target being treated, and further depending on the route through which the composition is administered. For example, a pharmaceutical composition may contain 0.1% to 100% (by weight) of one or more lipid nanoparticles (e.g., empty or filled LNPs).

[0238] In certain embodiments, the disclosed lipid nanoparticles (e.g., empty LNPs or filled LNPs) and / or pharmaceutical compositions are stored refrigerated or frozen for storage and / or shipment (e.g., at temperatures below 4°C, about -150°C to about 0°C, or about -80°C to about 20°C, etc. (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C)). For example, a pharmaceutical composition containing any compound from formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), and (Bc) should be stored at approximately -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C. It is a solution that is refrigerated for shipping. In certain embodiments, the disclosure also involves storing lipid nanoparticles (e.g., empty or filled LNPs) and / or pharmaceutical compositions at temperatures of 4°C or less, such as about -150°C to about 0°C or about -80°C to about -20°C, e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C). The present invention relates to a method for increasing the stability of lipid nanoparticles (e.g., empty LNPs or filled LNPs) and / or pharmaceutical compositions containing any compound from formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), and (Bc).For example, the lipid nanoparticles (e.g., empty LNPs or filled LNPs) and / or pharmaceutical compositions disclosed herein are stable at a temperature of 4°C or less (e.g., about 4°C to -20°C) for at least about 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months, for example, at a temperature of 4°C or less (e.g., about 4°C to -20°C). In some embodiments, the formulations are stabilized at about 4°C for at least 4 weeks. In certain embodiments, the pharmaceutical composition disclosed comprises the lipid nanoparticles (e.g., empty LNPs or filled LNPs) disclosed herein and a pharmaceutically acceptable carrier selected from one or more of Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), physiological saline, PBS, and sucrose. In certain embodiments, the disclosed pharmaceutical composition has a pH value of about 7–8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or 7.5–8, or 7–7.8). For example, the disclosed pharmaceutical composition comprises lipid nanoparticles disclosed herein (e.g., empty LNPs or filled LNPs), Tris, physiological saline, and sucrose, and has a pH of about 7.5–8, suitable for storage and / or shipment at about -20°C. For example, the disclosed pharmaceutical composition comprises lipid nanoparticles disclosed herein (e.g., empty LNPs or filled LNPs) and PBS, and has a pH of about 7–7.8, suitable for storage and / or shipment at about 4°C or below. In the context of this disclosure, “stability,” “stabilized,” and “stable” refer to the resistance of the lipid nanoparticles (e.g., empty LNPs or filled LNPs) and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size changes, aggregation, encapsulation changes) when subjected to stresses such as shear force, freeze / thaw stress, under given conditions during manufacturing, preparation, transport, storage, and / or use.

[0239] In some embodiments, the pharmaceutical composition of the disclosure comprises empty or filled LNPs, cryoprotectants, buffers, or combinations thereof.

[0240] In some embodiments, the cryoprotectant comprises one or more cryoprotectants, each of which is independently a polyol (e.g., diol or triol such as propylene glycol (i.e., 1,2-propanediol), 1,3-propanediol, glycerol, (+ / -)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), non-detergent sulfobetaine (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), osmolite (e.g., L-proline or trimethylamine N-oxide dihydrate), polymer (e.g., polyethylene glycol 200 (PEG200), PEG400, PEG600, PEG1000, PEG 2k-DMG, PEG3350, PEG4000, PEG8000, PEG10000, PEG20000, polyethylene glycol monomethyl ether 550 (mPEG550), mPEG600, mPEG2000, mPEG3350, mPEG4000, mPEG5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K15), pentaerythritol propoxylate, or polypropylene glycol P400), organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), sugar (e.g., D-(+)-sucrose, D- The cryoprotectant comprises sorbitol, trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, or D-(+)-glucose monohydrate), or salts (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof), or any combination thereof. In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the cryoprotectant and / or excipient is sucrose. In some embodiments, the cryoprotectant comprises sodium acetate. In some embodiments, the cryoprotectant and / or excipient is sodium acetate. In some embodiments, the cryoprotectant comprises sucrose and sodium acetate.

[0241] In some embodiments, the buffer is selected from the group consisting of acetate buffer, citrate buffer, phosphate buffer, Tris buffer, and combinations thereof.

[0242] Pharmaceutical compositions comprising lipid nanoparticles (e.g., empty LNPs or filled LNPs) and / or one or more lipid nanoparticles (e.g., empty LNPs or filled LNPs) may be administered to any patient or subject, including patients or subjects who can benefit from the therapeutic effects provided by the delivery of a therapeutic and / or prophylactic agent to one or more specific cells, tissues, organs, or systems or groups thereof. The descriptions provided herein of pharmaceutical compositions comprising lipid nanoparticles (e.g., empty LNPs or filled LNPs) and lipid nanoparticles (e.g., empty LNPs or filled LNPs) primarily concern compositions suitable for administration to humans, but it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammals. Modifications of compositions suitable for administration to humans are well understood for producing compositions suitable for administration to various animals, and such modifications can be designed and / or carried out by a veterinary pharmacologist of ordinary knowledge in simple, ordinary experiments (if any). The subjects to whom the composition is intended to be administered include, but are not limited to, humans, other primates, and other mammals, such as commercially relevant mammals like cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats. The lipid nanoparticles of the subject may also be used in vitro and ex vivo.

[0243] Pharmaceutical compositions comprising one or more lipid nanoparticles (e.g., empty LNPs or filled LNPs) can be prepared by any method known or to be developed in the field of pharmacology. Generally, such preparation methods involve associating the active ingredient with excipients and / or one or more other accessories, and then, if desired or required, dividing, shaping, and / or packaging the product into desired single or multi-dose units.

[0244] The pharmaceutical compositions described herein may be prepared, packaged, and / or sold in bulk as a single unit dose and / or as multiple unit doses. As used herein, “unit dose” is a distinct amount of a pharmaceutical composition containing a predetermined amount of an active ingredient (e.g., lipid nanoparticles). The amount of the active ingredient is generally equal to the dose of the active ingredient administered to a subject and / or a convenient portion of such dose, for example, half or one-third of such dose.

[0245] Pharmaceutical compositions can be prepared in various forms suitable for various routes and methods of administration. For example, pharmaceutical compositions can be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable dosage forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), topical and / or transdermal dosage forms (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.

[0246] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms may include inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters, and mixtures thereof. Beyond inert diluents, oral compositions may include additional agents such as additional therapeutic and / or prophylactic agents, wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and / or fragrances. In certain embodiments for parenteral administration, the composition is mixed with a solubilizer, such as Cremophor®, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or a combination thereof.

[0247] Preparations for injection, such as sterile aqueous or oily suspensions for injection, can be formulated according to known techniques using suitable dispersants, wetting agents, and / or suspending agents. Sterile preparations for injection may be sterile solutions, suspensions, and / or emulsions in non-toxic, parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Sterile fixatives are conventionally used as solvents or suspension media. For this purpose, any bland fixative containing synthetic monoglycerides or diglycerides can be used. Fatty acids, such as oleic acid, can be used in the preparation of injections.

[0248] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter and / or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0249] To extend the effects of the active ingredient, it is often desirable to slow down its absorption from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material with low water solubility. The absorption rate of the drug then depends on its dissolution rate, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered drug forms is achieved by dissolving or suspending the drug in an oil vehicle. Depot formulations for injection are prepared by forming a microencapsulation matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydrous). Depot injection formulations are prepared by encapsulating the drug in liposomes or microemulsions compatible with body tissues.

[0250] Compositions for rectal or vaginal administration can typically be prepared by mixing the composition with a suitable non-irritating excipient, such as cocoa butter, polyethylene glycol, or suppository wax, which is solid at ambient temperature but liquid at body temperature, and thus melts in the rectal or vaginal cavity, releasing the active ingredient.

[0251] Solid dosage forms for oral administration include capsules, tablets, pills, films, powders, and granules. The active ingredient in such solid dosage forms is at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate, and / or fillers or bulking agents (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g., glycerol), and disintegrants (e.g., agar, calcium carbonate, potato or It is mixed with tapioca starch (tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarders (e.g., paraffin), absorption enhancers (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g., kaolin and bentonite clay, silicates), and lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate), as well as mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include buffers.

[0252] Similar types of solid compositions can be used as fillers in soft and rigid gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol. Solid dosage forms of tablets, sugar coatings, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical field. They may optionally contain opaque agents and may be compositions that optionally release only or preferentially the active ingredient(s) in a delayed manner at a specific portion of the intestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. Similar types of solid compositions can be used as fillers in soft and rigid gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol.

[0253] Dosage forms for topical and / or transdermal administration of the composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. Generally, the active ingredient is mixed under sterile conditions with pharmaceutically acceptable excipients and / or any necessary preservatives and / or buffers, as needed. In addition, this disclosure often envisions the use of transdermal patches, which have the additional advantage of providing controlled delivery of the compound to the body. Such dosage forms may be prepared, for example, by dissolving and / or distributing the compound in a suitable culture medium. Alternatively or additionally, the rate may be controlled by either providing a rate-controlled membrane and / or dispersing the compound in a polymer matrix and / or gel.

[0254] Suitable devices for intradermal delivery of the pharmaceutical compositions described herein include short-needle devices. Intradermal compositions may be administered by devices that limit the effective penetration length of the needle into the skin. Jet injection devices are preferred for delivering liquid compositions to the dermis via a liquid jet injector and / or via a needle that penetrates the stratum corneum and generates a jet that reaches the dermis. Ballistic delivery powder / particle delivery devices are preferred for accelerating powder-form vaccines through the outer layers of the skin to the dermis using compressed gas. Alternatively or additionally, conventional syringes may be used in the classic Mantot method of intradermal administration.

[0255] Formulations suitable for topical administration include, but are not limited to, liquid and / or semi-liquid preparations such as ointments, lotions, oil-in-water and / or water-in-oil emulsions (e.g., creams, ointments and / or pastes), and / or solutions and / or suspensions. A formulation suitable for topical administration may contain, for example, about 1% to about 10% (by weight) of the active ingredient, but the concentration of the active ingredient may be comparable to the solubility limit of the active ingredient in the solvent. A formulation for topical administration may further contain one or more of the additional ingredients described herein.

[0256] Pharmaceutical compositions may be prepared, packaged, and / or sold in formulations suitable for pulmonary administration via the buccal cavity. Such formulations may contain dry particles containing the active ingredient. Conveniently, such compositions are in the form of dry powder for administration using a device including a dry powder storage container in which a flow of propellant can be directed to disperse the powder, and / or using a self-propelled solvent / powder dispensing container such as a device containing the active ingredient dissolved and / or suspended in a low-boiling propellant in a sealed container. Dry powder compositions may contain a solid fine powder diluent such as sugar and are conveniently provided in unit dose form.

[0257] Low-boiling propellants generally include liquid propellants having a boiling point of less than 65°F at atmospheric pressure. Generally, the propellant may constitute 50% to 99.9% (by weight) of the composition, and the active ingredient may constitute 0.1% to 20% (by weight) of the composition. The propellant may further include additional components such as liquid nonionic and / or solid anionic surfactants and / or solid diluents (which may have a particle size similar to that of the particles containing the active ingredient).

[0258] Pharmaceutical compositions formulated for pulmonary delivery may provide the active ingredient in the form of droplets of solution and / or suspension. Such formulations may be prepared, packaged and / or sold as optionally sterile, aqueous and / or diluted alcoholic solutions and / or suspensions containing the active ingredient, and may be conveniently administered using any spray and / or spraying device. Such formulations may further contain one or more additional components, but are not limited to, flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants, and / or preservatives such as methyl hydroxybenzoate. The droplets delivered by this administration route may have an average diameter ranging from about 1 nm to about 200 nm.

[0259] Formulations described herein as useful for pulmonary delivery are useful for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration is a crude powder containing the active ingredient and having average particles of about 0.2 μm to 500 μm. Such formulations are administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passages from a container of powder held near the nose.

[0260] Formulations suitable for nasal administration may, for example, contain about 0.1% (weight / weight) and about 100% (weight / weight) of the active ingredient, and may also contain one or more additional ingredients described herein. Pharmaceutical compositions may be prepared, packaged, and / or sold in formulations suitable for buccal administration. Such formulations may, for example, be in the form of tablets and / or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (weight / weight) of the active ingredient, with the remainder being an orally soluble and / or degradable composition, and optionally one or more additional ingredients described herein. Alternatively, formulations suitable for buccal administration may include powders and / or aerosolized and / or sprayed solutions and / or suspensions containing the active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range of about 0.1 nm to about 200 nm, and may further contain one or more of any additional ingredients described herein.

[0261] Pharmaceutical compositions may be prepared, packaged, and / or sold in formulations suitable for ophthalmic administration. Such formulations may be in the form of eye drops comprising, for example, a 0.1 / 1.0% (weight / weight) solution and / or a suspension of the active ingredient in an aqueous or oily liquid excipient. Such eye drops may further contain one or more other components of buffers, salts, and / or any additional components described herein. Other useful ophthalmally administered formulations include those containing the active ingredient in microcrystalline form and / or liposomal preparations. Ear drops and / or eye drops are intended to be within the scope of this disclosure.

[0262] mRNA therapy mRNA as a drug modality has the potential to deliver secreted proteins, as well as intracellular and transmembrane proteins. mRNA as a drug modality has the potential to transmembrane and deliver targets into cells that are inaccessible when delivered in protein form, i.e., because standard biological agents lack the ability to transmembrane. One major challenge in realizing mRNA-based therapies is identifying the optimal delivery vehicle. Due to its size, chemical instability, and potential immunogenicity, mRNA requires a delivery vehicle that can provide protection from endonucleases and exonucleases, as well as protection of the cargo from immunosentinels. Lipid nanoparticles (LNPs) have been identified as a lead option in this regard.

[0263] A key performance criterion for lipid nanoparticle delivery systems is the ability to maximize cellular uptake and enable efficient release of mRNA from endosomes. In one embodiment, subjective LNPs comprising novel lipids disclosed herein demonstrate improvements in at least one of cellular uptake and endosomal release. At the same time, LNPs must provide a stable drug product and be able to be safely administered at therapeutically relevant levels. LNPs are typically multi-component systems consisting of aminolipids, phospholipids, cholesterol, and PEG lipids. Each component is necessary in terms of efficient delivery of nucleic acid cargo and particle stability. The key component thought to drive cellular uptake, endosomal escape, and tolerability is aminolipid. Cholesterol and PEG lipids contribute to the stability of the drug product both in vivo and at storage, while phospholipids provide additional fusionability to the LNP, thus aiding in the promotion of endosomal escape and making nucleic acids bioavailable in the cytoplasm of cells.

[0264] For oligonucleotide delivery, several aminolipid series, including the aminolipid MC3 (DLin-MC3-DMA), have been developed over the past few decades. MC3-based LNPs have been shown to be effective for mRNA delivery. When delivered intravenously, this class of LNPs is rapidly opsonized by apolipoprotein E (ApoE), enabling cellular uptake by the low-density lipoprotein receptor (LDLr). However, concerns remain that the long tissue half-life of MC3 may contribute to undesirable side effects that hinder its use in long-term therapy. In addition, extensive literature evidence suggests that long-term administration of lipid nanoparticles may produce several toxic side effects, including complement activation-related pseudoallergy (CARPA) and liver damage. Therefore, a class of LNPs with increased delivery efficiency, along with metabolic and toxicity profiles that would enable long-term administration in humans, is needed to unlock the potential of mRNA and other nucleic acid, nucleoptide, or peptide-based therapies in humans.

[0265] The ability to treat a wide range of diseases requires the flexibility to safely administer drugs over long periods at fluctuating dose levels. Through systematic optimization of aminolipid structures, the compounds of this disclosure were identified as having a good balance of chemical stability, improved delivery efficiency due to improved endosomal escape, rapid in vivo metabolism, and a clean toxicity profile. The combination of these features provides drug candidates that can be administered over long periods without activating the immune system. Initial rodent screening led to the identification of lead lipids with good delivery efficiency and pharmacokinetics. Advanced LNPs were further profiled in non-human primates for delivery efficiency after single and repeated doses. Finally, optimized LNPs were evaluated in 1-month repeated dose toxicity studies in rats and non-human primates. While we do not wish to be constrained by theory, the novel ionic lipids of this disclosure possess improved cell delivery, improved protein expression, and improved biodegradability, and may result in 2-fold, 5-fold, 10-fold, 15-fold, or more than 20-fold increases in mRNA expression in cells compared to LNPs lacking the lipids of the invention. In another embodiment, LNPs containing the lipids of the invention may result in specific (e.g., preferential) delivery to certain cell types or multiple cell types compared to other cell types, thereby resulting in a 2-fold, 5-fold, 10-fold, 15-fold, or more than 20-fold increase in mRNA expression in certain cells or tissues compared to LNPs lacking the lipids of the invention. These improvements over the prior art enable the safe and effective use of mRNA-based therapies in acute and chronic diseases.

[0266] method In some embodiments, the disclosure provides a method for delivering therapeutic and / or prophylactic agents to cells (e.g., mammalian cells). The method includes the step of contacting cells with a filled LNP or pharmaceutical composition of the disclosure, thereby delivering the therapeutic and / or prophylactic agent to the cells. In some embodiments, the cells are located within a subject, and the contact includes administering the cells to the subject. In some embodiments, the method includes administering lipid nanoparticles comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), along with a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents, thereby delivering the therapeutic and / or prophylactic agents to cells.

[0267] In some embodiments, the disclosure provides a method for delivering therapeutic and / or prophylactic agents to cells within a subject, the method comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), DSPC, cholesterol, and PEG 2k - The procedure includes the step of administering lipid nanoparticles to a target comprising DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the disclosure provides a method for delivering therapeutic and / or prophylactic agents to cells in a target, the method comprising a compound of formula (Ac), DSPC, cholesterol, and PEG 2k - The procedure includes the step of administering a lipid nanoparticle to a target comprising DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0268] In some embodiments, the disclosure provides a method for delivering a therapeutic and / or prophylactic agent to cells within a subject, the method comprising the step of administering lipid nanoparticles to a subject comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the disclosure provides a method for delivering a therapeutic and / or prophylactic agent to cells within a subject, the method comprising administering lipid nanoparticles to a subject comprising a compound of formula (Ac), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0269] In some embodiments, the disclosure provides a method for delivering (e.g., specifically) a therapeutic and / or prophylactic agent to a mammalian organ or tissue (e.g., the liver, kidney, spleen, or lung). The method includes the step of contacting cells with a LNP or pharmaceutical composition filled with the disclosure, thereby delivering the therapeutic and / or prophylactic agent to the target organ or tissue. In some embodiments, the method includes administering lipid nanoparticles comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), along with a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents, thereby delivering the therapeutic and / or prophylactic agents to a target organ or tissue.

[0270] In some embodiments, the disclosure provides a method for specifically delivering therapeutic and / or prophylactic agents to an organ of interest, the method comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), DSPC, cholesterol, and PEG 2k - The procedure includes the step of administering lipid nanoparticles to a target, comprising DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the disclosure provides a method for specifically delivering a therapeutic and / or prophylactic agent to a target organ, the method comprising a compound of formula (Ac), DSPC, cholesterol, and PEG 2k - The procedure includes the step of administering a lipid nanoparticle to a target comprising DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0271] In some embodiments, the disclosure provides a method for specifically delivering therapeutic and / or prophylactic agents to an organ of interest, the method comprising the step of administering lipid nanoparticles to an interest comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the disclosure provides a method for specifically delivering a therapeutic and / or prophylactic agent to an organ of interest, the method comprising the step of administering lipid nanoparticles to an interest comprising a compound of formula (Ac), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0272] In some embodiments, the disclosure features a method for improved delivery of therapeutic and / or prophylactic agents (e.g., mRNA) to target tissue (e.g., liver, spleen, or lung). The method includes the step of contacting cells with a LNP or pharmaceutical composition filled with the disclosure, thereby delivering the therapeutic and / or prophylactic agent to target tissue (e.g., liver, kidney, spleen, or lung). In some embodiments, the method includes administering lipid nanoparticles comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), along with a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents, thereby delivering the therapeutic and / or prophylactic agents to a target tissue (e.g., liver, kidney, spleen, or lung).

[0273] In some embodiments, the disclosure provides a method for improved delivery of therapeutic and / or prophylactic agents to target tissues, the method comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), DSPC, cholesterol, and PEG 2k - The procedure includes the step of administering lipid nanoparticles to a target, comprising DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the disclosure provides a method for improved delivery of therapeutic and / or prophylactic agents to a target tissue, the method comprising a compound of formula (Ac), DSPC, cholesterol, and PEG 2k - The procedure includes the step of administering a lipid nanoparticle to a target comprising DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0274] In some embodiments, the disclosure provides a method for improved delivery of therapeutic and / or prophylactic agents to a target tissue, the method comprising the step of administering a lipid nanoparticle to a target comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the disclosure provides a method for improved delivery of therapeutic and / or prophylactic agents to a target tissue, the method comprising the step of administering lipid nanoparticles to a target comprising a compound of formula (Ac), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0275] In some embodiments, the disclosure provides a method for generating a polypeptide of interest in cells (e.g., mammalian cells). The method comprises the step of contacting cells with a filled LNP or pharmaceutical composition of the disclosure, the filled LNP or pharmaceutical composition comprising mRNA, thereby enabling the mRNA to be translated within the cells to generate a polypeptide. In some embodiments, the cells are located within a subject, and the contact involves administering the cells to the subject. In some embodiments, the method includes the step of administering a lipid nanoparticle containing a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc) along with a phospholipid, a structural lipid, a PEG lipid, and mRNA, thereby enabling the mRNA to be translated in the cell to produce a polypeptide.

[0276] In some embodiments, the disclosure provides a method for generating a polypeptide of interest in cells, the method comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), DSPC, cholesterol, and PEG 2k - The procedure includes the step of administering lipid nanoparticles containing DMG and mRNA to a target. For example, in some embodiments, the disclosure provides a method for generating a polypeptide of interest in cells, the method comprising the compounds of Table 1, DSPC, cholesterol, and PEG 2k - The procedure includes the step of administering lipid nanoparticles containing DMG and mRNA to a target. For example, in some embodiments, the disclosure provides a method for generating a polypeptide of interest in cells, the method comprising a compound of formula (Ac), DSPC, cholesterol, and PEG 2k The present invention provides a method comprising the step of administering lipid nanoparticles containing DMG and mRNA to a target.

[0277] In some embodiments, the disclosure provides a method for generating a polypeptide of interest in cells, the method comprising the step of administering lipid nanoparticles comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), DSPC, cholesterol, PEG-1, and mRNA to a target. For example, in some embodiments, the disclosure provides a method for generating a polypeptide of interest in cells, the method comprising the step of administering lipid nanoparticles comprising a compound of formula (Ac), DSPC, cholesterol, PEG-1, and mRNA to a target. For example, in some embodiments, the disclosure provides a method for generating a target polypeptide in cells, the method comprising the step of administering lipid nanoparticles comprising the compounds of Table 1, DSPC, cholesterol, PEG-1, and mRNA to the target.

[0278] In some embodiments, the disclosure provides a method for treating a disease or disorder in a mammal (e.g., human) that requires it. The method comprises the step of administering a therapeutically effective amount of a LNP or pharmaceutical composition filled with the disclosure to a mammal. In some embodiments, the method comprises the step of administering lipid nanoparticles comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc) along with a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents, thereby delivering the therapeutic and / or prophylactic agents to cells. In some embodiments, the disease or disorder is characterized by dysfunction or abnormal protein or polypeptide activity. For example, the disease or disorder is selected from the group consisting of rare diseases, infectious diseases, cancer and proliferative disorders, genetic disorders, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renovascular diseases, and metabolic diseases.

[0279] In some embodiments, the disclosure provides a method for treating a disease or disorder in a subject, the method comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), DSPC, cholesterol, and PEG 2k - The procedure includes the step of administering lipid nanoparticles to a subject, comprising DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the disclosure provides a method for treating a disease or disorder in a subject, wherein the method comprises a compound of formula (Ac), DSPC, cholesterol, and PEG 2k- The procedure includes the step of administering lipid nanoparticles to a subject, comprising DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the disclosure provides a method for treating a disease or disorder in a subject, the method comprising the compounds of Table 1, DSPC, cholesterol, and PEG 2k - The procedure includes the step of administering a lipid nanoparticle to a target comprising DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0280] In some embodiments, the disclosure provides a method for treating a disease or disorder in a subject, the method comprising the step of administering lipid nanoparticles to a subject comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the disclosure provides a method for treating a disease or disorder in a subject, the method comprising administering lipid nanoparticles to the subject, comprising a compound of formula (Ac), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0281] In yet another embodiment, the disclosure provides a method for reducing immunogenicity, comprising introducing the filled LNP or pharmaceutical composition of the disclosure into cells, wherein the filled LNP or pharmaceutical composition reduces the induction of a cellular immune response to the filled LNP or pharmaceutical composition compared to the induction of a cellular immune response in cells induced by a reference composition. In some embodiments, the cells are present in a subject, and the contact comprises administering the cells to the subject. In some embodiments, the method includes the step of administering a lipid nanoparticle comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA), to a target. Lipid nanoparticles containing compounds of formula (1-1), (2-1), (1 It reduces the induction of cellular immune responses in cells to lipid nanoparticles containing compounds of (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc). For example, cellular immune responses can be innate immune responses, adaptive immune responses, or both.

[0282] In some embodiments, the disclosure provides a method for reducing immunogenicity in a subject, the method comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), DSPC, cholesterol, and PEG 2k - The procedure includes the step of administering lipid nanoparticles to a target comprising DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the disclosure provides a method for reducing immunogenicity in a target, the method comprising a compound of formula (Ac), DSPC, cholesterol, and PEG 2k - The procedure includes the step of administering lipid nanoparticles to a target comprising DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the disclosure provides a method for reducing immunogenicity in a target, the method comprising the compounds of Table 1, DSPC, cholesterol, and PEG 2k - The procedure includes the step of administering a lipid nanoparticle to a target comprising DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0283] In some embodiments, the disclosure provides a method for reducing immunogenicity in a subject, the method comprising the step of administering lipid nanoparticles to a subject comprising a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the disclosure provides a method for reducing immunogenicity in a subject, the method comprising administering lipid nanoparticles to a subject comprising a compound of formula (Ac), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0284] The disclosure also includes methods for synthesizing compounds of formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), and methods for synthesizing compounds of formulas (1-1), (2-1), (Ia The method includes producing lipid nanoparticles (e.g., empty LNPs or filled LNPs) containing lipid components comprising compounds of (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc).

[0285] Method for generating polypeptides in cells This disclosure provides a method for generating a target polypeptide in mammalian cells. The method for generating the polypeptide involves contacting cells with lipid nanoparticles (e.g., empty LNPs or filled LNPs) containing mRNA encoding the target polypeptide. Upon contact between the nanoparticle composition and cells, the mRNA may be taken up into the cells and translated to generate the target polypeptide.

[0286] Generally, the step of contacting mammalian cells with lipid nanoparticles (e.g., empty LNPs or filled LNPs) containing mRNA encoding the polypeptide of interest can be carried out in vivo, ex vivo, in culture, or in vitro. The amount of lipid nanoparticles (e.g., empty LNPs or filled LNPs) and / or the amount of mRNA within them that are contacted with the cells may depend on the type of cells or tissue being contacted, the means of administration, the physiological and chemical characteristics of the lipid nanoparticles (e.g., empty LNPs or filled LNPs) and the mRNA within them (e.g., size, charge, and chemical composition), as well as other factors. Generally, an effective amount of lipid nanoparticles (e.g., empty LNPs or filled LNPs) will enable efficient polypeptide production in the cells. Metrics for efficiency may include polypeptide translation (indicated by polypeptide expression), mRNA degradation levels, and immune response indicators.

[0287] The step of bringing mRNA-containing lipid nanoparticles (e.g., empty or filled LNPs) into contact with cells may involve or induce transfection. The phospholipids contained in the lipid components of the lipid nanoparticles (e.g., empty or filled LNPs) may facilitate transfection and / or increase transfection efficiency by interacting with and / or fusing with, for example, the cell membrane or intracellular membrane. Transfection may enable translation of mRNA within the cell.

[0288] In some embodiments, the lipid nanoparticles described herein (e.g., empty LNPs or filled LNPs) may be used therapeutically. For example, mRNA contained in lipid nanoparticles (e.g., empty LNPs or filled LNPs) may encode a therapeutic polypeptide (e.g., in a translatable region) and, upon contact with and / or entry into cells (e.g., transfection), may generate the therapeutic polypeptide. In other embodiments, mRNA contained in lipid nanoparticles (e.g., empty LNPs or filled LNPs) may encode a polypeptide that can improve or enhance the immune response of a target. For example, the mRNA may encode a granulocyte colony-stimulating factor or trastuzumab.

[0289] In certain embodiments, mRNA contained in lipid nanoparticles (e.g., empty or filled LNPs) may encode recombinant polypeptides that can replace one or more polypeptides that may be substantially absent in the contacted cell with the nanoparticle composition. These substantially absent polypeptides may be absent due to genetic mutations in the coding gene or its regulatory pathway. Alternatively, recombinant polypeptides produced by mRNA translation may antagonize the activity of endogenous proteins present in cells, on the cell surface, or secreted from cells. Antagonistic recombinant polypeptides may be desirable to counteract adverse effects caused by the activity of endogenous proteins, such as altered activity or localization caused by mutations. In another alternative example, recombinant polypeptides produced by mRNA translation may indirectly or directly antagonize the activity of biological parts present in cells, on the cell surface, or secreted from cells. Examples of antagonized biological parts include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoproteins), nucleic acids, carbohydrates, and small molecule toxins. Recombinant polypeptides produced by mRNA translation can be manipulated for intracellular localization, such as within specific compartments like the nucleus, or for secretion from cells, or for translocation to the cell's plasma membrane.

[0290] In some embodiments, the innate immune response of cells to exogenous nucleic acids can be reduced by contacting cells with lipid nanoparticles containing mRNA (e.g., empty LNPs or filled LNPs). Cells may be contacted with first lipid nanoparticles (e.g., empty LNPs or filled LNPs) containing a first amount of first exogenous mRNA including a translatable region, and the level of the cell's innate immune response to the first exogenous mRNA can be determined. Subsequently, cells may be contacted with a second composition containing a second amount of first exogenous mRNA, the second amount being less than the first amount. Alternatively, the second composition may contain a first amount of second exogenous mRNA distinct from the first exogenous mRNA. The step of contacting cells with the first and second compositions may be repeated one or more times. In addition, the efficiency of intracellular polypeptide production (e.g., translation) may be optionally determined, and cells may be repeatedly re-contacted with the first and / or second compositions until the efficiency of target protein production is achieved.

[0291] Methods for delivering therapeutic drugs to cells and organs This disclosure provides a method for delivering therapeutic and / or prophylactic agents to mammalian cells or organs. Delivering therapeutic and / or prophylactic agents to cells involves targeting and administering lipid nanoparticles (e.g., empty LNPs or filled LNPs) containing the therapeutic and / or prophylactic agent, while administering a composition involves contacting the composition with the cells. For example, proteins, cytotoxic agents, radioactive ions, chemotherapeutic agents, or nucleic acids (RNA, e.g., mRNA) can be delivered to cells or organs. If the therapeutic and / or prophylactic agent is mRNA, contacting the nanoparticle composition with cells may cause the translatable mRNA to be translated within the cell to produce the polypeptide of interest. However, substantially untranslatable mRNA can also be delivered to cells. Substantially untranslatable mRNA may be useful as a vaccine and / or may blockade the cellular translational components, thereby reducing the expression of other species within the cell.

[0292] In some embodiments, lipid nanoparticles (e.g., empty LNPs or filled LNPs) may target specific types or classes of cells (e.g., cells of a specific organ or system). For example, lipid nanoparticles (e.g., empty LNPs or filled LNPs) containing the therapeutic and / or prophylactic agent of interest may be specifically delivered to the liver, kidney, spleen, or lungs of a mammal. Specific delivery to a specific class of cells, organs, or systems or groups thereof means that a higher proportion of lipid nanoparticles (e.g., filled LNPs) containing the therapeutic and / or prophylactic agent are delivered to the target site (e.g., tissue) compared to other locations. In some embodiments, specific delivery of mRNA-containing filled LNPs may result in a 2-fold, 5-fold, 10-fold, 15-fold, or more than 20-fold increase in mRNA expression in cells at the target site (e.g., the target tissue, such as the liver) compared to cells at another location (e.g., the spleen). In some embodiments, the target tissue is selected from the group consisting of the liver, kidney, lung, spleen, and tumor tissue (e.g., via intratumor injection).

[0293] In some embodiments, specific delivery of mRNA contained in LNPs filled with the Disclosure (i.e., lipid nanoparticles formulated with the Disclosure compound) may result in a 2-fold, 5-fold, 10-fold, 15-fold, or more than 20-fold increase in mRNA expression compared to delivery of mRNA contained in LNPs formulated with another lipid (i.e., none of the lipids of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc).

[0294] Another example of targeted or specific delivery is that a protein-binding partner (e.g., an antibody or its functional fragment, a backbone protein, or a peptide) or mRNA encoding a receptor on the cell surface may be included in the nanoparticle composition. mRNA may be used in addition to, or instead of, in order to induce the synthesis and extracellular localization of lipids, carbohydrates, or other biological parts. Alternatively, other therapeutic and / or prophylactic agents or elements (e.g., lipids or ligands) of lipid nanoparticles (e.g., empty LNPs or filled LNPs) may be selected based on their affinity for a particular receptor (e.g., a low-density lipoprotein receptor) so that the lipid nanoparticles (e.g., empty LNPs or filled LNPs) can interact more readily with the target cell population containing the receptor. For example, ligands may include, but are not limited to, members of specific binding pairs, antibodies, monoclonal antibodies, Fv fragments, single-stranded Fv(scFv) fragments, Fab' fragments, F(ab')2 fragments, single-domain antibodies, camelized antibodies and their fragments, humanized antibodies and their fragments, and their polyvalent versions; polyvalent binding reagents including single or bispecific antibodies such as disulfide-stabilized Fv fragments, scFv tandems, diabodies, tribodies, or tetrabodies; as well as aptamers, receptors, and fusion proteins.

[0295] In some embodiments, the ligand may be a surface-bound antibody that allows for the modification of cell targeting specificity. This is particularly useful because it can enhance the specificity of a highly specific antibody against a desired epitope at a desired targeting site. In some embodiments, multiple antibodies are expressed on the cell surface, and each antibody may have a different specificity for the desired target. Such an approach can increase the affinity and specificity of the targeting interaction.

[0296] Ligands can be selected, for example, by those skilled in the field of biotechnology, based on the desired localization or function of the cell.

[0297] Target cells, though not limited to them, may include hepatocytes, epithelial cells, hematopoietic cells, endothelial cells, lung cells, osteocytes, stem cells, mesenchymal cells, nerve cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0298] In some embodiments, lipid nanoparticles (e.g., empty or filled LNPs) can target hepatocytes. Apolipoproteins, such as apolipoprotein E (apoE), have been shown to associate with lipid nanoparticles (e.g., empty or filled LNPs) containing neutral or near-neutral lipids in the body and are known to associate with receptors such as low-density lipoprotein receptors (LDLRs) found on the surface of hepatocytes. Therefore, lipid nanoparticles (e.g., empty or filled LNPs) containing neutral or near-neutral charged lipid components administered to a subject may acquire apoE in the subject's body and subsequently deliver therapeutic and / or prophylactic agents (e.g., RNA) to hepatocytes containing LDLRs in a targeted manner.

[0299] Methods of treating diseases and disorders Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may be useful for treating diseases, disorders, or conditions. In particular, such compositions may be useful for treating diseases, disorders, or conditions characterized by deficient or abnormal protein or polypeptide activity. For example, lipid nanoparticles (e.g., empty LNPs or filled LNPs) containing mRNA encoding a deficient or abnormal polypeptide may be administered or delivered to cells. Subsequent translation of the mRNA may produce a polypeptide, thereby reducing or eliminating problems caused by the absence of the polypeptide or abnormal activity caused by the polypeptide. Since translation can occur rapidly, the methods and compositions may be useful for treating acute diseases, disorders, or conditions such as sepsis, stroke, and myocardial infarction. Therapeutic and / or prophylactic agents contained in lipid nanoparticles (e.g., empty LNPs or filled LNPs) may also be capable of altering the transcription rate of a given species, thereby affecting gene expression.

[0300] Diseases, disorders, and / or conditions characterized by dysfunctional or abnormal protein or polypeptide activity to which the composition may be administered include, but are not limited to, rare diseases, infectious diseases (both as vaccines and therapeutic agents), cancer and proliferative disorders, genetic disorders, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renovascular diseases, and metabolic diseases. Several diseases, disorders, and / or conditions may be characterized by a deficiency (or substantially reduced) of protein activity, resulting in a lack of proper protein function. Such proteins may be absent or essentially non-functional. This disclosure provides a method for treating such diseases, disorders and / or conditions in a subject by administering lipid nanoparticles (e.g., empty LNPs or filled LNPs) comprising RNA and a lipid component including lipids, phospholipids (optionally unsaturated), PEG lipids, and structural lipids according to formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), wherein the RNA may be mRNA encoding a polypeptide that antagonizes or overcomes abnormal protein activity present in the cells of the subject.

[0301] The disclosure provides lipid nanoparticles (e.g., empty LNPs or filled LNPs) comprising one or more therapeutic and / or prophylactic agents, and methods involving the administration of pharmaceutical compositions comprising them. The terms therapeutic and prophylactic agents may be used synonymously herein with respect to the features and embodiments of the disclosure. Therapeutic compositions, or their imaging, diagnostic, or prophylactic compositions, may be administered to a subject using any reasonable amount and any route of administration that is effective in preventing, treating, diagnosing, or imaging a disease, disorder, and / or condition and / or any other purpose. The specific amount administered to a given subject may vary depending on the subject's species, age, and general condition, the purpose of administration, the specific composition, the mode of administration, etc. The compositions according to the disclosure may be formulated in unit dosage forms for ease of administration and uniformity of dosage. However, it will be understood that the total daily dose of the compositions according to the disclosure will be determined by the attending physician within the bounds of sound medical judgment. A specific therapeutically effective, prophylactically effective, or otherwise appropriate dose level (e.g., for imaging) for any particular patient will depend on a variety of factors, including the severity and nature of the disorder being treated (if any); one or more therapeutic and / or prophylactic agents used; the specific composition used; the patient's age, weight, overall health, sex, and diet; the timing, route of administration, and rate of excretion of the specific pharmaceutical composition used; the duration of treatment; drugs used in combination with or concurrently with the specific pharmaceutical composition used; and similar factors well known in the medical field.

[0302] The filled LNPs may be administered by any route. In some embodiments, compositions comprising a prophylactic, diagnostic, or imaging composition comprising one or more filled LNPs as described herein may be administered by one or more of the following routes: orally, intravenously, intramuscularly, intra-arterially, subcutaneously, percutaneously or intradermally, interdermally, intraperitoneally, mucosally, nasally, intratumorally, intranasally; inhalation; as an oral spray and / or powder, nasal spray and / or aerosol, and / or via a portal vein catheter. In some embodiments, the compositions may be administered by intravenously, intramuscularly, intradermally, intra-arterially, intratumorally, subcutaneously, or any other parenteral administration route, or by inhalation. However, this disclosure encompasses the delivery or administration of the compositions described herein by any suitable route, taking into account possible advances in the science of drug delivery. Generally, the most suitable route of administration will depend on a variety of factors, including the properties of the filled LNPs comprising one or more therapeutic and / or prophylactic agents (e.g., their stability in various bodily environments such as blood flow and the gastrointestinal tract), the patient's condition (e.g., whether the patient is able to tolerate a particular route of administration), and so on.

[0303] In a particular embodiment, the composition according to this disclosure is, at a given dose, approximately 0.0001 mg / kg to approximately 10 mg / kg, approximately 0.001 mg / kg to approximately 10 mg / kg, approximately 0.005 mg / kg to approximately 10 mg / kg, approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.05 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 1 mg / kg to approximately 10 mg / kg, approximately 2 mg / kg to approximately 10 mg / kg, approximately 5 mg / kg to approximately 10 mg / kg, and approximately 0.0001 mg / kg. ~5mg / kg, 0.001mg / kg~5mg / kg, 0.005mg / kg~5mg / kg, 0.01mg / kg~5mg / kg, 0.05mg / kg~5mg / kg, 0.1mg / kg~5mg / kg, 1m g / kg~about 5mg / kg, about 2mg / kg~about 5mg / kg, about 0.0001mg / kg~about 2.5mg / kg, about 0.001mg / kg~about 2.5mg / kg, about 0.005mg / kg~about 2.5mg / kg, about 0.01mg / kg~about 2 .5mg / kg, about 0.05mg / kg to about 2.5mg / kg, about 0.1mg / kg to about 2.5mg / kg, about 1mg / kg to about 2.5mg / kg, about 2mg / kg to about 2.5mg / kg, about 0.0001mg / kg to about 1mg / kg, about 0 .001mg / kg~about 1mg / kg, about 0.005mg / kg~about 1mg / kg, about 0.01mg / kg~about 1mg / kg, about 0.05mg / kg~about 1mg / kg, about 0.1mg / kg~about 1mg / kg, about 0.0001mg / kg~about The therapeutic and / or prophylactic agent (e.g., mRNA) may be administered at a dosage level sufficient to deliver 0.25 mg / kg, approximately 0.001 mg / kg to approximately 0.25 mg / kg, approximately 0.005 mg / kg to approximately 0.25 mg / kg, approximately 0.01 mg / kg to approximately 0.25 mg / kg, approximately 0.05 mg / kg to approximately 0.25 mg / kg, or approximately 0.1 mg / kg to approximately 0.25 mg / kg, with a dose of 1 mg / kg (mpk) providing 1 mg of the therapeutic and / or prophylactic agent per kg of body weight of the subject. In some embodiments, doses of the therapeutic and / or prophylactic agent of filled LNPs ranging from approximately 0.001 mg / kg to approximately 10 mg / kg may be administered. In other embodiments, doses of the therapeutic and / or prophylactic agent ranging from approximately 0.005 mg / kg to approximately 2.5 mg / kg may be administered.In certain embodiments, doses of approximately 0.1 mg / kg to approximately 1 mg / kg may be administered. In other embodiments, doses of approximately 0.05 mg / kg to approximately 0.25 mg / kg may be administered. The dose may be administered once or more times daily in the same or different amounts to obtain the desired level of mRNA expression and / or therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered, for example, three times daily, twice daily, once daily, every other day, every three days, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple doses (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen times, or more doses). In some embodiments, a single dose may be administered, for example, before or after a surgical procedure, or in the case of an acute disease, disorder, or condition.

[0304] Lipid nanoparticles (e.g., empty LNPs or filled LNPs) containing one or more therapeutic and / or prophylactic agents may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. The phrase "in combination with" is not intended to imply that the agents must be administered simultaneously and / or formulated together for delivery, although these delivery methods are within the scope of this disclosure. For example, one or more lipid nanoparticles (e.g., empty LNPs or filled LNPs) containing one or more different therapeutic and / or prophylactic agents may be administered in combination. The composition may be administered simultaneously with, before, or after one or more other desired therapeutic or medical treatments. Generally, each agent is administered in a dose and / or time schedule determined for that agent. In some embodiments, this disclosure encompasses the delivery of compositions, or imaging, diagnostic, or prophylactic compositions, in combination with agents that improve bioavailability, reduce and / or alter metabolism, inhibit excretion, and / or alter distribution in the body.

[0305] It will be further understood that therapeutic, prophylactic, diagnostic, or imaging activators used in combination may be administered together in a single composition or separately in different compositions. Generally, it is expected that drugs used in combination will be utilized at levels not exceeding those used individually. In some embodiments, the combined level may be lower than the individual levels.

[0306] The specific combination of therapies (medicines or procedures) used in a combination regimen will take into account the compatibility between the desired drug and / or procedure and the desired therapeutic effect to be achieved. It will also be understood that the therapies used may achieve the desired effect against the same disorder (for example, a composition useful for treating cancer may be administered concurrently with a chemotherapeutic agent) or they may achieve different effects (for example, control of any adverse effects such as infusion-related reactions).

[0307] Lipid nanoparticles (e.g., empty LNPs or filled LNPs) may be used in combination with agents to increase the efficacy and / or therapeutic time frame of the composition. Such agents may be, for example, anti-inflammatory compounds, steroids (e.g., corticosteroids), statins, estradiol, BTK inhibitors, S1P1 agonists, glucocorticoid receptor modulators (GRMs), or antihistamines. In some embodiments, lipid nanoparticles (e.g., empty LNPs or filled LNPs) may be used in combination with dexamethasone, methotrexate, acetaminophen, H1 receptor blockers, or H2 receptor blockers. In some embodiments, methods for treating subjects requiring treatment of a disease or disorder, or for delivering therapeutic and / or prophylactic agents to subjects (e.g., mammals), may involve pre-treating the subject with one or more agents before administering the nanoparticle composition. For example, the subject may be pre-treated with a useful dose (e.g., 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, or any other useful dose) of dexamethasone, methotrexate, acetaminophen, an H1 receptor blocker, or an H2 receptor blocker. The pre-treatment may be performed 24 hours or less before the administration of lipid nanoparticles (e.g., empty LNPs or filled LNPs) (e.g., 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or 10 minutes), and may be performed, for example, once, twice, or more times with increased doses.

[0308] Those skilled in the art will be able to recognize or confirm, through mere routine experimentation, many equivalents to the specific embodiments of this disclosure described herein. The scope of this disclosure is not intended to be limited to the embodiments for carrying out the invention described above, but rather to those described in the appended claims.

[0309] In the claims, articles such as “a,” “an,” and “the” may mean one or more unless otherwise indicated or made clear from the context. Any claim or description containing “or” between one or more members of a group is deemed satisfied unless otherwise indicated or made clear from the context if one, two or more, or all of the members of the group are present, used, or otherwise related to a given product or process. The disclosure includes embodiments in which exactly one member of the group is present, used, or otherwise related to a given product or process. The disclosure includes embodiments in which two or more, or all, of the members of the group are present, used, or related to a given product or process. As used herein, expressions such as "one or more of A, B, or C," "one or more A, B, or C," "one or more of A, B, and C," "one or more A, B, and C," "selected from A, B, and C," and "selected from the group consisting of A, B, and C" are used synonymously and, unless otherwise specified, all refer to a selection from the group consisting of A, B, and / or C, i.e., one or more A's, one or more B's, one or more C's, or any combination thereof.

[0310] It should be noted that the term “comprising” is intended to be open and allows for, but does not require, the inclusion of additional elements or steps. Therefore, where the term “comprising” is used herein, the terms “consisting essentially of” and “consisting of” are also encompassed and disclosed. Throughout this specification, where a composition is described as having, containing, or containing certain components, the composition is also intended to consist essentially of or comprising the enumerated components. Similarly, where a method or process is described as having, containing, or containing certain process steps, the process is also intended to consist essentially of or comprising the enumerated processing steps. Furthermore, it should be understood that the order or sequence of steps for performing a particular act is not important as long as the invention remains operational. Moreover, two or more steps or acts can be performed simultaneously.

[0311] If a range is specified, it includes the endpoints. Furthermore, unless otherwise indicated or is evident from the context and the understanding of those skilled in the art, it should be understood that any value expressed as a range may be any specific value or subrange within the range described in different embodiments of the disclosure, as one-tenth of the lower limit unit of the range, unless the context otherwise clearly indicates.

[0312] The disclosed synthesis process can accommodate a wide variety of functional groups, and therefore various substituted starting materials can be used. The process generally provides the desired final compound at the end or near the end of the entire process, but in certain specific cases, it may be desirable to further convert the compound to its pharmaceutically acceptable salt.

[0313] The compounds of this disclosure can be prepared in various ways using commercially available starting materials, compounds known in the literature, or readily prepared intermediates by employing standard synthetic methods and procedures that are known to those skilled in the art or would be apparent to those skilled in the art in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from relevant scientific literature or standard textbooks in the field. Not limited to any one or more sources, but incorporated herein by reference, is Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 th edition, John Wiley&Sons: New York, 2001, Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3 rd John Wiley & Sons, New York, 1999; R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) are useful and recognized reference texts for organic synthesis known to those skilled in the art. The following description of the synthetic methods is designed to illustrate, rather than limit, the general procedures for the preparation of the compounds of this disclosure.

[0314] Compounds of this disclosure having any of the formulas described herein may be prepared from commercially available starting materials or starting materials that can be prepared using literature procedures, according to the procedures shown in Schemes 1, 2, and 3 below. Variable elements in the schemes (e.g., R 1 , R 2 , and R 3 (These are defined herein.) Those skilled in the art will note that the order of certain steps in the reaction sequence and synthesis scheme described herein, such as the introduction and removal of protecting groups, may be changed.

[0315] Those skilled in the art will recognize that certain groups may require protection from reaction conditions through the use of protecting groups. Furthermore, similar functional groups within a molecule can be modified using protecting groups. A list of protecting groups, as well as methods for introducing and removing these groups, can be found in Greene, TW, Wuts, PGM, Protective Groups. in Organic Synthesis, 3 rd This can be found in edition, John Wiley & Sons: New York, 1999.

[0316] Preferred protecting groups include, but are not limited to, the following:

[0317] The hydroxyl group consists of TBS, benzyl, THP, and Ac.

[0318] Carboxylic acids include benzyl esters, methyl esters, ethyl esters, and allyl esters.

[0319] Amines include Fmoc, Cbz, BOC, DMB, Ac, Bn, Tr, Ts, trifluoroacetyl, phthalimide, and benzylideneamine.

[0320] Diols are used with Ac(x2)TBS(x2) or acetonide.

[0321] For thiols, use AC.

[0322] Benzimidazole is also known as SEM, benzyl, PMB, or DMB.

[0323] Aldehydes include dimethoxyacetals or dialkylacetals such as diethylacetyl.

[0324] The reaction schemes described herein may produce multiple stereoisomers. Where a specific stereoisomer is not shown, it should be understood that this means all possible stereoisomers that may be produced from the reaction. Those skilled in the art will recognize that the reaction may be optimized to preferentially yield one isomer, or that a new scheme may be devised to produce a single isomer. If a mixture is produced, the isomers may be separated using techniques such as preparative thin-layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC.

[0325] [ka] As shown in Scheme 1 above, 8-bromooctanoic acid is reacted with alcohol a1 (e.g., heptadecan-9-ol) to obtain ester b1 (e.g., heptadecan-9-yl 8-bromooctanoate). Step 1 can be carried out in an organic solvent (e.g., dichloromethane) in the presence of, for example, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine, and DMAP. Step 1 can be carried out at room temperature for 18 hours. Next, ester b1 is reacted with 2-aminoethane-1-ol to obtain amine c1 (e.g., heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate). Step 2 can be carried out in ethanol at a temperature of about 60°C, for example. Then, amine c1 is reacted with bromoalkyl R 1-Br (e.g., 1-bromotetradecane) is reacted to obtain compound d1 (e.g., heptadecan-9-yl-8-((2-hydroxyethyl)(tetradecyl)amino)octanoate). Step 3 can be carried out in ethanol in the presence of N,N-diisopropylethylamine.

[0326] [ka] As shown in scheme 2 above, acid a2(x 3 is an integer from 1 to 7, for example, 8-bromooctanoic acid) is reacted with alcohol b2 (e.g., nonan-1-ol) to obtain ester c2 (e.g., nonyl-8-bromooctanoate). Step 1 can be carried out in an organic solvent (e.g., dichloromethane) in the presence of, for example, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine, and DMAP. Alcohol e2 (e.g., heptadecane-9-ol) reacts with aldehyde d2 (e.g., nonanol) via Grignard reagent R in Step 2. 3 -MgX(for example, n-C8H) 17It can be obtained by reacting with MgBr. Next, 8-bromooctanoic acid is reacted with alcohol e2 (e.g., heptadecan-9-ol) to obtain ester f2 (e.g., heptadecan-9-yl 8-bromooctanoate). Step 3 can be carried out in an organic solvent (e.g., dichloromethane) in the presence of, for example, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine, and DMAP. Next, ester f2 is reacted with 2-aminoethane-1-ol to obtain amine g2 (e.g., heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate). Step 4 can be carried out in ethanol in the presence of i-Pr2EtN. Next, amine g2 is reacted with ester c2 (e.g., nonyl-8-bromooctanoate) to obtain compound h2 (e.g., heptadecan-9-yl-8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate). Step 5 can be carried out, for example, at a high temperature (about 70-90°C, e.g., about 80°C) in an organic solvent (e.g., a mixture of CPME and MeCN) in the presence of a base (inorganic base (e.g., K2CO3) or a non-nucleophilic organic base (e.g., i-Pr2EtN)) and a catalyst (e.g., an iodide such as KI or NaI).

[0327] [ka] As shown in scheme 3 above, haloalkanol (x 3 is an integer from 1 to 12, for example, 6-bromohexane-1-ol) as the starting material a3(x 2is an integer from 1 to 6, and is reacted with, for example, 4-(hexyloxy)-4-oxobutanoic acid to obtain halogenated diester b3 (e.g., 6-bromohexylhexyl succinate). Compound a3 can be obtained by the reaction of an alcohol (e.g., hexane-1-ol) with an acid anhydride (e.g., succinic anhydride, dihydro-2H-pyran-2,6(3H)-dione, 3-(tert-butoxy)-3-oxopropanoic acid, 4-(tert-butoxy)-3-methyl-4-oxobutanoic acid, or 4-(tert-butoxy)-2-methyl-4-oxobutanoic acid). Step 1 can be carried out in an organic solvent (e.g., dichloromethane) in the presence of, for example, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine, and DMAP. Next, halogenated diester b3 is converted to amine c3(x 4 x is an integer between 5 and 13. 5 is an integer from 1 to 5, and is reacted with, for example, heptadecan-9-yl-8-((2-hydroxyethyl)amino)octanoate to obtain product d3. Step 2 can be carried out at a high temperature (e.g., about 90°C) in an organic solvent (e.g., a mixture of CPME and MeCN) in the presence of a base (inorganic base (e.g., K2CO3)), a catalyst (e.g., an iodide such as KI), and an ether solvent (e.g., cyclopentyl methyl ether).

[0328] Those skilled in the art will recognize that the order of certain steps in the above scheme may be interchangeable.

[0329] In certain embodiments, the disclosure also includes any compound of formulas (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), and (Bc), and methods for synthesizing intermediates(s) for synthesizing the compounds.

[0330] In some embodiments, a method for synthesizing the disclosed compound is given by formula (X2): [ka] The compound R 1 -The process involves reacting with Br to obtain the disclosed compound, where each variable element is as defined herein. For example, m is 5, 6, 7, 8, or 9, preferably 5, 7, or 9. For example, R 5 , R 6 , and R 7 Each of these is H. For example, M is -C(O)O- or -OC(O)-. For example, R 4 is unsubstituted C 1-3 Alkyl or -(CH2) n Q is OH, n is 2, 3, or 4, and Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, or -N(R)S(O)2R. For example, compound (X2) and R 1 The reaction with -Br takes place in the presence of a base (an inorganic base (e.g., K2CO3) or a non-nucleophilic organic base (e.g., i-Pr2EtN)). For example, the reaction takes place in the presence of an inorganic base (e.g., K2CO3) and a catalyst (e.g., an iodide such as KI or NaI). For example, the reaction takes place at high temperatures (e.g., about 50-100°C, 70-90°C, or about 80°C).

[0331] The method is also given by equation (X1): [ka] The compound R 4 This may include reacting with NH2 to obtain a compound of formula (X2), where each variable element is as defined herein.

[0332] In some embodiments, the intermediate(s) are given by formulas (X1) and (X2): [ka] This includes having any of the following, where each variable element is as defined herein. For example, intermediates include heptadecan-9-yl-8-bromooctanoate and heptadecan-9-yl-8-((2-hydroxyethyl)amino)octanoate, as well as their structural forms (e.g., crystalline form).

[0333] In addition, it should be understood that any particular embodiment of this disclosure that constitutes prior art may be expressly excluded from one or more of the claims. Since such embodiments are considered to be known to those skilled in the art, they may be excluded even if the exclusion is not expressly stated herein.

[0334] Even if not explicitly stated in the cited references, all cited references, such as reference documents, publications, databases, database entries, and the technical fields cited herein are incorporated by reference into this application. In the event of any conflict between the cited references and the descriptions in this application, the descriptions in this application shall prevail. [Examples]

[0335] Example 1: Synthesis of the compounds in Table 1 A. General considerations Unless otherwise noted, all solvents and reagents used were commercially available and used as is. 1 The 1H NMR spectrum was recorded in CDCl3 using a Bruker Ultrashield 300MHz instrument at 300K. The chemical shifts were: 1The purity is reported as parts per million (ppm) compared to TMS (0.00) in 1H. Silica gel chromatography was performed using ISCO CombiFlash Rf + Lumen Instruments with ISCO RediSep Rf Gold Flash Cartridges (particle size: 20-40 microns). Reverse-phase chromatography was performed using ISCO CombiFlash Rf + Lumen Instruments with RediSep Rf Gold C18 High Performance columns. All final compounds were determined to have a purity of over 85% via analysis by reverse-phase UPLC-MS (retention time, RT, in minutes) using a Waters Acquity UPLC instrument with DAD and ELSD, and ZORBAX Rapid Resolution High Definition (RRHD) SB-C18 LC columns, 2.1 mm, 50 mm, 1.8 μm, and a gradient of 65% to 100% acetonitrile in water containing 0.1% TFA, at 1.2 mL / min for 5 minutes. The injection volume was 5 μL, and the column temperature was 80°C. Detection was based on positive-mode electrospray ionization (ESI) using a Waters SQD mass spectrometer (Milford, MA, USA) and an evaporative light scattering detector. LCMS method: Equipment information: HPLC / MS-Agilent1100 Column: Agela Technologies Durashell C18 3.5μm, 100Å, 4.6×50mm Mobile phase A: Water / 0.1% trifluoroacetic acid Mobile phase B: Acetonitrile / 0.1% trifluoroacetic acid Flow rate: 1mL / min Gradient: 70% to 100% B in 5 minutes, hold at 100% B for 10 minutes, then decrease from 100% B to 70% B in 1 minute, then stop. Column temperature: ambient temperature Detector: ELSD

[0336] The procedure described below is useful for synthesizing the compounds in Table 1.

[0337] The following abbreviations are used in this specification: THF: Tetrahydrofuran MeCN: Acetonitrile LAH: Lithium aluminum hydride DCM: Dichloromethane DMAP: 4-dimethylaminopyridine LDA: Lithium diisopropylamide rt: room temperature DME: 1,2-dimethoxyethane n-BuLi:n-butyllithium CPME: Cyclopentyl methyl ether i-Pr2EtN:N,N-diisopropylethylamine

[0338] Representative synthesis of compounds 7, 12, and 13 [ka] Typical procedure A: 1,4-addition of Grignard reagent RMgX to methyl(E)-nona-2-enoate 1. A1. Compound 2a: Methyl 3-butylnonanoate [ka] In a 100 mL oven-dried round-bottom flask, copper(I) bromide (421.3 mg, 2.93 mmol) and lithium chloride (249 mg, 5.87 mmol) were added, followed by the addition of anhydrous THF (15 mL). The mixture was stirred for 10 minutes, during which time the solids were dissolved. The flask was placed in an ice bath, and methyl(E)-nona-2-enoate 1 (5 g, 29.37 mmol) was added, followed by the addition of TMSCl (4 mL, 32.31 mmol). The reaction mixture was stirred for 15 minutes. A THF solution of butylmagnesium bromide (17.6 mL, 35.2 mmol, 2.0 M in THF) was slowly added, and the reaction mixture was stirred for 2 hours. The reaction mixture was quenched with saturated NH4Cl (10 mL), extracted with diethyl ether (100 mL), and dried over anhydrous sodium sulfate. After removing the solvent, the crude product was purified by flash chromatography (SiO2:ethyl acetate / hexane 0-100%) to obtain a colorless oil product 2a (3g, 45%). 1 HNMR (300 MHz, CDCl3): δ ppm 3.64(s, 3H);2.21 (d, 2H, J = 6.9 Hz);1. 85-1.81(m,1H);1.23-1.20(m,16H);0.86-0.84 (m, 6H).

[0339] A2. Compound 2b: Methyl 3-isopropyl nonanoate [ka] The procedure is the same as in A1, but isopropylmagnesium bromide is used. Yield = 2.4g (38%). 1 HNMR(300 MHz, CDCl3): δ ppm 3.65 (s, 3H);2.25(dd, 1H, J = 15.1, 6.1Hz);2.14 (dd, 1H, J = 15.1, 7.1 Hz);1.23-1.20 (m, 12 H); 0.87-0.84 (m, 9H).

[0340] A3. Compound 2c: Methyl 3-propyl nonanoate [ka] The procedure is the same as in A1, but n-propylmagnesium bromide is used. Yield = 2.2g (35%). 1 HNMR(300 MHz, CDCl3): δ ppm 3.64 ( s, 3H);2.23(d, 2H, J = 6.8 Hz);1.85-1.84(m ,1H);1.23-1.20(m,14H);0.87-0.84 (m, 6H).

[0341] Typical procedure B: LAH reduction B1. Compound 3a: 3-butylnonan-1-ol [ka] A solution of methyl 3-butylnonanoate 2a (2.2 g, 9.63 mmol) in THF (10 mL) was added dropwise to a stirred suspension of LiAlH4 (0.73 g, 19.27 mmol) in THF (10 mL) under N2 conditions. The mixture was heated under reflux for 5 hours. The reaction product was cooled to room temperature. 0.7 mL of H2O, 0.7 mL of 15% NaOH, and 2.1 mL of H2O were added sequentially under an ice bath at 0°C. The white precipitate was filtered, and the filtrate was concentrated. The crude product was purified by flash chromatography (SiO2:ethyl acetate / hexane 0-100%) to obtain a colorless oil product 3a (980 mg, 51%). 1 HNMR(300 MHz, CDCl3): δ ppm 3.64 (t, 2H, J = 6.8 Hz) ;1.52 (q,2H,J = 7.1 Hz);1.32-1.30 (m, 1H); 1.23-1.20(m, 17H);0.88-0.84 (m, 6H).

[0342] B2. Compound 3b: 3-Isopropylnonan-1-ol [ka] The procedure is the same as B1, but methyl 3-isopropyl nonanoate, 2b is used. Yield = 1.7g (81%). 1 HNMR (300 MHz, CDCl3): δ ppm 3.64 (m,2H);1.55(m, 2H);1.43-1.41 (m, 1H) ;1.23-1.20(m, 12H);0.88-0.84 (m, 9H).

[0343] B3. Compound 3c: 3-Propylnonan-1-ol [ka] The procedure is the same as in B1, but methyl 3-propyl nonanoate, 2c is used. Yield = 1.28g (67%). 1 HNMR(300 MHz, CDCl3): δ ppm 3 .64 (t,2H,J = 6.6 Hz);1.52 (q, 2H, J = 6.3 H z);1.23-1.20(m, 16H);0.88-0.84 (m, 6H).

[0344] Typical procedure C for the esterification of 8-bromooctanoic acid, 4 C1. Compound 5a: 3-butylnonyl 8-bromooctanoate [ka] To a solution of 3-butylnonan-1-ol 3a (458 mg, 2.28 mmol), 8-bromooctanoic acid 4 (611.9 mg, 2.74 mmol), and DMAP (55.9 mg, 0.46 mmol) in dichloromethane (30 mL) at 0°C, EDCI (657.3 mg, 3.43 mmol) was added, and the reaction mixture was stirred overnight at room temperature. TLC showed completion of the reaction. The reaction mixture was cooled to 0°C, 1N hydrochloric acid (3 mL) was slowly added, and the mixture was extracted with diethyl ether (100 mL) to separate the layers. The organic layer was washed with saturated sodium bicarbonate (100 mL), water, and brine. The organic layer was separated and concentrated. The crude product was purified by flash chromatography (SiO2:hexane / diethyl ether 0-100%) to obtain a colorless oil product 5a (680 mg, 73%). 1 HNMR(300 MHz, CDCl3): δ ppm 4.07 (t, 2H, J = 6.8Hz);3.39 (t, 2H, J =6.8 Hz);2.28 (t, 2H, J = 7.6 Hz);1.88-1.79 (m, 2H);1.70-1.4 2 (m, 6H);1.38-1.17(m,21H);0.88-0.82 (m, 6H).

[0345] C2. Compound 5b: 3-Isopropylnonyl 8-bromooctanoate [ka] The procedure is the same as C1, but 3-isopropylnonan-1-ol and 3b are used. Yield = 297 mg (71%). 1 HNMR(300 MHz, CDCl3): δ ppm 4.05 (dd, 2H, J = 14.3, 6.6 Hz);3.39 (t,2H, J = 6.8Hz);2.28 (t, 2H, J = 7.7Hz);1.86-1. 81 (m, 2H);1.70-1.42(m, 6H);1.38-1.17(m, 16H); 0.84-0.82 (m, 9H).

[0346] C3. Compound 5c: 3-Propylnonyl 8-bromooctanoate [ka] The procedure is the same as C1, but 3-propylnonan-1-ol, 3c is used. Yield = 430 mg (68%). 1 HNMR(300 MHz, CDCl3): δ ppm 3 .96 (d,2H,J = 5.8 Hz);3.38 (t, 2H, J = 5.5 H z);2.27(t, 2H, J = 7.4 Hz);1.88-1.79 (m,2H );1.70-1.42(m, 6H);1.38-1.17 (m, 19H);0.88-0.82 (m, 6H).

[0347] Typical procedure D: N-alkylation of heptadecan-9-yl8-((2-hydroxyethyl)amino)octanoate, 6 D1. Compound 7: 3-Butylnonyl 8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoate (86g-nBu) [ka] In a 500 mL round-bottom flask connected to a condenser, heptadecan-9-yl-8-((2-hydroxyethyl)amino)octanoate 6 (601 mg, 1.36 mmol), 3-butylnonyl-8-bromooctanoate 5a (606 mg, 1.49 mmol), potassium carbonate (676 mg, 4.9 mmol), and potassium iodide (248.4 mg, 1.49 mmol) were mixed in cyclopentyl methyl ether (30 mL) and acetonitrile (30 mL). The reaction mixture was heated to 85 °C for 18 hours. MS showed a clean conversion, and the mixture was cooled to room temperature and diluted with hexane. The mixture was filtered through a Celite pad. After washing with hexane, the filtrate was concentrated to obtain a brown oil, which was purified by flash chromatography (SiO2:hexane / diethyl ether 0-100%) to obtain compound 7 as a colorless oil (588 mg, 56%). HPLC / ELSD: RT=7.07 min, MS(CI): m / z(MH + )C 48 H 95 766.7 compared to NO5. 1 HNMR(300 MHz, CDCl3) δ: ppm 4.85 (quintet, 1H, J = 6.1 Hz);4.07 (t, 2H, J= 6.9Hz);3.50 (t, 2H, J =5.5Hz);2.98 (bs, 1H);2.55 (t, 2H, J = 5.2 Hz);2.41(t, 4H, J = 7.4 Hz);2.26 (t,4 H, J = 7.4Hz);1.65-1.48 (m, 19H);1.26 (br. m, 48H);0.88-0.84 (m, 12H).

[0348] D2. Compound 12: Heptadecan-9-yl 8-((2-hydroxyethyl)(8-((3-isopropylnonyl)oxy)-8-oxooctyl)amino)octanoate(86-g-iPr) [ka] The procedure is the same as D1, but 3-isopropylnonyl 8-bromooctanoate, 5b is used. Yield = 258 mg (50%). HPLC / ELSD: RT = 6.98 min, MS (CI): m / z (MH) + )C 47 H 93 752.6 compared to NO5. 1 HNMR(300 MHz, CDCl3) δ: ppm 4.85 (quintet, 1H, J = 6.1 Hz);4.07(m,2H);3.50 (t, 2H, J = 5.2 Hz);3. 01 (bs,1H);2.55 (t, 2H, J = 5.2 Hz);2.41 (t , 4H, J=7.4 Hz);2.26 (dd, 4H, J = 7.6, 2.7 H z);1.65-1.48(m, 14H);1.26 (br. m, 48H);0. 88-0.84 (m, 15H).

[0349] D3. Compound 13: Heptadecan-9-yl 8-((2-hydroxyethyl)(8-oxo-8-((3-propylnonyl)oxy)octyl)amino)octanoate(86-g-nPr) [ka] The procedure is the same as D1, but 3-propylnonyl 8-bromooctaato, 5c is used. Yield = 510 mg (68%). HPLC / ELSD: RT = 7.01 min, MS (CI): m / z (MH) + )C 47 H 93 752.6 compared to NO5. 1 HNMR (300 M) Hz, CDCl3)δ:ppm 4.85 (quintet, 1H, J = 6.3 Hz); 4.07 (t,2H, J = 7.1 Hz);3.50 (t, 2H, J = 5.5 Hz);2.98(bs, 1H);2.55 (t, 2H, J = 5.2 Hz);2.41 (t, 4H, J = 7.4 Hz);2.26 (t, 4H,J = 7.4 H z);1.65-1.48(m, 17H);1.26 (br. m, 48H);0. 88-0.84 (m, 12H).

[0350] Synthesis scheme for the preparation of compound 8 [ka] C4. Compound 5d: 2-Propylnonyl-8-bromooctanoate [ka] The procedure is the same as C1, but 2-propylnonan-1-ol 15a is used. Yield = 1.67g (79%). 1 HNMR(300 MHz, CDCl3): δ ppm 3 .96 (d,2H,J = 5.8 Hz);3.38 (t, 2H, J = 5.5 H z);2.27(t, 2H, J = 7.4 Hz);1.88-1.79 (m,2H );1.70-1.42(m, 6H);1.38-1.17 (m, 19H);0.88-0.82 (m, 6H).

[0351] D4. Compound 8: Heptadecan-9-yl 8-((2-hydroxyethyl)(8-oxo-8-((2-propylnonyl)oxy)octyl)amino)octanoate(86-b-nPr) [ka] The procedure is the same as D1, but 2-propylnonyl 8-bromooctanoate, 5d is used. Yield = 355 mg (68%). HPLC / ELSD: RT = 7.0 min, MS (CI): m / z (MH) + )C47 H 93 752.6 compared to NO5. 1 HNMR (300 MHz, CDCl3)δ:ppm 4.85 (quintet, 1H, J = 6.3 Hz) ;3.95 (d,2H, J = 5.8 Hz);3.50 (t, 2H, J = 5. 5 Hz);3.02(bs,1H);2.55 (t, 2H, J = 5.5 Hz) ;2.41 (t,4H,J = 7.7 Hz);2.26 (dd, 4H, J = 1 3.9, 6.6Hz);1.65-1.48(m, 17H);1.26 (br. m , 48H); 0.88-0.84(m, 12H).

[0352] Synthesis of intermediates: Intermediate AA: Ethyl 3-propylhexa-2-enoate [ka] To a suspension of sodium hydride (2.28 g, 56.9 mmol) in THF (17 mL), triethyl phosphonoacetate (11.3 mL, 56.9 mmol) was added dropwise over 20 minutes, and the mixture was stirred at room temperature until gas evolution ceased (approximately 30 minutes). The reaction mixture was cooled to 0°C, and 4-heptanone (6.12 mL, 43.8 mmol) was added gradually. The reaction mixture was gradually warmed to room temperature and stirred under reflux for 24 hours. After the reaction mixture cooled to room temperature, it was quenched with saturated sodium bicarbonate aqueous solution. The aqueous phase was extracted with diethyl ether, the organic extract was washed with brine, dried, and concentrated in (MgSO4). The crude product was purified by silica gel chromatography (0-20% siRNA:hexane) to obtain ethyl 3-propylhexa-2-enoate (8.07 g, 43.8 mmol, 100%) as a clear oil and as a mixture of positional isomers. 1 HNMR (300 MHz, CDC) l3) As a mixture of positional isomers, δ:ppm 5.63 (s, 1H); 5.38-5.25 (m, 0.74H); 4.19-4.07 (m, 3.40H); 3.02 (s , 0.81H);2.96(s,0.59H);2.57 (ddd, 2H, J = 6.0, 6.0,3.0 Hz);2.16-1.98 (m, 4.87H);1.5 7-1.35 (m,6.10H);1.34-1.21(m, 7.59H);1.0 1-0.82 (m, 12.9H).

[0353] Intermediate AB: Ethyl 3-propylhexanoate [ka] A steel Parr reactor equipped with a stirring bar was packed with ethyl 3-propylhexa-2-enoate (8.07 g, 43.8 mmol) in ethanol (44 mL). Palladium carbon hydroxide (922 mg, 6.57 mmol) was added, the vessel was sealed, evacuated, refilled with H2 gas (3×), and the pressure was set to 200 psi. The reaction mixture was stirred at room temperature for 2 hours at 500 rpm under H2 gas at 200 psi. The vessel was then evacuated, refilled with N2 gas, and opened. The crude reaction mixture was filtered through a Celite pad. The Celite pad was washed with EtOH, and the crude substance was concentrated to obtain ethyl 3-propylhexanoate (6.55 g, 35.2 mmol, 80%) as a clear oil. Without further purification, the compound was moved to the next step. 1 HNMR(300 MHz, CDCl3) δ: ppm 4 .12 (q,2H,J = 6.0 Hz);2.22 (d, 2H, J = 9.0 H z);1.95-1.81(m, 1H);1.38-1.18 (m, 11H);0.89(br. t, 6H, J = 6.0 Hz).

[0354] Intermediate AC: 3-propylhexane-1-ol [ka] Under N2 conditions at 0°C, ethyl 3-propylhexanoate (6.55 g, 35.2 mmol) in 28 mL of anhydrous ether was added dropwise to a mixture of lithium aluminum hydride (1.60 g, 42.2 mmol) in 42 mL of anhydrous ether. The mixture was stirred at room temperature for 2.5 hours and then cooled to 0°C. Water (1 mL per 4 g of LiAlH) was added dropwise to the solution, followed by the slow addition of 15% sodium hydroxide (1 mL per 4 g of LiAlH) and water (3 mL per 4 g of LiAlH). The solution was stirred at room temperature for several minutes and filtered through a Celite pad. The Celite pad was washed with diethyl ether, and the filtrate was concentrated. The crude substance was purified by silica gel chromatography (0-40% SiO:hexane) to obtain 3-propylhexane-1-ol (4.82 g, 33.4 mmol, 95%) as a clear oil. 1 HNMR (300 MHz, CDCl3) δ: pp m 3.67 (t,2H, J = 6.0 Hz);1.57-1.39 (m, 3H) ;1.37-1.18(m,9H);0.88 (t, 6H, J = 6.0 Hz).

[0355] Intermediate AD: Ethyl 3-butylhepta-2-enoate [ka] To a suspension of sodium hydride (1.83 g, 45.7 mmol) in THF (14 mL), triethyl phosphonoacetate (9.07 mL, 45.7 mmol) was added dropwise over 20 minutes, and the mixture was stirred at room temperature until gas generation ceased (approximately 30 minutes). The reaction mixture was cooled to 0°C, and 5-nonanone (6.05 mL, 35.2 mmol) was added gradually. The reaction mixture was gradually warmed to room temperature and stirred under reflux for 24 hours. After the reaction mixture cooled to room temperature, it was quenched with saturated sodium bicarbonate aqueous solution. The aqueous phase was extracted with diethyl ether, the organic extract was washed with brine, dried (MgSO4), and concentrated. The crude product was purified by silica gel chromatography (0-20% siRNA:hexane) to obtain ethyl 3-butylhepta-2-enoate (5.27 g, 24.8 mmol, 71%) as a clear oil. 1 HNMR(300 MHz, CDCl3) δ: ppm 5.62 (s, 1H);4.14 (q, 2H, J = 6.0 Hz);2.59(t,2H, J = 6.0 Hz);2.14(t,2H, J = 6.0 Hz);1.50-1.23 (m, 11H);0.99-0.82 (m, 6H).

[0356] Intermediate AE: Ethyl 3-butylheptanoate [ka] A steel Parr reactor equipped with a stirring bar was packed with ethyl 3-butylhepta-2-enoate (10.5 g, 49.5 mmol) in ethanol (50 mL). Palladium carbon hydroxide (1.04 g, 7.42 mmol) was added, the vessel was sealed, evacuated, refilled with H2 gas (3 ×), and the pressure was set to 200 psi. The reaction mixture was stirred at room temperature for 2 hours at 500 rpm under H2 gas at 200 psi. The vessel was then evacuated, refilled with N2 gas, and opened. The crude reaction mixture was filtered through a Celite pad. The Celite pad was washed with EtOH, and the crude substance was concentrated to obtain ethyl 3-butylheptanoate (9.69 g, 45.2 mmol, 91%) as a clear oil. Without further purification, the compound was moved to the next step. 1 HNMR(300 MHz, CDCl3) δ: ppm 4.1 2 (q, 2H,J= 9.0 Hz);2.22 (d, 2H, J = 6.0 Hz) ;1.90-1.76(m, 1H);1.38-1.19 (m, 15H);0.88 (br.t, 6H, J = 6.0 Hz).

[0357] Intermediate AF: 3-butylheptan-1-ol [ka] Under N2 conditions at 0°C, ethyl 3-butylheptanoate (4.00 g, 18.7 mmol) in anhydrous ether (15 mL) was added dropwise to a mixture of lithium aluminum hydride (850 mg, 22.4 mmol) in anhydrous ether (23 mL). The mixture was stirred at room temperature for 2.5 hours and then cooled to 0°C. Water (1 mL per 1 g of LiAlH4) was added dropwise to the solution, followed by the slow addition of 15% sodium hydroxide (1 mL per 1 g of LiAlH4) and water (3 mL per 1 g of LiAlH4). The solution was stirred at room temperature for several minutes and filtered through a Celite pad. The Celite pad was washed with diethyl ether, and the filtrate was concentrated. The crude substance was purified by silica gel chromatography (0-40% SiO:hexane) to obtain 3-butylheptan-1-ol (3.19 g, 18.5 mmol, 99%) as a clear oil. 1 HNMR (300 MHz, CDCl3) δ: ppm 3.66 (t,2H,J = 6.0 Hz);1.53 (q, 2H, J = 6.0 Hz);1.46-1.36(m, 1H);1.35-1.21 (m, 12H);1.18(br. s, 1H);0.89 (br. t, 6H, J = 6.0 Hz).

[0358] Intermediate AG: Ethyl 3-pentyl octa-2-enoate [ka] To a suspension of sodium hydride (2.13 g, 53.4 mmol) in THF (16 mL), triethyl phosphonoacetate (10.6 mL, 53.4 mmol) was added dropwise over 20 minutes, and the mixture was stirred at room temperature until gas evolution ceased (approximately 30 minutes). The reaction mixture was cooled to 0°C, and 6-undecanone (8.42 mL, 41.1 mmol) was added gradually. The reaction mixture was gradually warmed to room temperature and stirred under reflux for 60 hours. After the reaction mixture cooled to room temperature, it was quenched with saturated sodium bicarbonate aqueous solution. The aqueous phase was extracted with diethyl ether, the organic extract was washed with brine, dried (MgSO4), and concentrated. The crude product was purified by silica gel chromatography (0-20% toluene:hexane) to obtain ethyl 3-pentyl octa-2-enoate (8.76 g, 36.5 mmol, 89%) as a clear oil. 1 HNMR(300 MHz, CDCl3) δ: ppm 5.61 (s, 1H);4.14(q, 2H, J = 6.0 Hz);2.58(ddd, 2H, J =9.0,9.0, 6.0 Hz);2.13 (ddd, 2H, J = 6.0, 6.0,3.0Hz);1.52-1.38 (m, 3H);1.38-1 0.23 (m, 12H); 0.93-0.86 (m, 6H).

[0359] Intermediate AH: Ethyl 3-pentyl octanoate [ka] A steel Parr reactor equipped with a stirring bar was packed with ethyl 3-pentyl octa-2-enoate (8.76 g, 36.5 mmol) in ethanol (37 mL). Palladium carbon hydroxide (768 mg, 5.47 mmol) was added, the vessel was sealed, evacuated, refilled with H2 gas (3×), and the pressure was set to 200 psi. The reaction mixture was stirred at room temperature for 2 hours at 500 rpm under H2 gas at 200 psi. The vessel was then evacuated, refilled with N2 gas, and opened. The crude reaction mixture was filtered through a Celite pad. The Celite pad was washed with EtOH, and the crude substance was concentrated to obtain ethyl 3-pentyl octanoate (8.45 g, 34.9 mmol, 96%) as a clear oil. Without further purification, the compound was moved to the next step. 1 HNMR(300 MHz, CDCl3) δ: ppm 4 .12 (q,2H,J = 6.0 Hz);2.22 (d, 2H, J = 6.0 H z);1.92-1.77(br. m, 1H);1.37-1.19 (m, 19H );0.88 (t,6H,J = 6.0 Hz).

[0360] Intermediate AI: 3-Pentyloctane-1-Oarl [ka] Under N2 conditions at 0°C, ethyl 3-pentyl octanoate (8.45 g, 34.9 mmol) in 28 mL of anhydrous ether was added dropwise to a mixture of lithium aluminum hydride (1.59 g, 41.8 mmol) in 42 mL of anhydrous ether. The mixture was stirred at room temperature for 2.5 hours and then cooled to 0°C. Water (1 mL per 4 g of LiAlH) was added dropwise to the solution, followed by the slow addition of 15% sodium hydroxide (1 mL per 4 g of LiAlH) and water (3 mL per 4 g of LiAlH). The solution was stirred at room temperature for several minutes and filtered through a Celite pad. The Celite pad was washed with diethyl ether, and the filtrate was concentrated. The crude substance was purified by silica gel chromatography (0-40% SiO:hexane) to obtain 3-pentyloctan-1-ol (6.98 g, 34.9 mmol, 100%) as a clear oil. 1 HNMR (300 MHz, CDCl3) δ: p pm 3.66(t, 2H, J = 6.0 Hz);1.53 (q, 2H, J =6 .0 Hz);1.47-1.37(br. s, 1H);1.36-1.15 (m, 17H);0.88 (t, 6H, J = 6.0 Hz).

[0361] Intermediate AJ: 3-Pentyloctanal [ka] 3-Pentyloctan-1-ol (6.98 g, 34.9 mmol) was added to a stirred suspension of pyridinium chlorochromate (9.02 g, 41.8 mmol) and silica gel (9.02 g per 1 g / g of pyridinium chlorochromate) in dichloromethane (90 mL) under an N2 atmosphere. The suspension was stirred at room temperature for 1 hour. The reaction product was then filtered through a Celite pad, the Celite pad was washed with dichloromethane, and the filtrate was concentrated. The crude product was purified by silica gel chromatography (0-20% siRNA:hexane) to obtain 3-pentyloctanal (4.66 g, 23.5 mmol, 67%) as a clear oil. 1HNMR(300 MHz, CDCl3) δ: ppm 9.76 (t, 1H, J = 3.0 Hz);2.33 (dd, 2H, J= 6.0,3.0 Hz);2.01-1.86 (br. m, 1H);1.40-1.19 (m, 16H) ;0.88 (t, 6H, J = 6.0 Hz).

[0362] Intermediate AK:6-allyl-undecane [ka] To a suspension of methyltriphenylphosphonium bromide (4.68 g, 13.1 mmol) in anhydrous ether (190 mL) under N2 conditions, tert-butoxide potassium (1.47 g, 13.1 mmol) was added all at once. The mixture was stirred at room temperature for 15 minutes, and then 3-pentyloctanal (2.00 g, 10.1 mmol) in anhydrous ether (26 mL) was added dropwise over 15 minutes. The resulting mixture was stirred at room temperature for 90 minutes. The reaction mixture was diluted with ice water, the layers were separated, and the organic layer was extracted with ether. The combined organic matter was dried (MgSO4), filtered, and concentrated. The crude substance was purified by silica gel chromatography (0-10% SiO:hexane) to obtain 6-allyl undecane (1.65 g, 8.38 mmol, 83%) as a clear oil. 1 HNMR (300 MHz, CDCl3) δ: ppm 5.77(dddd,1H, J = 15.0, 12.0, 9.0, 9 .0 Hz);5.03-4.94(m, 2H);2.02 (ddd, 2H, J = 9.0,6.0, 6.0 Hz);1.43-1.16 (m, 17H);0.88 ( d, 6H, J = 6.0 Hz).

[0363] Intermediate AL: 4-pentylnonane-1-ol [ka] A solution of sodium borohydride (131 mg, 3.46 mmol) in anhydrous diglyme (3.6 mL) was stirred under an N2 atmosphere. A solution of 6-allylundecane (2.26 g, 11.5 mmol) in anhydrous diglyme (2.3 mL) was added. Next, a solution of boron trifluoride ethretate (569 μL, 4.61 mmol) in 1.2 mL of anhydrous diglyme at room temperature was added over 15 minutes. The resulting mixture was stirred for 1 hour, after which water (1.2 mL) was added dropwise. When gas generation ceased, 2.3 mL of 3 M NaOH at room temperature was added, followed by 2.3 mL of 30% H2O2 at 40°C, added dropwise. After stirring at 40°C for 1 hour, the reaction mixture was poured into 10 mL of water. The reaction vessel was washed with additional water. The combined aqueous solution was extracted with ether (2 ×). The combined ether extract was washed with water (× 5). The etheric extract was dried (MgSO4), filtered, and concentrated. The crude residue was purified by silica gel chromatography (0-40% toluene:hexane) to obtain 4-pentylnonan-1-ol (1.88 g, 8.77 mmol, 76%) as a clear oil. 1 HNMR(300 MHz, CDCl3) δ: ppm 3.62 (t, 2H, J = 6.0 Hz); 1.60-1.48(m,2H);1.37-1.19 (m, 20H);0.88 (t, 6H, J = 6.0 Hz).

[0364] Intermediate AM: 3-Propylhexyl 8-bromooctanoate [ka] To a solution of 3-propylhexane-1-ol (4.82 g, 33.4 mmol), 8-bromooctanoic acid (8.94 g, 40.1 mmol), and DMAP (816 mg, 6.68 mmol) in methylene chloride (58 mL) at 0°C, EDCI (9.60 g, 50.1 mmol) was added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was then cooled to 0°C, and a solution of 10% hydrochloric acid (180 mL) was slowly added over 20 minutes. The layers were separated, and the organic layer was concentrated under vacuum to obtain crude oil. The oil was dissolved in hexane (180 mL) and washed with a mixture of acetonitrile (180 mL) and 5% sodium bicarbonate (180 mL). The hexane layer was separated, dried, filtered, and filtered. The solvent was removed under vacuum to obtain 3-propylhexyl 8-bromooctanoate (10.9 g, 31.2 mmol, 93%) as a clear oil. Without further purification, the compound was moved to the next step. 1 HNMR(300 MHz, CDCl3) δ: ppm 4.08 (t, 2 H, J = 6.0Hz);3.40(t, 2H, J = 6.0Hz);2.28 (t, 2H,J= 6.0 Hz);1.85 (quintet, 2H, J = 6.0 Hz );1.68-1.51(m,4H);1.49-1.18 (m, 15H);0.8 8 (t, 6H,J = 6.0 Hz).

[0365] Intermediate AN: 3-butylheptyl 8-bromooctanoate [ka] To a solution of 3-butylheptan-1-ol (3.19 g, 18.5 mmol), 8-bromooctanoic acid (4.96 g, 22.2 mmol), and DMAP (453 mg, 3.71 mmol) in methylene chloride (32 mL) at 0°C, EDCI (5.33 g, 27.8 mmol) was added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was then cooled to 0°C, and a solution of 10% hydrochloric acid (150 mL) was slowly added over 20 minutes. The layers were separated, and the organic layer was concentrated under vacuum to obtain crude oil. The oil was dissolved in hexane (150 mL) and washed with a mixture of acetonitrile (150 mL) and 5% sodium bicarbonate (150 mL). The hexane layer was separated, dried, filtered, and filtered. The solvent was removed under vacuum to obtain 3-butylheptyl 8-bromooctanoate (6.90 g, 18.3 mmol, 99%) as a clear oil. Without further purification, the compound was moved to the next step. 1 HNMR(300 MHz, CDCl3) δ: ppm 4.08 (t, 2H, J = 6.0Hz);3.40 (t, 2H, J =6.0 Hz);2.29 (t, 2H, J = 6.0 Hz);1.85 (quintet, 2H, J = 6.0 Hz);1 .69-1.52(m,4H);1.49-1.20 (m, 19H);0.89 ( (br. t, 6H,J = 6.0 Hz).

[0366] Intermediate AO: 3-pentyloctyl 8-bromooctanoate [ka] To a solution of 3-pentyloctan-1-ol (2.00 g, 9.98 mmol), 8-bromooctanoic acid (2.67 g, 12.0 mmol), and DMAP (244 mg, 2.00 mmol) in methylene chloride (18 mL) at 0°C, EDCI (2.87 g, 15.0 mmol) was added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was then cooled to 0°C, and a solution of 10% hydrochloric acid (70 mL) was slowly added over 20 minutes. The layers were separated, and the organic layer was concentrated under vacuum to obtain crude oil. The oil was dissolved in hexane (70 mL) and washed with a mixture of acetonitrile (70 mL) and 5% sodium bicarbonate (70 mL). The hexane layer was separated, dried, filtered, and filtered. The solvent was removed under vacuum to obtain 3-pentyloctyl 8-bromooctanoate (3.94 g, 9.72 mmol, 97%) as a clear oil. Without further purification, the compound was moved to the next step. 1 HNMR(300 MHz, CDCl3) δ: ppm 4.08 (t, 2H, J = 6.0 Hz);3.40(t,2H, J = 6.0 Hz);3.29 (t, 2 H, J = 6.0Hz);1.85 (quintet, 2H, J = 6.0 Hz);1.68-1.52(m, 4H);1.49-1.19 (m, 23H);0.88 (t, (6H, J = 6.0 Hz).

[0367] Intermediate AP: 4-pentylnonyl-8-bromooctanoate [ka] To a solution of 4-pentylnonan-1-ol (1.88 g, 8.77 mmol), 8-bromooctanoic acid (2.35 g, 10.5 mmol), and DMAP (214 mg, 1.75 mmol) in methylene chloride (15 mL) at 0°C, EDCI (2.52 g, 13.2 mmol) was added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was then cooled to 0°C, and a solution of 10% hydrochloric acid (60 mL) was slowly added over 20 minutes. The layers were separated, and the organic layer was concentrated under vacuum to obtain crude oil. The oil was dissolved in hexane (60 mL) and washed with a mixture of acetonitrile (60 mL) and 5% sodium bicarbonate (60 mL). The hexane layer was separated, dried, filtered, and filtered. The solvent was removed under vacuum to obtain 4-pentylnonyl-8-bromooctanoate (3.68 g, 8.77 mmol, 100%) as a clear oil. Without further purification, the compound was moved to the next step. 1 HNMR(300 MHz, CDCl3) δ: ppm 4.04 (t, 2H, J = 6.0 Hz);3.40(t,2H, J = 6.0 Hz);2.29 (t, 2 H, J = 6.0Hz);1.85 (quintet, 2H, J = 6.0 Hz);1.70-1.52(m, 4H);1.50-1.18 (m, 25H);0.88 (t, (6H, J = 6.0 Hz).

[0368] Intermediate AQ: Pentadecane-8-yl-8-bromooctanoate [ka] To a solution of 8-bromooctanoic acid (1.98 g, 8.87 mmol) in dichloromethane (30 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.13 g, 11.1 mmol), 4-(dimethylamino)pyridine (0.217 g, 1.77 mmol), and pentadecane-8-ol (2.03 g, 8.87 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was cooled to 0°C, and a 10% hydrochloric acid solution was slowly added. The organic layer was separated and evaporated under vacuum. The residue was dissolved in hexane and washed with a 1:1 mixture of acetonitrile and saturated NaHCO3 (aqueous solution). The hexane layer was separated, dried over MgSO4, then filtered, and evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% ethyl acetate in hexane) to obtain a mixture of pentadecane-8-yl-8-bromooctanoate and pentadecane-8-yl-8-chlorooctanoate in a ratio of approximately 13.2:1 (3.19 g, 83.1%) as a colorless liquid. 1 HNMR(300 MHz, CDCl3) δ: ppm 4. 89 (p, 1H);3.55(t,0.14H);3.42 (t, 1.86H); 2.31 (t,2H);1.88 (p, 2H);1.72-1.59 (m, 2H);1.59-1.42(m, 6H);1.42-1.18 (m, 24H);0.90 (t,6H).

[0369] Intermediate AR: Tridecane-7-yl-8-bromooctanoate [ka] To a solution of 8-bromooctanoic acid (1.96 g, 8.76 mmol) in dichloromethane (30 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.10 g, 10.9 mmol), 4-(dimethylamino)pyridine (0.234 g, 1.92 mmol), and tridecane-7-ol (1.75 g, 8.73 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with dichloromethane and extracted with saturated NaHCO3 (aqueous solution). The organic layer was separated, washed with brine, dried over MgSO4, filtered, and evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% ethyl acetate in hexane) to obtain a mixture of tridecane-7-yl-8-bromooctanoate and tridecane-7-yl-8-chlorooctanoate in a ratio of approximately 12.3:1 (2.10 g, 59.4%) as a colorless liquid. 1 HNMR (300 M) Hz, CDCl3)δ:ppm 4.89 (p, 1H);3.55 (t, 0.15 H);3.42(t, 1.85H);2.31 (t, 2H);1.88 (p, 2H);1.72-1.60(m, 2H);1.60-1.42 (m, 6H);1.42 -1.19 (m, 20H); 0.90 (t, 6H).

[0370] Intermediate AS: Undecane-6-yl-8-bromooctanoate [ka] To a solution of 8-bromooctanoic acid (4.00 g, 17.9 mmol) in dichloromethane (60 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (4.31 g, 22.5 mmol), 4-(dimethylamino)pyridine (0.438 g, 3.58 mmol), and 6-undecanol (3.09 g, 17.9 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was cooled to 0°C, and a 10% hydrochloric acid solution was slowly added. The organic layer was separated and evaporated under vacuum. The residue was dissolved in hexane and washed with a 1:1 mixture of acetonitrile and saturated NaHCO3 (aqueous solution). The hexane layer was separated, dried over MgSO4, then filtered, and evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% ethyl acetate in hexane) to obtain a mixture of undecane-6-yl-8-bromooctanoate and undecane-6-yl-8-chlorooctanoate in a ratio of approximately 19:1 (4.33 g, 64.01%) as a colorless liquid. 1 HNMR(300 MHz, CDCl3) δ: ppm 4.89 (p, 1 H);3.55(t,0.10H);3.42 (t, 1.90H);2.31 (t , 2H);1.88(p,2H);1.72-1.59 (m, 2H);1.59- 1.42 (m,6H);1.42-1.18(m, 16H);0.90 (t, 6H ).

[0371] Intermediate AT: Nonan-5-yl-8-bromooctanoate [ka] To a solution of 8-bromooctanoic acid (4.00 g, 17.9 mmol) in dichloromethane (60 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (4.31 g, 22.5 mmol), 4-(dimethylamino)pyridine (0.438 g, 3.59 mmol), and 5-nonanol (2.59 g, 17.9 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was cooled to 0°C, and a 10% hydrochloric acid solution was slowly added. The organic layer was separated and evaporated under vacuum. The residue was dissolved in hexane and washed with a 1:1 mixture of acetonitrile and saturated NaHCO3 (aqueous solution). The hexane layer was separated, dried over MgSO4, then filtered, and evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% ethyl acetate in hexane) to obtain a mixture of nonane-5-yl-8-bromooctanoate and nonane-5-yl-8-chlorooctanoate in a ratio of approximately 7:1 (5.23 g, 83.5%) as a colorless liquid. 1 HNMR(300 MHz, CDCl3) δ: ppm 4.90 (p, 1H);3.55 (t, 0.25H);3.42(t, 1.75H);2.31(t, 2H);1.88 (p, 2H);1.72-1.59 (m, 2H);1.59-1.19 (m, 18H);0.91 (t,6H).

[0372] Intermediate AU:3-Propylhexyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate [ka] To a solution of tert-butyl N-(3-aminopropyl)carbamate (23.9 g, 137 mmol) in EtOH (60 mL), 3-propylhexyl 8-bromooctanoate (8.00 g, 22.3 mmol) in EtOH (55 mL) was added over 20 minutes. The reaction mixture was heated to 60°C and stirred at this temperature for 16 hours. The solvent was evaporated during cooling, and the residue was diluted with ethyl acetate and washed with saturated NaHCO3 aqueous solution and brine (5×) until no white precipitate was observed in the aqueous layer. The organic layer was separated, washed with brine, dried in (MgSO4), filtered, and concentrated. The residue was purified by flash chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)) to obtain 3-propylhexyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate (5.81 g, 13.1 mmol, 57%) as a clear oil. 1 HNMR(300 MHz, CDCl3) δ: ppm 5.16 (br. s, 1H);4.08 (t , 2H, J=6.0 Hz);3.19 (br. q, 2H, J = 6.0 Hz) ;2.65 (t,2H, J = 6.0 Hz);2.56 (t, 2H, J =6. 0 Hz);2.27(t,2H, J = 6.0 Hz);1.70-1.51 (m, 6H);1.50-1.39 (m, 3H);1.43 (s, 9H);1.36-1 .17 (m,15H);0.88(t, 6H, J = 6.0 Hz).

[0373] Intermediate AV: 3-Propylhexyl 8-((2-hydroxyethyl)amino)octanoate [ka] 3-propylhexyl 8-bromooctanoate (2.82 g, 8.06 mmol), ethanolamine (14.6 mL, 242 mmol), and ethyl alcohol (6 mL) were added to a round-bottom flask equipped with a stirring bar. The resulting mixture was stirred at 40°C for 16 hours. The reaction product was diluted with dichloromethane, washed with water (2×), and the layers were separated. The organic layer was dried with (MgSO4), filtered, and concentrated. The crude product was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)) to obtain 3-propylhexyl 8-((2-hydroxyethyl)amino)octanoate (876 mg, 2.66 mmol, 33%) as a clear oil. 1 HNMR (300 MHz, CDCl3) δ: ppm 4.08 (t,2H,J = 6.0 Hz);3.63 (t, 2H, J = 6.0 Hz);2.77(t, 2H, J = 6.0 Hz);2.61 (t, 2H, J= 6.0 Hz);2.28 (t, 2H, J = 6.0 Hz);1.91 (br. s , 2H);1.68-1.39(m, 7H);1.38-1.18(m, 14H) ;0.88 (t, 6H, J = 6.0 Hz).

[0374] Intermediate AW: 3-Pentyloctyl 8-((3-((tert-butoxycarbonyl)aminopropyl)amino)octanoate [ka] To a solution of tert-butyl N-(3-aminopropyl)carbamate (15.5 g, 88.8 mmol) in EtOH (38 mL), 3-pentyloctyl 8-bromooctanoate (6.00 g, 14.8 mmol) in EtOH (36 mL) was added over 20 minutes. The reaction mixture was heated to 60°C and stirred at this temperature for 16 hours. The solvent was evaporated during cooling, and the residue was diluted with ethyl acetate and washed with saturated NaHCO3 aqueous solution and brine (5 ×) until no white precipitate was observed in the aqueous layer. The organic layer was separated, washed with brine, dried in (MgSO4), filtered, and concentrated. The residue was purified by flash chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)) to obtain 3-pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate (4.23 g, 8.49 mmol, 57%) as a clear oil. 1 HNMR(300 MHz, CDCl3) δ: ppm 5.17 (br. s, 1H);4.07 ( t, 2H, J=6.0 Hz);3.19 (br. q, 2H, J = 6.0 Hz) );2.66 (t,2H, J = 6.0 Hz);2.56 (t, 2H, J =6 .0 Hz);2.28(t,2H, J = 6.0 Hz);1.70-1.52 (m , 6H);1.51-1.39(m, 3H);1.44 (s, 9H);1.36- 1.19 (m,22H);0.88(t, 6H, J = 6.0 Hz).

[0375] Intermediate AX: 4-Pentylnonyl 8-((2-hydroxyethyl)amino)octanoate [ka] A round-bottom flask equipped with a stirring bar was charged with 4-pentylnonyl 8-bromooctanoate (600 mg, 1.43 mmol), ethanolamine (2.59 mL, 42.9 mmol), and ethyl alcohol (1 mL). The resulting mixture was stirred at 40 °C for 16 h. The reaction was diluted with dichloromethane, washed with water (2×), and the layers were separated. The organic layer was dried (MgSO4), filtered, and concentrated. The crude material was purified by silica gel chromatography (0 - 5 - 10 - 25 - 50 - 100% in dichloromethane (mixture of 1% NH4OH in dichloromethane, 20% MeOH)) to afford 4-pentylnonyl 8-((2-hydroxyethyl)amino)octanoate (306 mg, 0.77 mmol, 54%) as a clear oil. UPLC / ELSD: RT = 1.66 min, MS(ES): m / z(MH + )C 24 H 49 NO3 was 400.31. 1 HNMR(300 MHz, CDCl3) δ: ppm 3.97 (t, 2H, J = 6.0 Hz); 3.57 (br. t, 2H, J = 6.0 Hz); 2.81 (br. s, 2H), 2.67 (br. t, 2H, J = 6.0 Hz); 2.53 (t, 2H, J = 6.0 Hz); 2.22 (t, 2H, J = 6.0 Hz); 1.61 - 1.35 (m, 6 H); 1.32 - 1.10 (m, 25H); 0.81 (t, 6H, J = 6.0 Hz).

[0376] Intermediate AY: 3-pentyloctyl 8-((3-hydroxypropyl)amino)octanoate

Chemical Structure

[0377] Intermediate AZ: Heptadecan-9-yl-8-((3-hydroxypropyl)amino)octanoate [ka] Heptadecan-9-yl-8-bromooctanoate (1.00 g, 2.17 mmol), propanolamine (4.97 mL, 65.0 mmol), and ethyl alcohol (2 mL) were added to a round-bottom flask equipped with a stirring bar. The resulting mixture was stirred at 40°C for 16 hours. The reaction product was diluted with dichloromethane, washed with water (2×), and the layers were separated. The organic layer was dried with (MgSO4), filtered, and concentrated. The crude product was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)) to obtain heptadecan-9-yl-8-((3-hydroxypropyl)amino)octanoate (723 mg, 1.59 mmol, 73%) as a clear oil. UPLC / ELSD: RT=2.06 min, MS(ES): m / z(MH + )C 28 H 57456.17 with respect to NO3.

[0378] Intermediate BA: Heptadecan-9-yl 8-((4-hydroxybutyl)amino)octanoate

Chemical formula

[0379] Intermediate BB: 3-Pentyloctyl 8-((4-hydroxybutyl)amino)octanoate

Chemical formula

[0380] Intermediate BC: 3-Pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate [ka] Potassium carbonate (1.66 g, 12.0 mmol) and potassium iodide (366 mg, 2.21 mmol) were added to a solution of 3-propylhexyl 8-bromooctanoate (735 mg, 2.11 mmol) and 3-pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate (1.00 g, 2.01 mmol) in cyclopentyl methyl ether (9 mL) and actonitrile (9 mL). The reaction mixture was stirred at 80°C for 16 hours. During cooling, volatile substances were evaporated under vacuum. The residue was diluted with dichloromethane and washed with water. The organic layer was separated, washed with brine, dried, filtered, and concentrated. The crude residue was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)) to obtain 3-pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate (698 mg, 0.91 mmol, 45%) as a golden oil. UPLC / ELSD: RT=2.82 min, MS(ES): m / z(MH) + )C 46 H 90 767.59 relative to N2O6.

[0381] Intermediate BD: 3-Butylheptyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate [ka] Potassium carbonate (1.87 g, 13.6 mmol) and potassium iodide (412 mg, 2.49 mmol) were added to a solution of 3-butylheptyl 8-bromooctanoate (895 mg, 2.37 mmol) and 3-propylhexyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate (1.00 g, 2.26 mmol) in cyclopentyl methyl ether (10 mL) and actonitrile (10 mL). The reaction mixture was stirred at 80°C for 16 hours. During cooling, volatile substances were evaporated under vacuum. The residue was diluted with dichloromethane and washed with water. The organic layer was separated, washed with brine, dried, filtered, and concentrated. The crude residue was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)) to obtain 3-butylheptyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate (972 mg, 1.32 mmol, 58%) as a golden oil. UPLC / ELSD: RT=2.69 min, MS(ES): m / z(MH) + )C 44 H 86 739.46 relative to N2O6.

[0382] Intermediate BE: Bis(3-propylhexyl)8,8'-((3-((tert-butoxycarbonyl)amino)propyl)azandiyl)dioctanoate [ka] Potassium carbonate (1.87 g, 13.6 mmol) and potassium iodide (412 mg, 2.49 mmol) were added to a solution of 3-propylhexyl 8-bromooctanoate (829 mg, 2.37 mmol) and 3-propylhexyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate (1.00 g, 2.26 mmol) in cyclopentyl methyl ether (10 mL) and actonitrile (10 mL). The reaction mixture was stirred at 80°C for 16 hours. During cooling, volatile substances were evaporated under vacuum. The residue was diluted with dichloromethane and washed with water. The organic layer was separated, washed with brine, dried, filtered, and concentrated. The crude residue was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)) to obtain bis(3-propylhexyl)8,8'-((3-((tert-butoxycarbonyl)amino)propyl)azandiyl)dioctanoate (730 mg, 1.03 mmol, 45%) as a clear, viscous oil. UPLC / ELSD: RT=2.58 min, MS(ES): m / z(MH) + )C 42 H 82 711.59 relative to N2O6.

[0383] Intermediate BF: 3-Butylheptyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate [ka] Potassium carbonate (1.66 g, 12.0 mmol) and potassium iodide (366 mg, 2.21 mmol) were added to a solution of 3-butylheptyl 8-bromooctanoate (794 mg, 2.11 mmol) and 3-pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate (1.00 g, 2.01 mmol) in cyclopentyl methyl ether (9 mL) and actonitrile (9 mL). The reaction mixture was stirred at 80°C for 16 hours. During cooling, volatile substances were evaporated under vacuum. The residue was diluted with dichloromethane and washed with water. The organic layer was separated, washed with brine, dried, filtered, and concentrated. The crude residue was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)) to obtain 3-butylheptyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate (896 mg, 1.13 mmol, 56%) as a clear oil. UPLC / ELSD: RT=2.95 min, MS(ES): m / z(MH) + )C 48 H 94 795.59 relative to N2O6.

[0384] Intermediate BG: 3-Pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-(undecane-6-yloxy)octyl)amino)octanoate [ka] UPLC / ELSD: RT=2.93 minutes. MS(ESI):m / z C 48 H 95 N2O6 +Calculated value for (M+H) 795.288; measured value 795.71.1H NMR (300 MHz, CDCl3) δ: ppm 5.66 (br. s, 1H); 4.88 (p, 1H); 4.09 (t, 2H); 3.18 (br. d, 2H); 2.50 (br. d, 2H); 2.32 (br. d, 3H); 2.29 (t, 4H); 1 .65-1.46(m, 28H);1.27 (m,44H);0.90 (t, 12 H).

[0385] Intermediate BH: Nonane-5-yl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate [ka] Potassium carbonate (2.00 g, 14.4 mmol) and potassium iodide (439 mg, 2.65 mmol) were added to a solution of nonan-5-yl 8-bromooctanoate (882 mg, 2.53 mmol) and 3-pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate (1.20 g, 2.41 mmol) in cyclopentyl methyl ether (11 mL) and actonitrile (11 mL). The reaction mixture was stirred at 80°C for 16 hours. During cooling, volatile substances were evaporated under vacuum. The residue was diluted with dichloromethane and washed with water. The organic layer was separated, washed with brine, dried, filtered, and concentrated. The crude residue was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)) to obtain nonan-5-yl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate (1.06 g, 1.39 mmol, 58%) as a golden oil. 1HNMR(300 MHz, CDCl3) δ: ppm 5.65 (br. s, 1H);4.87 (quintet, 2H, J = 6 .0 Hz);4.08(t,2H, J = 6.0 Hz);3.18 (br. q, 2H, J =6.0 Hz);2.44 (br. s, 2H);2.35 (br. s , 2H);2.28(t,4H, J = 6.0 Hz);1.71-1.17 (m, 53H);1.43 (s, 9H);0.88 (t, 12H, J = 6.0 Hz) .

[0386] Intermediate BI: Pentadecane-8-yl-8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate [ka] UPLC / ELSD: RT=2.68 min, MS(ESI): m / z C 48 H 95 N2O6 + Calculated value for (M+H) 795.288; measured value 795.71.1H NMR (300 MHz, CDCl3) δ: ppm 5.66 (br. s, 1H); 4.89 (p, 1H); 4.10 (t, 2H); 3.19 (br.d, 2H); 2.56 -2.35 (br.d, 5H);2.30 (t, 5H);1.66-1.39 (m, 66H);0.90 (t, 12H).

[0387] Intermediate BJ: 3-Propylhexyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-(tridecane-7-yloxy)octyl)amino)octanoate [ka] To a solution of 3-propylhexyl 8-({3-[(tert-butoxycarbonyl)amino]propyl}amino)octanoate (1.38 g, 3.11 mmol) in acetonitrile (9 mL), potassium iodide (0.588 g, 3.54 mmol), potassium carbonate (1.73 g, 12.5 mmol), and tridecane-7-yl 8-bromooctanoate (1.26 g, 3.11 mmol) in CMPE (9 mL) were added. The reaction mixture was stirred at 77°C for 18 hours. The reaction mixture was cooled to room temperature, filtered, and the filtrate was evaporated under vacuum. The residue was purified by silica gel chromatography [0-70% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)] to obtain 3-propylhexyl 8-({3-[(tert-butoxycarbonyl)amino]propyl}[8-oxo-8-(tridecane-7-yloxy)octyl]amino)octanoate (1.32 g, 55.1%) as a yellowish oil. UPLC / ELSD: RT=2.70 min, measured value 767.34. 1 HNMR(300 MHz, CDCl3) δ: ppm 5.67 (br. s, 1H);4.89 (p, 1H);4.10 ( t, 2H);3.20(q,2H);2.61-2.43 (m, 2H);2.43 -2.35 (m,4H);2.30(dt, 4H);1.71-1.49 (m, 1 4H);1.49-1.40(m, 12H);1.40-1.19 (m, 36H);1.01-0.83(m, 12H).

[0388] Intermediate BK: 3-Propylhexyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-(undecane-6-yloxy)octyl)amino)octanoate [ka] To a solution of 3-propylhexyl 8-({3-[(tert-butoxycarbonyl)amino]propyl}amino)octanoate (1.50 g, 3.39 mmol) in acetonitrile (10 mL), potassium iodide (0.619 g, 3.73 mmol), potassium carbonate (1.87 g, 13.6 mmol), and a solution of undecane-6-yl 8-bromooctanoate (1.28 g, 3.39 mmol) in CPME (10 mL) were added. The reaction mixture was stirred at 77 °C for 18 hours. The reaction mixture was cooled to room temperature, filtered, and the filtrate was evaporated under vacuum. The residue was purified by silica gel chromatography [0-70% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)] to obtain 3-propylhexyl 8-({3-[(tert-butoxycarbonyl)amino]propyl}[8-oxo-8-(undecane-6-yloxy)octyl]amino)octanoate (1.53 g, 61.2%) as a yellowish oil. UPLC / ELSD: RT=2.56 min, measured value 739.46. 1 HNMR (300 MHz, CDCl) 3) δ: ppm 5.66 (br. s, 1H);4.89 (p, 1H);4.10 (t,2H);3.20(q, 2H);2.60-2.44 (m, 2H);2.4 4-2.35 (m,4H);2.30 (t, 4H);1.74-1.49 (m, 14H);1.49-1.39(m, 12H);1.39-1.19 (m, 32H); 0.91 (t, 12H).

[0389] Intermediate BL: Nonan-5-yl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate [ka] To a solution of 3-propylhexyl 8-({3-[(tert-butoxycarbonyl)amino]propyl}amino)octanoate (1.50 g, 3.39 mmol) in acetonitrile (10 mL), potassium iodide (0.619 g, 3.73 mmol), potassium carbonate (1.87 g, 13.6 mmol), and a solution of nonan-5-yl 8-bromooctanoate (1.18 g, 3.39 mmol) in CMPE (10 mL) were added. The reaction mixture was stirred at 77°C for 18 hours. The reaction mixture was cooled to room temperature, filtered, and the filtrate was evaporated under vacuum. The residue was purified by silica gel chromatography [0-70% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)] to obtain nonan-5-yl 8-({3-[(tert-butoxycarbonyl)amino]propyl}({8-oxo-8-[(3-propylhexyl)oxy]octyl})amino)octanoate (0.483 g, 20.1%) as a yellowish oil. UPLC / ELSD: RT=2.45 min, measured value 711.46. 1 HNMR (300 MHz, CDCl) 3) δ: ppm 5.66 (br. s, 1H);4.89 (p, 1H);4.11 (t,2H);3.28-3.11(m, 2H);2.60-2.44 (m, 2H) );2.44-2.35(m, 4H);2.30 (t, 4H);1.74-1.49 (m, 14H);1.49-1.39(m, 12H);1.39-1.20 (m, 28H); 0.91(t, 12H).

[0390] Intermediate BM: 3-Pentyloctyl 8-((3-aminopropyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate [ka] A solution of 3-pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate (698 mg, 0.91 mmol) in methylene chloride (18 mL) was mixed with trifluoroacetic acid (1.39 mL, 18.2 mmol). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was quenched with saturated aqueous NaHCO3 solution and extracted with dichloromethane. The organic layer was separated, washed with brine, dried (MgSO4), filtered, and concentrated. The crude substance was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)) to obtain 3-pentyloctyl 8-((3-aminopropyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate (378 mg, 0.57 mmol, 62%) as a clear oil. UPLC / ELSD: RT=2.26 min, MS(ES): m / z(MH) + )C 41 H 82 667.56 relative to N2O4.

[0391] Intermediate BN: 3-Butylheptyl 8-((3-aminopropyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate [ka] To a solution of 3-butylheptyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate (972 mg, 1.32 mmol) in methylene chloride (27 mL), trifluoroacetic acid (2.01 mL, 26.3 mmol) was added. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was quenched with saturated NaHCO3 aqueous solution and extracted with dichloromethane. The organic layer was separated, washed with brine, dried (MgSO4), filtered, and concentrated. The crude substance was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)) to obtain 3-butylheptyl 8-((3-aminopropyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate (503 mg, 0.79 mmol, 60%) as a clear oil. UPLC / ELSD: RT=2.13 min, MS(ES): m / z(MH) + )C 39 H 78 639.31 relative to N2O4.

[0392] Intermediate BO: Bis(3-propylhexyl)8,8'-((3-aminopropyl)azandiyl)dioctanoate [ka] To a solution of bis(3-propylhexyl)8,8'-((3-((tert-butoxycarbonyl)amino)propyl)azandiyl)dioctanoate (730 mg, 1.03 mmol) in methylene chloride (21 mL), trifluoroacetic acid (1.57 mL, 20.5 mmol) was added. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was quenched with saturated aqueous NaHCO3 solution and extracted with dichloromethane. The organic layer was separated, washed with brine, dried (MgSO4), filtered, and concentrated. The crude substance was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)) to obtain bis(3-propylhexyl)8,8'-((3-aminopropyl)azandiyl)dioctanoate (499 mg, 0.82 mmol, 80%) as a clear oil. UPLC / ELSD: RT=1.93 min, MS(ES): m / z(MH) + )C 37 H 74 611.44 for N2O4

[0393] Intermediate BP: 3-Butylheptyl 8-((3-aminopropyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate [ka] To a solution of 3-butylheptyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate (896 mg, 1.13 mmol) in methylene chloride (23 mL), trifluoroacetic acid (1.72 mL, 22.5 mmol) was added. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was quenched with saturated aqueous NaHCO3 solution and extracted with dichloromethane. The organic layer was separated, washed with brine, dried (MgSO4...

Claims

1. Formula (A): 【Chemistry 1】 A compound thereof, or its N-oxide, or its salt or isomer, In the formula, R' ａ However, R' 分岐状 or R' 環式 And, R' 分岐状 but, 【Chemistry 2】 And, R' 環式 but, 【Transformation 3】 And, 【Chemistry 4】 However, it indicates a connection point, R ａα is H, and R ａβ , R ａγ , and R ａδ are each independently selected from the group consisting of H, C ２－１２ alkyl, and C ２－１２ alkenyl, and at least one of R ａβ , R ａγ , and R ａδ is selected from the group consisting of C ２－１２ alkyl and C ２－１２ alkenyl, R ２ and R ３ However, each C １－１４ It is alkyl, R ４ However, - (CH ２ ) ２ OH, - (CH ２ ) ３ OH, - (CH ２ ) ４ OH, - (CH ２ ) ５ OH, and 【Transformation 5】 Selected from the group consisting of R １０ However, N(R) ２ And each R independently, C １－６ Alkyl, C ２－３ Selected from the group consisting of alkenyl and H, n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Each R ５ However, independently, OH, C １－３ Alkyl, C ２－３ Selected from the group consisting of alkenyls and H, Each R ６ However, independently, OH, C １～３ Alkyl, C ２－３ Selected from the group consisting of alkenyls and H, R ７ However, it is H, M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-, R' is C １－１２ Alkyl or C ２－１２ It is alkenyl, Y ａ However, C ３－６ It is a carbon ring, R ＊ " ａ However, C １－１５ Alkyl and C ２－１５ Selected from the group consisting of alkenils, l is selected from the group consisting of 1, 2, 3, 4, and 5. s is 2 or 3, A compound of formula (A), or its N-oxide, or a salt or isomer thereof, wherein m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

2. Formula (B): 【Transformation 6】 A compound thereof, or its N-oxide, or its salt or isomer, In the formula, R' ａ However, R' 分岐状 or R' 環式 And, R' 分岐状 but, 【Transformation 7】 And, R' 環式 but, 【Transformation 8】 And, 【Chemistry 9】 However, it indicates a connection point, R ａα and R ａβ However, each is H, and R ａγ and R ａδ However, H and C are independent of each other. ２－１２ Alkyl and C ２－１２ Selected from the group consisting of alkenyls, R ａγ and R ａδ At least one of them is C ２－１２ Alkyl and C ２－１２ Selected from the group consisting of alkenils, R ｂα , R ｂβ , R ｂγ , and R ｂδ However, H and C are independent of each other. ２－３０ Alkyl and C ５－２０ Selected from the group consisting of alkenyls, R ｂα , R ｂβ , R ｂγ , and R ｂδ At least one of them is C ２－３０ Alkyl and C ５－２０ Selected from the group consisting of alkenils, R ４ However, - (CH ２ ) ２ OH, - (CH ２ ) ３ OH, - (CH ２ ) ４ OH, - (CH ２ ) ５ OH, and 【Chemistry 10】 Selected from the group consisting of, 【Chemistry 11】 However, it indicates a connection point, R １０ However, N(R) ２ And each R independently, C １－６ Alkyl, C ２－３ Selected from the group consisting of alkenyl and H, n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Each R ５ is independently selected from the group consisting of OH, C １－３ alkyl, C ２－３ alkenyl, and H Each R ６ However, independently, OH, C １～３ Alkyl, C ２～３ Selected from the group consisting of alkenyls and H, M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-, R' is C １－１２ Alkyl or C ２－１２ It is alkenyl, Y ａ is a C ３－６ carbon ring, and R ＊ " ａ However, C １－１５ Alkyl and C ２－１５ Selected from the group consisting of alkenils, l is selected from the group consisting of 1, 2, 3, 4, and 5. s is 2 or 3, A compound of formula (B), or its N-oxide, or a salt or isomer thereof, wherein m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

3. Formula (1-1): 【Chemistry 12】 A compound thereof, or its N-oxide, or its salt or isomer, In the formula, R' ａ However, R' 分岐状 or R' 環式 And, R' 分岐状 but, 【Chemistry 13】 And, R' 環式 but, 【Chemistry 14】 And, R' ｂ but, 【Chemistry 15】 And, 【Chemistry 16】 However, it indicates a connection point, R ａγ and R ｂγ However, each is independent of C ２－１２ Alkyl or C ２－１２ It is alkenyl, R ２ and R ３ However, each is independent of C １－１４ Alkyl and C ２－１４ Selected from the group consisting of alkenils, R ４ However, - (CH ２ ) ２ OH, Each R' independently, C １－１２ Alkyl or C ２－１２ It is alkenyl, Y ａ However, C ３－６ It is a carbon ring, R ＊ " ａ However, C １－１５ Alkyl and C ２－１５ Selected from the group consisting of alkenils, A compound of formula (1-1), or its N-oxide, or a salt or isomer thereof, wherein s is 2 or 3.

4. The following structure: 【Chemistry 17】 A compound according to any one of the prior claims, having one of the following.

5. R ａγ However, C ２－６ A compound according to any one of the prior claims, wherein it is alkyl.

6. R ｂγ However, C ２－６ A compound according to any one of the prior claims, wherein it is alkyl.

7. R ａγ and R ｂγ However, each is independent of C ２－６ A compound according to any one of the prior claims, wherein it is alkyl.

8. R ｂγ However, C ４－６ A compound according to any one of the prior claims, wherein it is alkyl.

9. R ２ and R ３ However, each C ８ A compound according to any one of the prior claims, wherein it is alkyl.

10. Y ａ The compound according to any one of the prior claims, wherein the compound is cyclohexyl or cyclopentyl.

11. R ＊ " ａ C ２ - Alkyl or C ３ - A compound according to any one of the prior claims, wherein it is alkyl.

12. Each R ’ However, independently, C ２－５ A compound according to any one of the prior claims, wherein it is alkyl.

13. A compound selected from the following: Table 1

14. An empty lipid nanoparticle (empty LNP) comprising a compound according to any one of the prior claims, a phospholipid, a structural lipid, and a PEG lipid.

15. An empty LNP according to any one of the prior claims, comprising about 40 mol% to about 60 mol% of the compound, about 0 mol% to about 20 mol% of phospholipids, about 30 mol% to about 50 mol% of structural lipids, and about 0 mol% to about 5 mol% of PEG lipids.

16. The phospholipids mentioned above are 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-Dimiristoyl-sn-glycerophosphocholine (DMPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Diundecanoyl-sn-glycerophosphocholine (DUPC), 1-Palmitoyl-2-Oleoyl-sn-Glycerol-3-Phosphocholine (POPC), 1,2-Di-O-Octadecenyl-sn-Glycerol-3-Phosphocholine (18:0 DietherPC), 1-Oleoyl-2-Cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-Dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), An empty LNP according to any one of the prior claims, selected from the group consisting of sphingomyelin and mixtures thereof.

17. An empty LNP according to any one of the prior claims, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brazicasterol, and mixtures thereof.

18. An empty LNP according to any one of the prior claims, wherein the PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

19. The aforementioned PEG lipid is PEG ２ｋ - DMG and PEG-1: [Chemistry 18] An empty LNP according to any one of the prior claims, selected from and mixtures thereof.

20. A filled lipid nanoparticle (filled LNP) comprising an empty LNP as described in any one of the prior claims and one or more therapeutic and / or prophylactic agents.

21. The filled LNP according to any one of the prior claims, wherein the one or more therapeutic and / or prophylactic agents are nucleic acids.

22. A packed LNP according to any one of the prior claims, wherein the nucleic acid is RNA, and the RNA is selected from the group consisting of short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecules, microRNA (miRNA), antagomyl, antisense RNA, ribozyme, dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

23. The packed LNP according to any one of the prior claims, wherein the RNA is mRNA.

24. A pharmaceutical composition comprising a filled LNP according to any one of the prior claims and a pharmaceutically acceptable carrier.

25. A method for delivering a therapeutic and / or prophylactic agent to cells within a subject, wherein the method comprises administering a filled LNP according to any one of the prior claims to the subject.

26. A method for specifically delivering a therapeutic and / or prophylactic agent to a target organ, wherein the method comprises administering a filled LNP according to any one of the prior claims to the target.

27. A method for generating a target polypeptide in cells within a subject, wherein the method comprises administering a filled LNP according to any one of the prior claims to the subject.

28. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a filled LNP according to any one of the prior claims.

29. The method according to any one of the prior claims, wherein the organ is selected from the group consisting of the liver, kidney, lung, and spleen.

30. The method according to any one of the prior claims, wherein the administration is performed parenterally, intramuscularly, intradermally, subcutaneously, and / or intravenously.

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