Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents - Patents.com

Abstract

The present application relates to lipids of Formula (A), Formula (B), and Formula (1-1), as well as lipid nanoparticles (LNPs) (empty or filled) comprising such lipids and additional lipids, such as phospholipids, structural lipids, and PEG-lipids. Lipid nanoparticles further comprising a therapeutic and / or prophylactic agent, such as RNA, are useful for delivery of the therapeutic and / or prophylactic agent to mammalian cells or organs, for example, to modulate polypeptide, protein, or gene expression. [Formula 1] JPEG2022548304000424.jpg108170

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 902,927, filed September 19, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure provides novel compounds, compositions comprising such compounds, and methods involving lipid nanoparticle compositions for delivering one or more therapeutic and / or prophylactic agents to mammalian cells or organs and / or producing polypeptides. In addition to the novel lipids, the disclosed lipid nanoparticle compositions may contain, in specific fractions, one or more cationic and / or ionic amino lipids, phospholipids including polyunsaturated lipids, PEG-lipids, structured lipids, and / or therapeutic and / or prophylactic agents. [Background technology]

[0003] Effective targeted delivery of biologically active substances, such as small molecule drugs, proteins, and nucleic acids, represents a continuing medical challenge. In particular, delivery of nucleic acids into cells is made difficult by the relative instability and poor cell permeability of such species. Thus, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and / or prophylactic agents, such as nucleic acids, into cells.

[0004] Lipid-containing nanoparticle compositions, liposomes, and lipoplexes have proven effective as delivery 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 polyethylene glycol-containing lipids (PEG-lipids). Examples of cationic and / or ionic lipids include amine-containing lipids that can be readily protonated. While various lipid-containing nanoparticle compositions have been demonstrated, improvements in safety, efficacy, and specificity remain lacking. Summary of the Invention

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

[0006] In some embodiments, the disclosure provides a compound of formula (1-1): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is 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] indicates the point of attachment, R aγ and R bγ are each independently C 2-12 Alkyl or C 2-12 is alkenyl, R 2 and R 3 are each independently C 1-14 Alkyl and C 2-14 alkenyl, R 4 is -(CH2)2OH, Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl, Y a is C 3-6 is a carbocyclic ring, R * ” a is C 1-15 Alkyl and C 2-15 alkenyl, s is 2 or 3.

[0007] In some embodiments, the disclosure provides a compound of formula (2-1): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is 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] indicates the point of attachment, R aγ and R bγ are each independently C 2-12 Alkyl or C 2-12 is alkenyl, R 2 and R 3 are each independently C 1-14 Alkyl and C 2-14 alkenyl, R 4 teeth, [ka] and [ka] indicates the attachment point, and R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 independently C 1-12 Alkyl or C 2-12 is alkenyl, Y a is C 3-6 is a carbocyclic ring, R * ” a is C 1-15 Alkyl and C 2-15 alkenyl, s is 2 or 3.

[0008] In some embodiments, the disclosure provides a compound of formula (A): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' 分岐状 or R' 環式 and R' 分岐状 teeth, [ka] and R' 環式 teeth, [ka] and [ka] indicates the point of attachment, R aα is H and R aβ , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aβ , R aγ , and R aδ At least one of the 2-12 Alkyl and C 2-12 alkenyl, R 2 and R 3 are C 1-14 is alkyl, R 4 are -(CH2)2OH, -(CH2)3OH, -(CH2)4OH, -(CH2)5OH, and [ka] is selected from the group consisting of [ka] indicates the point of attachment, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 are independently OH, C 1-3 Alkyl, C2-3 selected from the group consisting of alkenyl, and H; Each R 6 are independently OH, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and 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 is alkenyl, Y a is C 3-6 is a carbocyclic ring, 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 provides a compound of formula (B): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' 分岐状 or R' 環式 and R' 分岐状 teeth, [ka] and R' 環式 teeth, [ka] and [ka] indicates the point of attachment, R aα and R aβ are H and R aγ and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aγ and R aδ At least one of the 2-12 Alkyl and C 2-12 alkenyl, R bα , R bβ , R bγ , and R bδ are each independently H, C 2-30 Alkyl, and C 5-20 alkenyl; R bα , R bβ , R bγ , and R bδ At least one of the 2-30 Alkyl and C 5-20 alkenyl, R 4 are -(CH2)2OH, -(CH2)3OH, -(CH2)4OH, -(CH2)5OH, and [ka] is selected from the group consisting of [ka] indicates the point of attachment, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R5 are independently OH, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R 6 are independently OH, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, 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 is alkenyl, Y a is C 3-6 is a carbocyclic ring, 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.

[0010] In some embodiments, the disclosure provides a compound of formula (Aa): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R aβ , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aβ , R aγ , and R aδ At least one of the 2-12 Alkyl and C 2-12 alkenyl, R 4is selected from the group consisting of —(CH2)2OH, —(CH2)3OH, —(CH2)4OH, —(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 alkenyl.

[0011] In some embodiments, the disclosure provides a compound of formula (Ab): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R aβ , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aβ , R aγ , and R aδ At least one of the 2-12 Alkyl and C 2-12 alkenyl, R 4 is selected from the group consisting of —(CH2)2OH, —(CH2)3OH, —(CH2)4OH, —(CH2)5OH; R' is C 1-12 Alkyl or C 2-12 It is alkenyl. DETAILED DESCRIPTION OF THE INVENTION

[0012] The disclosure relates to novel lipids and lipid nanoparticles (e.g., empty LNPs or filled LNPs) comprising the novel lipids. 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 producing a polypeptide of interest in mammalian cells, methods for improving the level of a protein produced in mammalian cells compared to LNPs comprising other lipids, and methods for treating a disease or disorder in a mammal in need of such treatment. For example, a method for producing a polypeptide of interest in a cell can involve contacting nanoparticles comprising mRNA with mammalian cells, thereby translating the mRNA to produce the polypeptide of interest. A method for delivering a therapeutic and / or prophylactic agent to mammalian cells or organs can involve administering a nanoparticle composition comprising the therapeutic and / or prophylactic agent to a subject, wherein the administration involves contacting the cells or organ with the composition, thereby delivering the therapeutic and / or prophylactic agent to the cells or organ. Such delivery methods can be in vitro or in vivo.

[0013] The present disclosure provides lipids comprising a central amine moiety and at least one biodegradable group. The lipids described herein can be advantageously used in lipid nanoparticles (e.g., empty or filled LNPs) to deliver therapeutic and / or prophylactic agents to mammalian cells or organs. For example, the lipids described herein have little or no immunogenicity. For example, lipid 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) have reduced immunogenicity compared to reference lipids (e.g., MC3, KC2, or DLinDMA). For example, a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent has an increased therapeutic index compared to a corresponding formulation comprising a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.

[0014] In some embodiments, the disclosure provides a compound of formula (A-1): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' 分岐状 or R' 環式 and R' 分岐状 teeth, [ka] and R' 環式 teeth, [ka] and [ka] indicates the point of attachment, R aα , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aβ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 2 and R 3 are C 1-14 is alkyl, R 4 is -(CH2)2OH or [ka] and [ka] indicates the point of attachment, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 are 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 is alkenyl, Y a is C 3-6 is a carbocyclic ring, 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.

[0015] In some embodiments, the disclosure provides a compound of formula (A-2): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' 分岐状 or R' 環式 and R' 分岐状 teeth, [ka] and R' 環式 teeth, [ka] and [ka] indicates the point of attachment, R aα , R aβ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aγ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 2 and R 3 are C 1-14 is alkyl, R 4 is -(CH2)2OH or [ka] and [ka] indicates the point of attachment, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 are 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 is alkenyl, Y a is C3-6 is a carbocyclic ring, 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.

[0016] In some embodiments, the disclosure provides a compound of formula (A-3): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' 分岐状 or R' 環式 and R' 分岐状 teeth, [ka] and R' 環式 teeth, [ka] and [ka] indicates the point of attachment, R aα , R aγ , and R aβ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aδ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 2 and R 3are C 1-14 is alkyl, R 4 is -(CH2)2OH or [ka] and [ka] indicates the point of attachment, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 are 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 is alkenyl, Y a is C 3-6 is a carbocyclic ring, 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.

[0017] In some embodiments, the disclosed compounds have one of the following structures: [ka]

[0018] In some embodiments, the disclosure provides a compound of formula (B-1): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' 分岐状 or R' 環式 and R' 分岐状 teeth, [ka] and R' 環式 teeth, [ka] and [ka] indicates the point of attachment, R aα , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aβ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R bα , R bγ , and R bδ are each independently H, C 2-30 Alkyl, and C 5-20 alkenyl; R bβ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 4 teeth, [ka] and [ka] indicates the point of attachment, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 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 is alkenyl, Y a is C 3-6 is a carbocyclic ring, 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.

[0019] In some embodiments, the disclosure provides a compound of formula (B-2): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' 分岐状 or R' 環式 and R' 分岐状 teeth, [ka] and R' 環式 teeth, [ka] and [ka] indicates the point of attachment, R aα , R aβ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aγ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R bα , R bβ , and R bδ are each independently H, C 2-30 Alkyl, and C 5-20 alkenyl; R bγ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 4 is -(CH2)2OH or [ka] and [ka] indicates the point of attachment, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 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 is alkenyl, Y a is C 3-6 is a carbocyclic ring, 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.

[0020] In some embodiments, the disclosure provides a compound of formula (B-3): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' 分岐状 or R' 環式 and R' 分岐状 teeth, [ka] and R' 環式 teeth, [ka] and [ka] indicates the point of attachment, R aα , R aβ , and R aγ are each independently H, C 2-12Alkyl, and C 2-12 alkenyl; R aδ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R bα , R bβ , and R bγ are each independently H, C 2-30 Alkyl, and C 5-20 alkenyl; R bδ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 4 is -(CH2)2OH or [ka] and [ka] indicates the point of attachment, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 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 is alkenyl, Y a is C 3-6 is a carbocyclic ring, 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.

[0021] In some embodiments, the disclosure provides a compound of formula (A-a1): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R aγ and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aβ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 4 is -(CH2)2OH or [ka] and [ka] indicates the point of attachment, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 alkenyl.

[0022] In some embodiments, the disclosure provides a compound of formula (A-a2): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R aβ and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aγ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 4 is -(CH2)2OH or [ka] and [ka] indicates the point of attachment, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 alkenyl.

[0023] In some embodiments, the disclosure provides a compound of formula (A-a3): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R aβ and R aγ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aδis a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 4 is -(CH2)2OH or [ka] and [ka] indicates the point of attachment, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 alkenyl.

[0024] In some embodiments, the disclosure provides a compound of formula (A-b1): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R aγ and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aβ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 4 is -(CH2)2OH or [ka] and [ka] indicates the point of attachment, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 alkenyl.

[0025] In some embodiments, the disclosure provides a compound of formula (A-b2): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R aβ and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aγ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 4 is -(CH2)2OH or [ka] and [ka] indicates the attachment point, and R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 alkenyl.

[0026] In some embodiments, the disclosure provides a compound of formula (A-b3): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R aβ and R aγ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R aδ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 4 is -(CH2)2OH or [ka] and [ka] indicates the point of attachment, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 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 alkenyl.

[0027] In some embodiments, the disclosure provides a compound of formula (Ac): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R aγis a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 4 is -(CH2)2OH or [ka] and [ka] indicates the point of attachment, and R' is C 1-12 It is alkyl.

[0028] In some embodiments, the disclosure provides a compound of formula (Bc): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R aγ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R bγ is a C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl; R 4 is -(CH2)2OH or [ka] and [ka] indicates the point of attachment, and R' is C 1-12 It is alkyl.

[0029] In some embodiments, the disclosure provides a compound of formula (Ia): [ka] In the compound of formula R 2and R 3 are each independently C 1-14 Alkyl and C 2-14 alkenyl; R aγ and R bγ are each independently C 2-6 It is alkyl.

[0030] Any 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), and (Bc) may include one or more of the following features, where applicable:

[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] and n2 is 4.

[0032] In some embodiments, R 10 is -NH. In some embodiments, R 10 is -NH(C 1-6 In some embodiments, R 10 is -N(C 1-6 In some embodiments, R 10 is —NH(CH). In some embodiments, R10 is -N(CH3)2.

[0033] In some embodiments, R 4 is —(CH)OH, —(CH)OH, or —(CH)OH, and M and M′ are each —C(O)O—. In some embodiments, R 4 is —(CH)OH, and M and M′ are each —C(O)O—. In some embodiments, R 4 is —(CH)OH, and M and M′ are each —C(O)O—. In some embodiments, R 4 is —(CH 2 ) 4 OH, and M and M′ are each —C(O)O—.

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

[0035] In some embodiments, R 4 teeth, [ka] and M and M' are each -C(O)O-. 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] wherein 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 are each H and m is 5, 6, 7, 8, or 9. In some embodiments, R 5 , R 6 、 and R 7 are each H and m is 5. In some embodiments, R 5 , R 6 、 and R 7 are each H and m is 7.

[0039] In some embodiments, R 2 and R 3 are each independently C 1-14 Alkyl or C 2-14 In some embodiments, R is alkenyl. 2 and R 3 are each independently C 3-14 Alkyl or C 3-14 In some embodiments, R is alkenyl. 2and R 3 independently, C 5-14 Alkyl or C 5-14 It is alkenyl.

[0040] In some embodiments, R 2 and R 3 are each independently C 1-14 In some embodiments, R 2 and R 3 are each independently C 3-14 In some embodiments, R 2 and R 3 are each independently C 7-9 It is alkyl.

[0041] In some embodiments, R 2 and R 3 are each C alkyl. In some embodiments, R 2 and R 3 are each C alkyl. In some embodiments, R 2 and R 3 are each C9 alkyl.

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

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

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

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

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

[0047] In some embodiments, R' is C 1-12 Alkyl or C 2-12 In some embodiments, R' is alkenyl. In some embodiments, R' is C2 alkyl. In some embodiments, R' is C3 or C4 alkyl. In some embodiments, R' is C3 alkyl. In some embodiments, R' is C4 alkyl. In some embodiments, R' is 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 In some embodiments, R' is C 11 Alkyl or C 11 It is alkenyl.

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

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

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

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

[0055] In some embodiments, Y a R * ” a teeth, [ka] In some embodiments, Y a R * ” a teeth, [ka] is.

[0056] In some embodiments, s is 2 and Y a R * ” a teeth, [ka] and R * ” a is C2 alkyl or C3-alkyl. In some embodiments, s is 2 and Y a R * ”a teeth, [ka] and R * ” a is C2 alkyl or C3-alkyl.

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

[0058] In some embodiments, the 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) is selected from the compounds of Table 1, and N-oxides, salts, or isomers thereof. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0059] The central amine moiety of lipids 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) can be protonated at physiological pH. Thus, the lipids can have a positive or partial positive charge at physiological pH. Such lipids are sometimes referred to as cationic or ionic (amino) lipids. Lipids can also be zwitterionic, i.e., neutral molecules with both positive and negative charges.

[0060] definition As used herein, the term "alkyl" or "alkyl group" means a linear or branched saturated hydrocarbon containing one or more 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), which is optionally substituted. 1-14 The term "alkyl" means an optionally substituted linear or branched saturated hydrocarbon containing 1 to 14 carbon atoms. Unless otherwise specified, alkyl groups described herein refer to both unsubstituted and substituted alkyl groups.

[0061] As used herein, the term "alkenyl" or "alkenyl group" means an optionally substituted linear or branched hydrocarbon containing two or more 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. Alkenyl groups can contain 1, 2, 3, 4, or more carbon-carbon double bonds. For example, C 18 Alkenyl may contain one or more double bonds. C containing two double bonds18 The alkenyl group can be a linoleyl group. Unless otherwise specified, the alkenyl groups described herein refer to both unsubstituted and substituted alkenyl groups.

[0062] As used herein, the term "alkynyl" or "alkynyl group" means an optionally substituted linear or branched hydrocarbon containing two or more 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. Alkynyl groups can contain 1, 2, 3, 4, or more carbon-carbon triple bonds. For example, C 18 An alkynyl may contain one or more carbon-carbon triple bonds. Unless otherwise specified, alkynyl groups described herein refer to both unsubstituted and substituted alkynyl groups.

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

[0064] "Aryl" includes "composite," or polycyclic, systems having at least one aromatic ring that does not contain heteroatoms in the ring structure. Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthalenyl, and the like. In some embodiments, "aryl" refers to C 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 3 6-10 A carbocyclic ring (e.g., "aryl" is C 6-10 (aryl).

[0065] As used herein, the term "heterocycle" or "heterocyclic group" means an optionally substituted monocyclic or polycyclic ring system containing one or more rings, wherein at least one ring contains at least one heteroatom. The heteroatom can be, for example, a nitrogen, oxygen, or sulfur atom. The ring can be 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered. The heterocycle can contain one or more double or triple bonds and can be non-aromatic or aromatic (e.g., a heterocycloalkyl or heteroaryl group). 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" refers to a non-aromatic heterocycle, which may or may not contain double or triple bonds. Unless otherwise specified, heterocycles described herein refer to both unsubstituted 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 the ring structure contains one to four heteroatoms, and may also be referred to as an "aryl heterocycle" or "heteroaromatic." As used herein, the term "heteroaryl" is intended to include a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic heteroaromatic ring composed of carbon atoms and one or more heteroatoms, e.g., one, or one to two, or one to three, or one to four, or one to five, or one to six heteroatoms, independently selected from the group consisting of, e.g., nitrogen, oxygen, sulfur, and boron. The nitrogen atom can be substituted or unsubstituted (i.e., N or NR, where R is H or other substituent as defined). The nitrogen and sulfur heteroatoms can 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, pyrimidine, and the like.

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

[0069] As used herein, a "biodegradable group" is a group that can promote faster metabolism of lipids in mammalian organisms. The biodegradable group may be selected from the group consisting of, but is 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)-, an aryl group, and a heteroaryl group. As used herein, an "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, a "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 the non-limiting group consisting of optionally substituted phenyl, oxazole, and thiazole. In the formulas herein, M and M' can be independently selected from the list of biodegradable groups above. Unless otherwise specified, the aryl or heteroaryl groups described herein refer to both unsubstituted and substituted, i.e., optionally substituted, aryl or heteroaryl groups.

[0070] Alkyl, alkenyl, and cyclyl (eg, carbocyclyl and heterocyclyl) groups may be optionally substituted, unless otherwise specified. Optional substituents include, but are 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), alkoxy (e.g., —OR), acetals (e.g., —C(OR)R″ where each OR is an alkoxy group, which may be the same or different, and R″″ is an alkyl or alkenyl group), phosphates (e.g., P(O) 3- ), thiol (e.g., -SH), sulfoxide (e.g., -S(O)R), sulfinic acid (e.g., -S(O)OH), sulfonic acid (e.g., -S(O)2OH), thial (e.g., -C(S)H), sulfate (e.g., S(O)4 2-), sulfonyl (e.g., -S(O)2-), amido (e.g., -C(O)NR2 or -N(R)C(O)R), azido (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, In some embodiments, the substituent may be selected from the group consisting of: -OC(O)NRH, or -OC(O)NH), sulfonamide (e.g., -S(O)NRH, -S(O)NH, -N(R)S(O)R, -N(H)S(O)R, -N(R)S(O)H, or -N(H)S(O)H), alkyl, alkenyl, and cyclyl (e.g., carbocyclyl or heterocyclyl) groups. In any of the foregoing, R is an alkyl or alkenyl group as defined herein. In some embodiments, the substituent itself may be further substituted, for example, with 1, 2, 3, 4, 5, or 6 substituents as defined herein. For example, C 1-6 Alkyl groups may be further substituted with 1, 2, 3, 4, 5, or 6 substituents described herein.

[0071] The disclosed compounds containing nitrogen can be converted to N-oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (mCPBA) and / or hydrogen peroxide) to provide other disclosed compounds. Accordingly, all nitrogen-containing compounds shown and claimed may be converted to N-oxide derivatives (N→O or N) of the shown compounds, where permitted by valency and structure. + -O -(which may be designated as aryl, ...

[0072] About, Approximately: As used herein, the terms "approximately" and "about," when applied to one or more values ​​of interest, refer to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls 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 of the stated reference value, unless otherwise stated or apparent from the context (except when such numerical value exceeds 100% of the possible values). For example, when used in the context of the amount of a given compound in the lipid component of a nanoparticle composition, "about" can mean ±10% of the recited value. For example, a nanoparticle composition containing a lipid component with about 40% of a given compound may contain 30-50% of the compound.

[0073] As used herein, the term "compound" is intended to include all isomers and isotopes of the structure shown. "Isotopes" refer to atoms with the same atomic number but different mass numbers resulting from different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. Furthermore, compounds, salts, or complexes of the present disclosure can be prepared in combination with solvents or water molecules by conventional methods to form solvates and hydrates.

[0074] As used herein, the term "contacting" means establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and the nanoparticle are made to share a physical connection. Methods for contacting cells with external entities, both in vivo and ex vivo, are well known in the biological arts. For example, contacting a nanoparticle composition with mammalian cells placed within a mammal can be performed by various administration routes (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can involve various amounts of lipid nanoparticles (e.g., empty LNPs or loaded LNPs). Furthermore, two or more mammalian cells may be contacted with the nanoparticle composition.

[0075] As used herein, the term "delivering" means providing an entity to a target location. For example, delivering a therapeutic and / or prophylactic agent to a subject can involve administering to the subject (e.g., via intravenous, intramuscular, intradermal, or subcutaneous routes) a nanoparticle composition comprising the therapeutic and / or prophylactic agent. Administering a nanoparticle composition to a mammal or cells of a mammal can involve contacting one or more cells with the nanoparticle composition.

[0076] As used herein, the term "enhanced delivery" refers to increased (e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold) delivery of a therapeutic and / or prophylactic agent by a nanoparticle to a target cell of interest (e.g., the liver of a mammal) compared to the level of delivery of a therapeutic and / or prophylactic agent by a control nanoparticle to a target cell of interest (e.g., MC3, KC2, or DLinDMA). The level of nanoparticle delivery to a particular tissue can 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. It will be understood that enhanced delivery of nanoparticles to target cells need not be determined in the subject being treated, but can be determined in a surrogate animal model (e.g., a rat model), etc. In certain embodiments, nanoparticle compositions comprising a 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) have substantially the same level of enhanced delivery regardless of the route of administration. For example, certain compounds disclosed herein exhibit similar enhanced delivery whether used to deliver therapeutic and / or prophylactic agents intravenously or intramuscularly. In other embodiments, certain compounds disclosed herein exhibit even greater levels of delivery enhancement when used to deliver therapeutic and / or prophylactic agents intramuscularly rather than intravenously.

[0077] As used herein, the terms "specific delivery," "specifically deliver," or "specifically delivering" refer to a greater (e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold) delivery of a therapeutic and / or prophylactic agent by a nanoparticle to a target tissue of interest (e.g., the liver of a mammal) compared to non-target cells (e.g., the spleen of a mammal). The level of nanoparticle delivery to a particular tissue can 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, in renal vascular targeting, a therapeutic and / or prophylactic agent is specifically provided to the kidney of a mammal relative to the liver and spleen if 1.5, 2, 3, 5, 10, 15, or 20 times more therapeutic and / or prophylactic agent is delivered to the kidney per gram of tissue compared to that delivered to the liver or spleen following systemic administration of the therapeutic and / or prophylactic agent. It will be understood that the ability of nanoparticles to be specifically delivered to a target tissue need not be determined in the subject being treated, but can be determined in a surrogate such as an animal model (e.g., a rat model).

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

[0079] As used herein, "encapsulation," "encapsulated," "loaded," and "associated" can refer to complete, substantial, or partial enclosure, confinement, surrounding, or packing. As used herein, "encapsulation" or "association" can refer to the process of confining individual nucleic acid molecules within a nanoparticle and / or establishing a physiochemical relationship between an individual nucleic acid molecule and a nanoparticle. As used herein, "empty nanoparticles" can refer to nanoparticles that are substantially free of therapeutic or prophylactic agents. As used herein, "empty nanoparticles" or "empty lipid nanoparticles" can refer to nanoparticles that are substantially free of nucleic acids. As used herein, "empty nanoparticles" or "empty lipid nanoparticles" can refer to nanoparticles that are substantially free of nucleotides or polypeptides. As used herein, "empty nanoparticles" or "empty lipid nanoparticles" can refer to nanoparticles that are substantially composed only of lipid components. As used herein, "loaded nanoparticles" or "loaded lipid nanoparticles" (also referred to as "full nanoparticles" or "full lipid nanoparticles") can refer to nanoparticles that contain the components of an empty nanoparticle and a therapeutic or prophylactic agent. As used herein, "loaded nanoparticles" or "loaded lipid nanoparticles" (also referred to as "full nanoparticles" or "full lipid nanoparticles") may refer to nanoparticles that contain the components of an empty nanoparticle and a nucleotide or polypeptide. As used herein, "loaded nanoparticles" or "loaded lipid nanoparticles" (also referred to as "full nanoparticles" or "full lipid nanoparticles") may refer to nanoparticles that contain the components of an empty nanoparticle and a nucleic acid.

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

[0081] As used herein, the term "in vitro" refers to events that occur not in a living organism (e.g., an animal, plant, or microorganism) but in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, in a Petri dish, etc.

[0082] As used herein, the term "in vivo" refers to events that occur within an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof).

[0083] As used herein, the term "ex vivo" refers to an event that occurs outside of an organism (e.g., an animal, plant, or microorganism or cells or tissues thereof). An ex vivo event can occur in an environment that is minimally altered from the natural (e.g., in vivo) environment.

[0084] As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterion, 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 stereoisomers, such as double bond isomers (i.e., geometric E / Z isomers), or diastereomers (e.g., enantiomers (i.e., (+) or (-)), or cis / trans isomers). The present 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 stereoisomers, e.g., racemates. Enantiomeric and stereoisomeric mixtures of compounds and means for resolving them into their component enantiomers or stereoisomers are well known.

[0085] A "tautomer" is one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another. This conversion involves the formal migration of a hydrogen atom accompanied by the switching of adjacent conjugated double bonds. Tautomers exist as mixtures of pairs of tautomers in solution. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. The concept of tautomers that are interconvertible by tautomerization is called tautomerism.

[0086] Of the various types of tautomerism possible, two are commonly observed: keto-enol tautomerism, in which a simultaneous shift of an electron and a hydrogen atom occurs; and ring-chain tautomerism, which occurs when an aldehyde group (-CHO) on a sugar molecule reacts with one of the hydroxyl groups (-OH) on the same molecule, resulting in the cyclic (ring-shaped) form assumed by glucose.

[0087] Common tautomeric pairs are ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomers, imine-enamine, and enamine-enamine in heterocyclic rings (e.g., in nucleobases such as guanine, thymine, and cytosine). Examples of tautomers in disubstituted guanidines are shown below. [ka]

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

[0089] As used herein, " lipid component " refers to the component of nanoparticle composition that comprises one or more lipids.For example, lipid component can include one or more cationic / ionic lipids, PEGylated lipids, structural lipids, or other lipids such as phospholipids.

[0090] As used herein, a "linker" refers to a moiety that connects two moieties, such as the connection between two nucleosides of a capped species. Linkers can include one or more groups, including, but not limited to, phosphate groups (e.g., phosphate, boranophosphate, thiophosphate, selenophosphate, and phosphonate), alkyl groups, amidates, or glycerol. For example, the two nucleosides of a capped analog can be linked at their 5' positions by a triphosphate group or by a chain containing two phosphate moieties and a boranophosphonate moiety.

[0091] As used herein, "administration method" includes intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. The administration method can be selected to target (e.g., specifically deliver) delivery to a particular area or system of the body.

[0092] As used herein, "modified" means non-natural. For example, RNA can be modified RNA. That is, RNA can contain one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers. "Modified" species can also be referred to herein as "altered" species. Species can be chemically, structurally, or functionally modified or altered. For example, modified nucleobase species can contain one or more non-naturally occurring substitutions.

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

[0094] As used herein, a "nanoparticle composition" refers to a composition containing one or more lipids. Nanoparticle compositions are typically on the order of a few micrometers or less in size and may contain a lipid bilayer. Nanoparticle compositions 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 existing in nature without artificial assistance.

[0096] As used herein, "patient" refers to a subject who may seek or need treatment, who needs treatment, who is receiving treatment, who is receiving treatment, or who is being treated by a professional trained in a particular disease or condition.

[0097] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that includes a polyethylene glycol moiety.

[0098] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0099] The term "pharmaceutically acceptable excipient" as used herein refers to any component other than the compounds described herein (e.g., a vehicle capable of suspending, complexing, or dissolving an active compound), which has substantially non-toxic and non-inflammatory properties in patients. Excipients may include, for example, anti-adhesives, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (coloring agents), softeners, emulsifiers, fillers (diluents), film-forming or coating agents, flavors, flavoring agents, glidants (gliding agents), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, 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 species disclosed herein.

[0100] In this specification, the structural formula of a compound may conveniently represent a specific isomer, but the present disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, etc., and it is understood that not all isomers may have the same level of activity.In addition, the compound represented by formula may have crystalline polymorphism.It should be noted that any crystalline form, crystalline mixture, or their anhydride or hydrate are included in the scope of the present disclosure.

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

[0102] The composition may also contain one or more salts of the compounds. The salt may be a pharmaceutically acceptable salt. As used herein, "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound, in which the parent compound is modified by converting an existing acid or base moiety into 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, alkali salts or organic salts of acidic residues such as carboxylic acids, etc. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfonate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy- Salts include ethanesulfonate, lactobionate, lactate, laurate, sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with an appropriate amount of stoichiometric base or acid in water or an organic solvent, or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0103] As used herein, "phospholipid" refers to a lipid that contains 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). Certain phospholipids can promote membrane fusion. For example, cationic phospholipids can interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or an intracellular membrane). By fusing a phospholipid with a membrane, one or more components of a lipid-containing composition can pass through the membrane, for example, allowing one or more components to be delivered to a cell.

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

[0105] As used herein, the term "polypeptide" or "polypeptide of interest" typically refers to a polymer of amino acid residues joined by peptide bonds, which can be naturally (e.g., isolated or purified) or synthetically produced. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can contain modified amino acids. The term also encompasses amino acid polymers that are naturally occurring or modified by intervention, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides containing one or more analogs of an amino acid (including unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include encoded polypeptide products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. Polypeptides can be monomers or multimolecular complexes, such as dimers, trimers, or tetramers. They can also include single-chain or multi-chain polypeptides. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids. In some embodiments, a "peptide" can be 50 amino acids or less in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0106] As used herein, "RNA" refers to ribonucleic acid, which may be natural or non-natural. For example, RNA may contain modified and / or non-naturally occurring components, such as one or more nucleic acid bases, nucleosides, nucleotides, or linkers. RNA may contain a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence that encodes a polypeptide of interest.

[0107] As used herein, "DNA" refers to deoxyribonucleic acid, which may be naturally occurring or non-naturally occurring. 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, a "recombinant DNA molecule" refers to a DNA molecule that does not occur as a natural product but is produced using molecular biology techniques.

[0108] As used herein, a "single unit dose" is a dose of any therapeutic agent administered in one dose / one time / single route / single point of contact, i.e., a single administration event.

[0109] As used herein, a "split dose" is the division of a single unit dose or total daily dose into two or more doses.

[0110] As used herein, a "total daily dose" is the amount given or prescribed in a 24-hour period. It 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 term "subject" or "patient" refers to any organism to which a composition according to the disclosure can be administered, e.g., 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 cell" refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ, or in the tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

[0114] As used herein, "target tissue" refers to any one or more tissue types of interest where delivery of a therapeutic and / or prophylactic agent will produce a desired biological and / or pharmacological effect. Examples of target tissues of interest include specific tissues, organs, and systems or groups thereof. In particular applications, the target tissue may be the kidney, lung, spleen, vascular endothelium in a blood vessel (e.g., a coronary artery or femoral artery), or tumor tissue (e.g., via intratumoral injection). "Non-target tissue" refers to any one or more tissue types where expression of the encoded protein does not produce a desired biological and / or pharmacological effect. In particular applications, non-target tissues may include the liver and spleen.

[0115] The term "therapeutic agent" or "prophylactic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. Therapeutic agents are also referred to as "active agents" or "active agents." 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 "therapeutically effective amount" means an amount of an agent (e.g., nucleic acid, drug, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) delivered 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, ameliorate the symptoms thereof, diagnose, prevent, and / or delay the onset thereof.

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

[0118] As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, ameliorating, mitigating, delaying the onset of, inhibiting progression, reducing the severity, and / or reducing the incidence of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer can refer to inhibiting tumor survival, growth, and / or spread. Treatment can be administered to subjects who do not exhibit symptoms of the disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0119] As used herein, "zeta potential" is the electrokinetic potential of, for example, lipids in a particle composition.

[0120] Nanoparticle Composition The disclosure also features lipid nanoparticles comprising a 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) described herein.

[0121] In some embodiments, the largest dimension of the nanoparticle composition is 1 μm or less (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, for example, by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. Nanoparticle compositions include, for example, lipid nanoparticles (LNPs, e.g., empty or filled LNPs), liposomes, lipid vesicles, and lipoplexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In certain embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or crosslinked to each other. The lipid bilayers can comprise one or more ligands, proteins, or channels.

[0122] The nanoparticle composition comprises 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 can comprise one or more of the compounds in Table 1. The nanoparticle composition can also comprise a variety of other components. For example, the lipid component of the nanoparticle composition can include one or more other lipids in addition to a lipid 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).

[0123] Cationic / Ionic Lipids Lipid nanoparticles (e.g., empty or filled LNPs) can include one or more cationic and / or ionic lipids (e.g., lipids that can have a positive charge or partial positive charge at physiological pH) in addition to lipids 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).Cationic and / or ionic lipids include 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethyl 2,2-Dilinoleyl-4-dimethylaminopropane (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-Dilinoleyl Oxy-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 , 12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)), and (2S)-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(2S)). In addition, the cationic lipid may also be a lipid containing a cyclic amine group.

[0124] structural lipids Lipid nanoparticles (e.g., empty or filled LNPs) may comprise one or more structural lipids. The structural lipid may be selected from the group consisting of, but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid comprises cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. In some embodiments, the structural lipid is [ka] is.

[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, the phospholipids may contain a phospholipid moiety and one or more fatty acid moieties. For example, the phospholipid may be represented by the formula (IV): [ka] wherein R p represents the phospholipid moiety, and R A and R Brepresents a fatty acid moiety, with or without unsaturation, which may be the same or different. The phospholipid moiety may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting 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-naturally occurring species, including naturally occurring species with modifications and substitutions including branching, oxidation, cyclization, and alkynes, are also contemplated. 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 with triple bonds). Under appropriate reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition when exposed to azides. Such reactions can be useful for functionalizing the lipid bilayer of lipid nanoparticles (e.g., empty or filled LNPs) to facilitate membrane penetration or cell recognition, or for conjugating lipid nanoparticles (e.g., empty or filled LNPs) to useful components such as targeting or imaging moieties (e.g., dyes).

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

[0127] PEG lipids Lipid nanoparticles (e.g., empty or filled LNPs) can contain one or more PEGs or PEG-modified lipids. Such species can alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. The PEG lipid can be selected from the 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, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[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-distearic acid glycerol (PEG-DSG), PEG-dipalmetyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (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 certain embodiments, the PEG lipid has the formula (PL-I): [ka] The compound or a salt thereof, wherein R 3PL1 -OR OPL1 and R OPL1 is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r PL1 is an integer between 1 and 100 (inclusive), L 1 is an optionally substituted C 1-10 alkylene and optionally substituted C 1-10 At least one methylene of the alkylene is independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, O, N(R NPL1 ), S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R NPL1 ), NR NPL1 C(O)O, or NR NPL1 C(O)N(R NPL1 ) and D is a moiety obtained by click chemistry or cleavable under physiological conditions; m PL1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, A is a group having the formula: [ka] It is of L 2 Each instance of is independently a bond or an optionally substituted C 1-6 alkylene and optionally substituted C 1-6 One methylene unit of the alkylene is 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 ) and R 2SL Each instance of is independently an optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl, and optionally R 2SL one or more methylene units of are independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, N(R NPL1 ), O, S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), NR NPL1 C(O)N(R NPL1 ), C(O)O, OC(O), OC(O)O, OC(O)N(R NPL1 ), NR NPL1 C(O)O, C(O)S, SC(O), C(=NR NPL1 ), C(=NR NPL1 )N(R NPL1 ), NR NPL1 C(=NR NPL1 ), NR NPL1 C(=NR NPL1 )N(R NPL1 ), C(S), C(S)N(R NPL1 ), NR NPL1 C(S), NR NPL1 C(S)N(R NPL1 ), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R NPL1 )S(O), S(O)N(R NPL1 ), N(R NPL1 )S(O)N(R NPL1 ), OS(O)N(R NPL1 ), N(R NPL1 )S(O)O, S(O)2, N(R NPL1)S(O)2, S(O)2N(R NPL1 ), N(R NPL1 )S(O)2N(R NPL1 ), OS(O)2N(R NPL1 ), or N(R NPL1 )S(O)2O, R NPL1 is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p SL is either 1 or 2.

[0131] In certain embodiments, the PEG lipid has the formula (PL-I-OH): [ka] or a salt thereof.

[0132] In certain embodiments, the PEG lipid has the formula (PL-II-OH): [ka] or a salt or isomer thereof, wherein R 3PEG -OR O and R O is hydrogen, C 1-6 alkyl, or oxygen protecting group, r PEG is an integer between 1 and 100, R 5PEG is C 10-40 Alkyl, C 10-40 Alkenyl, or C 10-40 alkynyl, and optionally R 5PEG One or more methylene groups in 3-10 Carbocyclylene, 4-10 membered heterocyclylene, C6-10 Arylene, 4- to 10-membered heteroarylene, -N(R NPEG )-, -O-, -S-, -C(O)-, -C(O)N(R NPEG )-, -NR NPEG C(O)-, -NR NPEG C(O)N(R NPEG )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R NPEG )-, -NR NPEG C(O)O-, -C(O)S-, -SC(O)-, -C(=NR NPEG )-, -C(=NR NPEG )N(R NPEG )-, -NR NPEG C(=NR NPEG )-, -NR NPEG C(=NR NPEG )N(R NPEG )-, -C(S)-, -C(S)N(R NPEG )-, -NR NPEG C(S)-, -NR NPEG C(S)N(R NPEG )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R NPEG )S(O)-, -S(O)N(R NPEG )-, -N(R NPEG )S(O)N(R NPEG )-, -OS(O)N(R NPEG )-, -N(R NPEG )S(O)O-, -S(O)2-, -N(R NPEG )S(O)2-, -S(O)2N(R NPEG )-, -N(R NPEG )S(O)2N(R NPEG )-, -OS(O)2N(R NPEG )-, or -N(R NPEG )S(O)2O-, R NPEG Each instance of is independently hydrogen, C 1-6 alkyl, or nitrogen protecting groups.

[0133] In certain embodiments, in 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 certain embodiments, in the PEG lipid of formula (PL-II-OH), R 5 is C 17 It is alkyl.

[0135] In certain embodiments, the PEG lipid has the formula (PL-II): [ka] wherein r PEG is an integer between 1 and 100.

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

[0137] In certain embodiments, the PEG lipid has the formula (PL-III): [ka] or a salt or isomer thereof, wherein s PL1 is an integer between 1 and 100.

[0138] In certain embodiments, the PEG lipid has the following formula: [ka] is a compound of

[0139] In certain embodiments, compounds of formula (PL-I), (PL-I-OH), (PL-II), (PL-II-OH), (PL-III), PEG 2k The incorporation of one of the lipids (PL-II-OH), (PL-IIa-OH), (PL-II), or PEG-1 into the nanoparticle formulation can improve the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulation. For example, the incorporation of one of the lipids (PL-II-OH), (PL-IIa-OH), (PL-II), or PEG-1 into the nanoparticle formulation can reduce the accelerated blood clearance (ABC) effect.

[0140] Adjuvants In some embodiments, lipid nanoparticles (e.g., empty LNPs or loaded LNPs) comprising one or more lipids described herein may further comprise one or more adjuvants, such as glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxynucleotide (e.g., class A or B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0141] Treatment drugs The lipid nanoparticles (e.g., empty or loaded LNPs) can contain one or more therapeutic and / or prophylactic agents. The disclosure features methods for delivering a therapeutic and / or prophylactic agent to a mammalian cell or organ, methods for producing a polypeptide of interest in a mammalian cell, and methods for treating a disease or disorder in a mammal in need thereof, comprising administering to the mammal lipid nanoparticles (e.g., empty or loaded LNPs) containing a therapeutic and / or prophylactic agent and / or contacting mammalian cells with lipid nanoparticles (e.g., empty or loaded LNPs) containing a therapeutic and / or prophylactic agent.

[0142] Therapeutic and / or prophylactic agents include biologically active substances, alternatively referred to as "active agents." Therapeutic and / or prophylactic agents can be substances that, once delivered to a cell or organ, effect a desired change in the cell, organ, or other body tissue or system. Such species can be useful in the treatment of one or more diseases, disorders, or conditions. In some embodiments, therapeutic and / or prophylactic agents are small molecule drugs useful in the treatment of a particular disease, disorder, or condition.

[0143] In some embodiments, the therapeutic and / or prophylactic agent is a vaccine, a compound (e.g., a polynucleotide or nucleic acid molecule encoding a protein, polypeptide, or peptide, or a protein, polypeptide, or protein) that elicits an immune response, and / or another therapeutic and / or prophylactic agent. Vaccines include compounds and preparations capable of providing immunity against one or more conditions associated with an infectious disease and may include mRNA encoding antigens and / or epitopes derived from the infectious disease. 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 eliciting an immune response are administered intramuscularly via the disclosed compositions.

[0144] In other embodiments, the therapeutic and / or prophylactic agent is a protein, e.g., a protein needed to enhance or replace a naturally occurring protein of interest. Such proteins or polypeptides can be naturally occurring or can be modified, e.g., to increase half-life, using methods known in the art. Exemplary proteins are intracellular, transmembrane, or secreted proteins.

[0145] Polynucleotides and Nucleic Acids In some embodiments, the therapeutic agent is an agent that enhances (i.e., increases, stimulates, upregulates) protein expression. Non-limiting examples of 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). Agents that upregulate protein expression may upregulate the expression of naturally occurring or non-naturally occurring proteins (e.g., chimeric proteins modified to improve half-life or those containing 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 can 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 is triple-stranded or partially triple-stranded, i.e., having a triple-stranded portion and a double-stranded portion. The DNA molecule can be a circular DNA molecule or a linear DNA molecule.

[0147] A DNA therapeutic agent can be a DNA molecule capable of transcribing a gene into a cell, e.g., encoding and expressing a transcript. In other embodiments, the DNA molecule is a synthetic molecule, e.g., a synthetic DNA molecule generated in vitro. In some embodiments, the DNA molecule is a recombinant molecule. Non-limiting exemplary DNA therapeutic agents include plasmid expression vectors and viral expression vectors.

[0148] The DNA therapeutic agents described herein, such as DNA vectors, can include a variety of different features. The DNA therapeutic agents described herein, such as DNA vectors, can include non-coding DNA sequences. For example, the DNA sequence can 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 can have a non-coding DNA sequence operably linked to a transcriptionally active gene. In other embodiments, the DNA sequence described herein can have a non-coding DNA sequence that is not linked to a gene, i.e., the non-coding DNA does not regulate the gene on the DNA sequence.

[0149] In some embodiments, in the disclosed loaded LNPs, the one or more therapeutic and / or prophylactic agents are nucleic acids, hi some embodiments, the 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 loaded LNPs of the disclosure, the one or more therapeutic and / or prophylactic agents are selected from the group consisting of a plasmid expression vector, a viral expression vector, 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), antagomir, antisense RNA, ribozymes, dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), locked nucleic acid (LNA), and CRISPR / Cas9 technology, and 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 is an mRNA. In some embodiments, one or more therapeutic and / or prophylactic agents is a modified mRNA (mmRNA).

[0156] In some embodiments, the one or more therapeutic and / or prophylactic agents are mRNAs that incorporate a microRNA binding site (miR binding site). Further, in some embodiments, the mRNA comprises one or more of a stem-loop, a chain-terminating nucleoside, a polyA sequence, a polyadenylation signal, and / or a 5' cap structure.

[0157] mRNA can be natural or non-naturally occurring mRNA. mRNA can contain one or more modified nucleic acid bases, nucleosides, or nucleotides as described below, and in this case can be referred to as "modified mRNA" or "mmRNA". As used herein, a "nucleoside" is defined as a compound containing a sugar molecule (e.g., pentose or ribose) or its derivative, combined with an organic base (e.g., purine or pyrimidine) or its derivative (also referred to herein as "nucleobase"). As used herein, a "nucleotide" is defined as a nucleoside containing a phosphate group.

[0158] An mRNA can include a 5'-untranslated region (5'-UTR), a 3'-untranslated region (3'-UTR), and / or a coding region (e.g., an open reading frame). An mRNA can include 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., 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000) of base pairs. Any number (e.g., all, some, or none) of the nucleic acid bases, nucleosides, or nucleotides can be canonical species, substituted, modified, or otherwise non-naturally occurring analogs. In certain embodiments, all types of specific nucleic acid bases can be modified.In some embodiments, all uracil or uridine are modified.When all nucleic acid bases, nucleosides or nucleotides, for example, all uracil or uridine are modified, mRNA can be referred to as " fully modified " uracil or uridine.

[0159] In some embodiments, the mRNAs described herein may include a 5' cap structure, chain-terminating nucleotides, optionally a Kozak sequence (also known as a Kozak consensus sequence), a stem-loop, a polyA 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, non-naturally occurring caps or cap analogs, or inversion-preventing cap analogs (ARCAs). A CAP species can contain one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap can contain a guanine nucleotide and a guanine (G) nucleotide methylated at the 7th position joined by a triphosphate bond at the 5' position, such as m7G(5')ppp(5')G, commonly written as m7GpppG. A cap species can also be an inversion-preventing cap analog. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG, m27,O3'GppppG, m27,O2'GppppG, m7Gpppm7G, m73'dGpppG, m27,O3'GppppG, m27,O3'GppppG, and m27,O2'GppppG.

[0161] Alternatively, or in addition, mRNA may contain chain-terminating nucleosides. For example, chain-terminating nucleosides may include nucleosides 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, incorporation of chain-terminating nucleotides into mRNA, for example, at the 3' end, may result in stabilization of the mRNA.

[0162] Alternatively, or in addition, the mRNA may contain a stem loop, such as a histone stem loop. The stem loop may contain 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs. For example, the stem loop may contain 4, 5, 6, 7, or 8 nucleotide base pairs. The stem loop may be located in any region of the mRNA. For example, the stem loop may be located in, before, or after an untranslated region (5' untranslated region or 3' untranslated region), a coding region, a polyA sequence, or a tail. In some embodiments, the stem loop may affect one or more functions of the mRNA, such as translation initiation, translation efficiency, and / or transcription termination.

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

[0164] Alternatively or additionally, the mRNA may contain a microRNA binding site. MicroRNA binding sites (or miR binding sites) can be used to regulate mRNA expression in various tissues or cell types. In an exemplary embodiment, a miR binding site is engineered into the 3'UTR sequence of an mRNA to regulate, e.g., enhance, the degradation of the mRNA in cells or tissues that express the cognate miR. Such regulation is useful for regulating or controlling "off-target" expression of miR mRNA, i.e., in vivo expression in undesired cells or tissues. 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 that includes a first coding region and a second coding region, with an intervening sequence that includes an internal ribosome entry site (IRES) sequence that allows internal translation initiation between the first and second coding regions, or an intervening sequence that encodes a self-cleaving peptide, such as a 2A peptide. IRES sequences and 2A peptides are typically used to improve expression of multiple proteins from the same vector. For example, various IRES sequences, including the encephalomyomyositis virus IRES, are known and available in the art and can be used.

[0166] In some embodiments, the disclosed mRNAs comprise 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 a reference unmodified mRNA, including improved stability, intracellular retention, improved translation, and / or lack of substantial induction of the innate immune response of a cell into which the mRNA is introduced. Thus, the use of modified mRNA may improve the efficiency of protein production, intracellular retention of nucleic acids, and may have reduced immunogenicity.

[0167] In some embodiments, the mRNA comprises one or more (e.g., 1, 2, 3, or 4) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the mRNA comprises 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 nucleobases, nucleosides, or nucleotides. In some embodiments, the modified mRNA may be reduced in degradation in cells into which the mRNA is introduced, compared to the corresponding unmodified mRNA.

[0168] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-aminoallyl-uridine, 5-amino ... 5-Methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-aminomethyl-2-thiouridine (mcm5s2U), uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, l-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine,1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-meth oxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2 '-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (mcm5Um), Uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0169] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydro-cytidine (5-hydro-cytidine), 5-hydroxy-cytidine (5-hydroxy ... Hm5C, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine , Zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl These include 2'-F-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-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0170] In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having modified adenines 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-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenosine, 2-methoxy-adenosine, α-thiamine These include N6-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0171] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanosine include α-thio-guanosine, inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyobutosine (yW), peroxywyobutosine (o2yW), hydroxywyobutosine (OhyW), and hypomodified hydroxywyobutosine (OhyW). *), 7-deaza-guanosine, queosine (Q), epoxyqueosine (oQ), galactosyl-queosine (galQ), mannosyl-queosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (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), These include 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-ara-guanosine, and 2'-F-guanosine.

[0172] In some embodiments, the disclosed mRNAs comprise a combination of one or more of the foregoing modified nucleobases (e.g., a combination of two, three, or four of the foregoing modified nucleobases).

[0173] In some embodiments, the modified nucleobase is pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2'-0-methyluridine. In some embodiments, the disclosed mRNAs comprise a combination of one or more of the foregoing modified nucleobases (e.g., a combination of two, three, or four of the foregoing modified nucleobases). In some embodiments, the modified nucleobase is N1-methylpseudouridine (m1ψ), and the disclosed mRNA is fully modified with N1-methylpseudouridine (m1ψ). In some embodiments, N1-methylpseudouridine (m1ψ) represents 75-100% of the uracils in the mRNA. In some embodiments, N1-methylpseudouridine (m1ψ) represents 100% of the uracils in the mRNA.

[0174] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine. In some embodiments, the disclosed mRNAs comprise a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of two, three, or four of the aforementioned modified nucleobases).

[0175] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A). In some embodiments, the disclosed mRNAs comprise a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of two, three, or four of the aforementioned modified nucleobases).

[0176] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (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 disclosed mRNAs comprise a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of two, three, or four of the aforementioned modified nucleobases).

[0177] In some embodiments, the modified nucleobase is 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine, or α-thio-adenosine. In some embodiments, the disclosed mRNAs comprise a combination of one or more of the foregoing modified nucleobases (e.g., a combination of two, three, or four of the foregoing modified nucleobases).

[0178] In some embodiments, the mRNA comprises pseudouridine (ψ). In some embodiments, the mRNA comprises pseudouridine (ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2-thiouridine (s2U). In some embodiments, the mRNA comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2'-O-methyluridine. In some embodiments, the mRNA comprises 2'-O-methyluridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0179] In certain embodiments, the disclosed mRNA is uniformly modified (i.e., completely modified, modified throughout the entire sequence) for a particular modification. For example, the mRNA can be uniformly modified with N1-methylpseudouridine (m1ψ) or 5-methyl-cytidine (m5C), which means that all uridine or all cytosine nucleosides in the mRNA sequence are replaced with N1-methylpseudouridine (m1ψ) or 5-methyl-cytidine (m5C). Similarly, the disclosed mRNA for any type of nucleoside residue present in the sequence can be uniformly modified by substitution with modified residues such as those described above.

[0180] In some embodiments, the disclosed mRNAs may be modified in the coding region (e.g., the open reading frame encoding a polypeptide). In other embodiments, the mRNAs may be modified in regions other than the coding region. For example, in some embodiments, a 5'-UTR and / or a 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 can include combinations of modifications to the sugar, nucleobase, and / or internucleoside linkages, which can include any one or more of the modifications described herein.

[0182] When a single modification is listed, the listed nucleoside or nucleotide represents 100% of the modified A, U, G, or C nucleotides or nucleosides. When percentages are listed, they represent the percentage of that particular A, U, G, or C triphosphate nucleobase relative to 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 in which 25% of the cytosine triphosphates are 5-aminoallyl-CTP and 75% of the cytosines are CTP, while 25% of the uracils are 5-methoxy-UTP and 75% of the uracils are UTP. When no modified UTP is listed, naturally occurring ATP, UTP, GTP, and / or CTP are used at 100% of those nucleotide positions found in the polynucleotide. In this example, all of the GTP and ATP nucleotides remain unmodified.

[0183] The mRNA of the present disclosure, or a region thereof, can be codon-optimized. Codon optimization methods are known in the art and can be useful for a variety of purposes, including matching the codon frequency in a host organism to ensure proper folding, biasing the G / C content to increase mRNA stability or reduce secondary structures, minimizing tandem repeat codons or stretches of bases that may impair gene assembly or expression, customizing transcriptional and translational control regions, inserting or removing protein transport sequences, removing / adding post-translational modification sites (e.g., glycosylation sites) in the encoded protein, adding, removing, or shuffling protein domains, inserting or deleting restriction sites, modifying ribosome binding sites and mRNA degradation sites, adjusting the translation rate to allow various domains of a protein to fold properly, or reducing or eliminating problematic secondary structures within a polynucleotide. 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), 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 present disclosure includes polynucleotides having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the polynucleotide sequences set forth herein.

[0185] The mRNA of the present disclosure can be produced by means available in the art, including, but not limited to, in vitro transcription (IVT) and synthetic methods. Enzymatic (IVT), solid-phase, liquid-phase, combined synthesis, small-area synthesis, and ligation methods can be utilized. In some embodiments, the mRNA is produced using an IVT enzymatic synthesis method. Thus, the present disclosure also includes polynucleotides, such as DNA, constructs, and vectors, that can be used to in vitro transcribe the mRNA described herein.

[0186] Non-natural modified nucleobases can be introduced into polynucleotides, such as mRNA, during or after synthesis.In certain embodiments, modifications can be present on internucleoside linkages, purine or pyrimidine bases, or sugars.In certain embodiments, modifications can be introduced at the end of polynucleotide chain or at any other location within polynucleotide chain using chemical synthesis or polymerase enzyme.

[0187] Either enzymatic or chemical ligation methods can be used to conjugate polynucleotides or regions thereof with different functional moieties such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc.

[0188] Therapeutic Agents for Reducing Protein Expression In some embodiments, the therapeutic agent is one that reduces (i.e., reduces, inhibits, downregulates) protein expression. Non-limiting examples of types of therapeutic agents that can be used to reduce protein expression include microRNA binding site(s) (miR binding sites), microRNAs (miRNAs), antagomirs, small (short) interfering RNAs (siRNAs) (including shortmers and Dicer substrate RNAs), RNA interference (RNAi) molecules, antisense RNAs, ribozymes, small hairpin RNAs (shRNAs), locked 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 naturally occurring, e.g., isolated from a natural source. In other embodiments, the peptide or polypeptide is a synthetic molecule, e.g., a synthetic peptide or polypeptide generated 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 of the composition is a naturally occurring peptide or polypeptide. In some embodiments, the peptide or polypeptide therapeutic of the composition is a modified version of a naturally occurring peptide or polypeptide (e.g., containing fewer than 3, fewer than 5, fewer than 10, fewer than 15, fewer than 20, or fewer than 25 amino substitutions, deletions, or additions compared to its wild-type, naturally occurring peptide or polypeptide counterpart).

[0191] In some embodiments, in the loaded LNPs of the disclosure, the one or more therapeutic and / or prophylactic agents is a polynucleotide or a polypeptide.

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

[0193] Lipid nanoparticles (e.g., empty or filled LNPs) may also include 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] A polymer may be included and / or used to encapsulate or partially encapsulate the nanoparticle composition. The polymer may be biodegradable and / or biocompatible. The polymer may be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polyethacrylates, 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-glycolic acid) (PLGA), poly(L-lactic-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), and poly(D,L-lactide-co-PPO-co-D,L-lactide).L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose cellulose, 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, and Included may be mentioned polymers of acrylic acid, such as mixtures thereof, polydioxanone and copolymers thereof, polyhydroxyalkanoates, polypropylene fumarates, polyoxymethylenes, poloxamers, poloxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycols.

[0195] Surface-altering agents may 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, eprazinone, mesna, ambroxol, sobrerol, domiodol, letostein, stepronin, thioproline, gellin, thymosin, β4, dornase, alpha, neltenexin, and erdosteine), and DNases (e.g., rhDNase). Surface-altering agents may be disposed within the nanoparticle and / or on the surface of the lipid nanoparticle (e.g., empty or filled LNP) (e.g., by coating, adsorption, covalent attachment, or other process).

[0196] Lipid nanoparticles (e.g., empty 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 facilitating membrane penetration, cell recognition, or imaging. The surface of lipid nanoparticles (e.g., empty or filled LNPs) may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful in targeted cell delivery, imaging, and membrane penetration are well known in the art.

[0197] In addition to these components, lipid nanoparticles (e.g., empty or filled LNPs) can contain any substance useful in pharmaceutical compositions. For example, lipid nanoparticles (e.g., empty or filled LNPs) can contain one or more pharmaceutically acceptable excipients or accessory ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, fluids, liquid vehicles, binders, surfactants, isotonicity agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives, and other species. They may also contain excipients such as waxes, butters, colorants, coating agents, flavors, and fragrances.

[0198] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof. Granulating and dispersing agents may be selected from the 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 sponge, 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, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium raudyl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0199] Surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene ethers), sorbitan 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 ethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(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, docusate sodium, and / or combinations thereof may be included.

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

[0201] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl formate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antibacterial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal 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, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopheryl acetate, distoxime 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 buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, aminosulfonic acid 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 the 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, black current seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, linseed, geraniol, gourd, grapeseed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savory, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, camellia, vetiver, walnut, and wheat germ oils, as well as butyl stearate, caprylic triglyceride, glyceryl tricaprate, 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 or filled LNPs) can include a lipid component and one or more additional components, such as a therapeutic agent and / or a prophylactic agent. Lipid nanoparticles (e.g., empty or filled LNPs) can be designed for one or more specific applications or targets. The components of a lipid nanoparticle (e.g., empty or filled LNPs) can be selected based on the specific application or target and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more components. Similarly, a particular formulation of a nanoparticle composition can be selected for a particular application or target, depending, for example, on the efficacy and toxicity of a particular combination of components.

[0205] The lipid component of the nanoparticle composition can include, for example, lipids 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), phospholipids (unsaturated lipids, such as DOPE or DSPC), PEG lipids, and structural lipids. The elements of the lipid component can be provided in specific fractions.

[0206] In some embodiments, the lipid component of the nanoparticle composition comprises a lipid 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), a phospholipid, a PEG lipid, and a structural lipid. In certain embodiments, 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 a phospholipid, about 18.5 mol% to about 48.5 mol% of a structural lipid, and about 0 mol% to about 10 mol% of a PEG lipid, 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 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 5 mol% to about 25 mol% of a phospholipid, about 30 mol% to about 40 mol% of a structural lipid, and about 0 mol% to about 10 mol% of a PEG lipid. In certain embodiments, the lipid component comprises about 50 mol% of the compound, about 10 mol% of a phospholipid, about 38.5 mol% of a structural lipid, and about 1.5 mol% of a PEG lipid. In another specific embodiment, the lipid component comprises about 40 mol% of the compound, about 20 mol% of a phospholipid, about 38.5 mol% of a structural lipid, and about 1.5 mol% of a PEG lipid. In some embodiments, the phospholipid may be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-1 or PEG. 2k-DMG and / or the structural lipid may be cholesterol.

[0207] In some embodiments, the empty lipid nanoparticles (empty 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, and a PEG lipid.

[0208] In some embodiments, the loaded lipid nanoparticles (loaded 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 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) in an amount of about 40% to about 60%.

[0210] In some embodiments, empty or loaded LNPs contain phospholipids in an amount of about 0% to about 20%. For example, in some embodiments, empty or loaded LNPs contain DSPC in an amount of about 0% to about 20%.

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

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

[0213] In some embodiments, empty or filled LNPs comprise 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 a phospholipid, about 30 mol% to about 50 mol% of a structural lipid, and about 0 mol% to about 5 mol% of a PEG-lipid.

[0214] In some embodiments, empty or filled LNPs comprise 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% DSPC, about 30 mol% to about 50 mol% cholesterol, and about 0 mol% to about 5 mol% PEG. 2kIn some embodiments, empty or filled LNPs comprise about 40 mol% to about 60 mol% of a compound of Table 1, about 0 mol% to about 20 mol% DSPC, about 30 mol% to about 50 mol% cholesterol, and about 0 mol% to about 5 mol% PEG. 2k -DMG, including.

[0215] In some embodiments, empty or filled LNPs comprise 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% DSPC, about 30 mol% to about 50 mol% cholesterol, and about 0 mol% to about 5 mol% PEG-1. In some embodiments, empty or loaded LNPs comprise about 40 mol% to about 60 mol% of a compound in Table 1, about 0 mol% to about 20 mol% DSPC, about 30 mol% to about 50 mol% cholesterol, and about 0 mol% to about 5 mol% PEG-1.

[0216] In some embodiments, empty or 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, and a PEG-lipid, wherein the phospholipid is DSPC and the structural lipid is cholesterol. In some embodiments, empty or filled LNPs comprise a compound of Table 1, a phospholipid, a structural lipid, and a PEG-lipid, wherein the phospholipid is DSPC and the structural lipid is cholesterol.

[0217] In some embodiments, the empty or 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, and a PEG lipid, wherein the structural lipid is cholesterol and the PEG lipid is PEG. 2k In some embodiments, empty or filled LNPs comprise a compound of Table 1, a phospholipid, a structural lipid, and a PEG lipid, wherein the structural lipid is cholesterol and the PEG lipid is PEG-DMG. 2k -DMG.

[0218] In some embodiments, empty or 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, and a PEG lipid, wherein the structural lipid is cholesterol and the PEG lipid is PEG-1. In some embodiments, empty or filled LNPs comprise a compound of Table 1, a phospholipid, a structural lipid, and a PEG lipid, wherein the structural lipid is cholesterol and the PEG lipid is PEG-1.

[0219] In some embodiments, the empty or 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, and a PEG lipid, wherein the phospholipid is DSPC and the PEG lipid is PEG.2k In some embodiments, empty or filled LNPs comprise a compound of Table 1, a phospholipid, a structural lipid, and a PEG lipid, wherein the structural lipid is DSPC and the PEG lipid is PEG -DMG. 2k -DMG.

[0220] In some embodiments, empty or 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, and a PEG lipid, wherein the phospholipid is DSPC and the PEG lipid is PEG-1. In some embodiments, empty or filled LNPs comprise a compound of Table 1, a phospholipid, a structural lipid, and a PEG lipid, wherein the phospholipid is DSPC and the PEG lipid is PEG-1.

[0221] In some embodiments, the empty or 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, and a PEG lipid, wherein the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG. 2k In some embodiments, empty or filled LNPs comprise a compound of Table 1, 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-DMG. 2k -DMG.

[0222] In some embodiments, the empty or filled LNPs comprise a compound of formula (Ac), 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 In some embodiments, empty or filled LNPs comprise a compound of Table 1, 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-DMG. 2k -DMG.

[0223] In some embodiments, empty or 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, and a PEG lipid, wherein the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG-1.

[0224] In some embodiments, empty or filled LNPs comprise a compound of formula (Ac), 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-1. In some embodiments, empty or filled LNPs comprise a compound of Table 1, 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-1.

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

[0226] The amount of therapeutic and / or prophylactic agent in a nanoparticle composition can depend on the size, composition, desired target and / or use, or other characteristics of the nanoparticle composition, as well as the characteristics of the therapeutic and / or prophylactic agent. For example, the amount of RNA useful in a nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and / or prophylactic agent and other components (e.g., lipids) in the nanoparticle composition can also vary. In some embodiments, the weight / weight ratio of lipid component to therapeutic and / or prophylactic agent in the nanoparticle composition can be from about 5:1 to about 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 component to therapeutic and / or prophylactic agent can be 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 agent in the nanoparticle composition can be determined using, for example, absorption spectroscopy (eg, UV-visible spectroscopy).

[0228] In some embodiments, nanoparticle compositions include one or more RNAs, one or more RNAs, and lipids, the amounts of which can be selected to provide a specific N:P ratio. The N:P ratio of a composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. Generally, lower N:P ratios are preferred. The one or more RNAs, lipids, and their amounts can be selected to provide an N:P ratio of about 2:1 to about 30:1, e.g., 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 can 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 can be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. For example, the N:P ratio can be about 5.67:1.

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

[0230] Lipid nanoparticles (e.g., empty or loaded 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. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure several properties of the nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0231] The average size of lipid nanoparticles (e.g., empty or filled LNPs) can be tens to hundreds of nanometers, as measured, for example, by dynamic light scattering (DLS). For example, the average size can be about 40 nm to about 150 nm, e.g., about 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) can be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 150 nm, about 70 nm to about 130 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 150 nm, about 80 nm to about 130 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, about 90 nm to about 150 nm, about 90 nm to about 130 nm, or about 90 nm to about 100 nm. In certain embodiments, the average size of the lipid nanoparticles (e.g., empty or filled LNPs) can be about 70 nm to about 130 nm, or about 70 nm to about 100 nm. In certain embodiments, the average size can be about 80 nm. In other embodiments, the average size can be about 100 nm. In other embodiments, the average size can 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 the 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) can have a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the lipid nanoparticles (e.g., empty LNPs or filled LNPs) can be from about 0.10 to about 0.20.

[0233] The zeta potential of lipid nanoparticles (e.g., empty or filled LNPs) can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Lipid nanoparticles (e.g., empty or filled LNPs) with a relatively low positive or negative charge are generally desirable because more highly charged species may have undesirable interactions with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of lipid nanoparticles (e.g., empty or filled LNPs) is between about -10 mV and about +20 mV, about -10 mV and about +15 mV, about -10 mV and about +10 mV, about -10 mV and about +5 mV, about -10 mV and about 0 mV, about -10 mV and about -5 mV, about -5 mV and about +20 mV, or about It can be −5 mV to about +15 mV, about −5 mV to about +10 mV, about −5 mV to about +5 mV, about −5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0234] The efficiency of encapsulation of a therapeutic and / or prophylactic agent 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 desirably high (e.g., approaching 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) with one or more organic solvents or detergents before and after the lipid nanoparticles (e.g., empty or filled LNPs) are disassembled. Fluorescence may also be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in solution. In the lipid nanoparticles described herein (e.g., empty LNPs or loaded LNPs), the encapsulation efficiency of the therapeutic and / or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%. In some embodiments, the encapsulation efficiency of the therapeutic and / or prophylactic agent is between 80% and 100%.

[0235] Pharmaceutical Compositions Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be formulated in whole or in part as a pharmaceutical composition. A pharmaceutical composition can include one or more lipid nanoparticles (e.g., empty LNPs or loaded LNPs). In one embodiment, a pharmaceutical composition includes a population of lipid nanoparticles (e.g., empty LNPs or loaded LNPs). For example, a pharmaceutical composition can include one or more lipid nanoparticles (e.g., empty LNPs or loaded LNPs) containing one or more different therapeutic and / or prophylactic agents. A pharmaceutical composition can further include one or more pharmaceutically acceptable excipients or accessory ingredients, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and medicaments can be found, for example, in Remington's The Science and Practice of Pharmacy, 21 st Edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and accessory ingredients can be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more of the components of the nanoparticle composition. An excipient or accessory ingredient may be incompatible with a component of a nanoparticle (e.g., empty or filled LNP) if its combination with the component could result in any undesirable biological or other adverse effect.

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

[0237] The relative amounts of one or more lipid nanoparticles according to the present disclosure (e.g., empty or filled LNPs), one or more pharmaceutically acceptable excipients, and / or any additional components in a pharmaceutical composition will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. By way of example, a pharmaceutical composition may contain 0.1% to 100% (w / w) 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 refrigerated or frozen for storage and / or shipping (e.g., stored at temperatures below 4°C, between about -150°C and about 0°C, or between about -80°C and about 20°C, such as at 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 comprising any one 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), and (Bc) can be stored and maintained at, for example, about −20° C., −30° C., −40° C., −50° C., −60° C., −70° C., or −80° C. In certain embodiments, the disclosure also provides for the storage of lipid nanoparticles (e.g., empty or filled LNPs) and / or pharmaceutical compositions at temperatures below 4°C, such as temperatures between about -150°C to about 0°C or between 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. and a method for increasing the stability of lipid nanoparticles (e.g., empty or filled LNPs) and / or pharmaceutical compositions comprising a compound of any 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).For example, the lipid nanoparticles (e.g., empty or loaded LNPs) and / or pharmaceutical compositions disclosed herein are stable, e.g., at temperatures below 4°C (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. In some embodiments, the formulations are stabilized at about 4°C for at least 4 weeks. In certain embodiments, the disclosed pharmaceutical compositions comprise the lipid nanoparticles (e.g., empty or loaded 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), saline, PBS, and sucrose. In certain embodiments, the disclosed pharmaceutical compositions have a pH 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 compositions comprise lipid nanoparticles disclosed herein (e.g., empty or filled LNPs), Tris, saline, and sucrose, and have a pH of about 7.5-8, which is suitable for storage and / or shipping at, for example, about -20°C. For example, the disclosed pharmaceutical compositions comprise lipid nanoparticles disclosed herein (e.g., empty or filled LNPs) and PBS, and have a pH of about 7-7.8, which is suitable for storage and / or shipping at, for example, about 4°C or below. "Stability," "stabilized," and "stable," in the context of this disclosure, refer to the resistance of the lipid nanoparticles (e.g., empty or filled LNPs) and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size change, aggregation, change in encapsulation, etc.) under given conditions of manufacture, preparation, transportation, storage, and / or use, e.g., when stressed by shear forces, freeze / thaw stress, etc.

[0239] In some embodiments, the disclosed pharmaceutical compositions comprise empty or filled LNPs, a cryoprotectant, a buffer, or a combination thereof.

[0240] In some embodiments, the cryoprotectant comprises one or more cryoprotectants, each of which may independently be selected from the group consisting of a polyol (e.g., a 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), a non-detergent sulfobetaine (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), an osmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), a 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- 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. In some embodiments, the cryoprotectant and / or excipient is sodium acetate. In some embodiments, the cryoprotectant comprises sucrose and 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] Lipid nanoparticles (e.g., empty or filled LNPs) and / or pharmaceutical compositions comprising one or more lipid nanoparticles (e.g., empty or filled LNPs) can be administered to any patient or subject, including those who can benefit from a therapeutic effect provided by delivery of a therapeutic and / or prophylactic agent to one or more specific cells, tissues, organs, or systems or groups thereof. While the description provided herein of lipid nanoparticles (e.g., empty or filled LNPs) and pharmaceutical compositions comprising lipid nanoparticles (e.g., empty or filled LNPs) is primarily directed to compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammal. Modifications of compositions suitable for administration to humans to create compositions suitable for administration to various animals are well understood, and such modifications can be designed and / or implemented by a veterinary pharmacologist of ordinary skill with no more than routine experimentation (if any). Subjects to which the compositions are intended to be administered include, but are not limited to, humans, other primates, and other mammals, e.g., commercially relevant mammals such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats. The subject lipid nanoparticles can also be used for in vitro and ex vivo uses.

[0243] Pharmaceutical compositions containing one or more lipid nanoparticles (e.g., empty or filled LNPs) can be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparative methods involve bringing into association the active ingredient with an excipient and / or one or more other accessory ingredients, and then, as desired or necessary, dividing, shaping, and / or packaging the product into desired single or multiple dosage units.

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

[0245] Pharmaceutical compositions can be prepared in a variety of forms suitable for various routes and modes 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), dosage forms for topical and / or transdermal administration (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 contain inert diluents commonly used in the art, such as water or other solvents, solubilizing and emulsifying agents, 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 fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may contain additional agents, such as additional therapeutic and / or prophylactic agents, wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and / or perfuming agents. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent, such as Cremophor®, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or combinations thereof.

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

[0248] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0249] To prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends on its dissolution rate, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the drug-to-polymer ratio and the properties of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions that are 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 a suppository wax, which is solid at ambient temperature but liquid at body temperature and will therefore melt in the rectum or vaginal cavity and release 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 a mixture of at least one inert pharmaceutically acceptable excipient, such as sodium citrate or dicalcium phosphate, and / or a filler or extender (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid), a binder (e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), a humectant (e.g., glycerol), a disintegrant (e.g., agar, calcium carbonate, potato or The formulation may be mixed with additives such as tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarders (e.g., paraffin), absorption accelerators (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 laurate sulfate), and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may contain buffering agents.

[0252] Solid compositions of a similar type can be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They can optionally contain opacifying agents and can be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[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, the present disclosure often contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared, for example, by dissolving and / or dispensing the compound in an appropriate medium. Alternatively or additionally, the rate can be controlled by either providing a rate-controlling 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 can be administered using devices that limit the effective needle penetration length into the skin. Jet injection devices are suitable, 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 suitable, using compressed gas to accelerate a powder-form vaccine through the outer layer of the skin and into the dermis. Alternatively or additionally, conventional syringes can be used in the classic Mantoux method of intradermal administration.

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

[0256] Pharmaceutical compositions can be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such formulations may comprise dry particles containing the active ingredient. Such compositions are conveniently in the form of a dry powder for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder, and / or using a self-propelling 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 can include a solid fine powder diluent, such as sugar, and are conveniently provided in a unit dose form.

[0257] Low-boiling propellants generally comprise a liquid propellant having a boiling point below 65°F at atmospheric pressure. Generally, the propellant may comprise 50% to 99.9% (w / w) of the composition, and the active ingredient may comprise 0.1% to 20% (w / w) of the composition. The propellant may further comprise additional ingredients such as liquid nonionic and / or solid anionic surfactants and / or solid diluents (which may have a particle size on the same order as the particles containing the active ingredient).

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

[0259] The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 μm to 500 μm. Such formulations are administered in the same manner as snuff is taken, i.e., by rapid inhalation through the nasal passages from a container of powder held close to the nose.

[0260] Formulations suitable for nasal administration may contain, for example, between about 0.1% (w / w) and about 100% (w / w) of the active ingredient, and may include one or more of the additional ingredients described herein. Pharmaceutical compositions may be prepared, packaged, and / or sold in a formulation suitable for buccal administration. Such formulations may be, for example, in the form of tablets and / or lozenges made using conventional methods and may contain, for example, 0.1 to 20% (w / w) of the active ingredient, with the remainder comprising an orally dissolvable and / or disintegrable composition, and optionally one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise a powder and / or aerosolized and / or atomized solution and / or suspension comprising the active ingredient. Such powdered, aerosolized, and / or atomized 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 include any one or more of the additional ingredients described herein.

[0261] The pharmaceutical compositions may be prepared, packaged, and / or sold in a formulation suitable for ophthalmic administration. Such formulations may be in the form of, for example, eye drops comprising a 0.1 / 1.0% (w / w) solution and / or suspension of the active ingredient in an aqueous or oily liquid vehicle. Such eye drops may further comprise one or more other ingredients, such as a buffer, salt, and / or any of the additional ingredients described herein. Other useful ophthalmically administrable formulations include those comprising the active ingredient in microcrystalline form and / or in a liposomal preparation. Ear drops and / or eye drops are contemplated as being within the scope of the present 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 penetrate membranes and deliver intracellular targets that are inaccessible to standard biological agents due to their inability to cross the cell membrane when delivered in protein form. One major challenge to realizing mRNA-based therapy is identifying an 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 protect the cargo from immune sentinels. Lipid nanoparticles (LNPs) have been identified as a leading option in this regard.

[0263] Important performance criteria for lipid nanoparticle delivery systems are maximizing cellular uptake and enabling efficient release of mRNA from endosomes. In one embodiment, the subject LNPs containing the novel lipids disclosed herein exhibit improved cellular uptake and / or endosomal release. At the same time, the 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 amino lipids, phospholipids, cholesterol, and PEG lipids. Each component is necessary for efficient delivery of nucleic acid cargo and particle stability. The amino lipid is a key component believed to promote cellular uptake, endosomal escape, and tolerability. Cholesterol and PEG lipids contribute to the stability of the drug product both in vivo and during storage, while the phospholipid provides additional fusogenicity to the LNPs, thereby helping to promote endosomal escape and making the nucleic acid bioavailable in the cytoplasm of cells.

[0264] Several aminolipid series, including the aminolipid MC3 (DLin-MC3-DMA), have been developed over the past few decades for oligonucleotide delivery. 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 preclude its use in long-term therapy. In addition, extensive literature evidence suggests that long-term administration of lipid nanoparticles may result in several toxic side effects, including complement activation-associated pseudoallergy (CARPA) and liver damage. Therefore, to realize the potential of mRNA and other nucleic acid, nucleotide, or peptide-based therapies in humans, a class of LNPs with increased delivery efficiency combined with metabolic and toxicity profiles that would enable long-term administration in humans is needed.

[0265] The ability to treat a wide range of diseases requires the flexibility to safely administer at varying dose levels over the long term. Through systematic optimization of amino lipid structures, the disclosed compounds were identified as offering a good balance of chemical stability, improved delivery efficiency due to improved endosomal escape, rapid in vivo metabolism, and a clean toxicity profile. This combination of features provides drug candidates that can be administered chronically without activating the immune system. Initial rodent screening led to the identification of lead lipids with favorable delivery efficiency and pharmacokinetics. The advanced LNPs were further profiled in non-human primates for delivery efficiency after single and repeated administration. Finally, the optimized LNPs were evaluated in a 1-month repeat-dose toxicity study in rats and non-human primates. Without wishing to be bound by theory, the novel ionic lipids disclosed herein possess improved cellular delivery, improved protein expression, and improved biodegradability, and may result in a 2-fold, 5-fold, 10-fold, 15-fold, or even greater than 20-fold increase in mRNA expression in cells compared to LNPs lacking the inventive lipid. In another embodiment, LNPs comprising the lipids of the invention may result in specific (e.g., preferential) delivery to a particular cell type or types relative to other cell types, thereby resulting in a 2-fold, 5-fold, 10-fold, 15-fold, or even 20-fold increase in mRNA expression in a particular cell or tissue relative 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 aspects, the disclosure provides methods for delivering a therapeutic and / or prophylactic agent to a cell (e.g., a mammalian cell). The method includes contacting the cell with a disclosed loaded LNP or pharmaceutical composition, thereby delivering the therapeutic and / or prophylactic agent to the cell. In some embodiments, the cell is in a subject, and the contacting includes administering the cell to the subject. In some embodiments, the method includes administering to a subject 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), a phospholipid, a structured lipid, a PEG-lipid, and one or more therapeutic and / or prophylactic agents, thereby delivering the therapeutic and / or prophylactic agents to the cells.

[0267] In some embodiments, the disclosure provides a method of delivering a therapeutic and / or prophylactic agent to a cell in a subject, the method comprising: combining 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 and administering to a subject lipid nanoparticles comprising -DMG and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the disclosure provides a method of delivering a therapeutic and / or prophylactic agent to cells in a subject, the method comprising administering to a subject lipid nanoparticles comprising a compound of formula (Ac), DSPC, cholesterol, and PEG. 2k - Administering to a subject lipid nanoparticles 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 of delivering a therapeutic and / or prophylactic agent to cells in a subject, the method comprising administering to the subject 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 one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the disclosure provides a method for delivering a therapeutic and / or prophylactic agent to cells in a subject, the method comprising administering to the subject lipid nanoparticles comprising a compound of formula (Ac), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA).

[0269] In some aspects, the disclosure provides methods for delivering (e.g., specifically delivering) a therapeutic and / or prophylactic agent to a mammalian organ or tissue (e.g., liver, kidney, spleen, or lung), the method comprising contacting a cell with a disclosed loaded LNP or pharmaceutical composition, thereby delivering the therapeutic and / or prophylactic agent to the target organ or tissue. In some embodiments, the method includes administering to a subject 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), a phospholipid, a structured lipid, a PEG-lipid, and one or more therapeutic and / or prophylactic agents, whereby the therapeutic and / or prophylactic agents are delivered to the target organ or tissue.

[0270] 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: 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 and administering to a subject lipid nanoparticles comprising -DMG and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (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 a subject, the method comprising administering to a subject lipid nanoparticles comprising a compound of formula (Ac), DSPC, cholesterol, and PEG. 2k - Administering to a subject lipid nanoparticles 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 a therapeutic and / or prophylactic agent to an organ of a subject, the method comprising administering to the subject 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 one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (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 a subject, the method comprising administering to a subject lipid nanoparticles comprising a compound of formula (Ac), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA).

[0272] In some aspects, the disclosure features a method for enhanced delivery of a therapeutic and / or prophylactic agent (e.g., mRNA) to a target tissue (e.g., liver, spleen, or lung). The method includes contacting a cell with a disclosed loaded LNP or pharmaceutical composition, thereby delivering the therapeutic and / or prophylactic agent to the target tissue (e.g., liver, kidney, spleen, or lung). In some embodiments, the method includes administering to a subject 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), a phospholipid, a structured lipid, a PEG-lipid, and one or more therapeutic and / or prophylactic agents, thereby delivering the therapeutic and / or prophylactic agents to the target tissue (e.g., liver, kidney, spleen, or lung).

[0273] In some embodiments, the disclosure provides a method for enhanced delivery of a therapeutic and / or prophylactic agent to a target tissue, 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 and administering to a subject lipid nanoparticles comprising -DMG and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the disclosure provides a method for enhanced delivery of a therapeutic and / or prophylactic agent to a target tissue, the method comprising administering to a subject lipid nanoparticles comprising a compound of formula (Ac), DSPC, cholesterol, and PEG. 2k - Administering to a subject lipid nanoparticles 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 a therapeutic and / or prophylactic agent to a target tissue, the method comprising administering to a subject 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 one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (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 administering to a subject lipid nanoparticles comprising a compound of formula (Ac), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA).

[0275] In some aspects, the disclosure provides methods for producing a polypeptide of interest in a cell (e.g., a mammalian cell). The method includes contacting the cell with a disclosed loaded LNP or pharmaceutical composition, wherein the loaded LNP or pharmaceutical composition includes mRNA, whereby the mRNA can be translated within the cell to produce the polypeptide. In some embodiments, the cell is in a subject, and the contacting includes administering the cell to the subject. In some embodiments, the method includes administering to a subject 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), a phospholipid, a structural lipid, a PEG lipid, and mRNA, whereby the mRNA can be translated intracellularly to produce the polypeptide.

[0276] In some embodiments, the disclosure provides a method of producing a polypeptide of interest in a cell, the method comprising: combining 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 method includes administering to a subject lipid nanoparticles comprising -DMG and mRNA. For example, in some embodiments, the disclosure provides a method for producing a polypeptide of interest in a cell, the method comprising administering to a subject lipid nanoparticles comprising a compound of Table 1, DSPC, cholesterol, and PEG. 2k and administering to a subject lipid nanoparticles comprising -DMG and mRNA. For example, in some embodiments, the disclosure provides a method for producing a polypeptide of interest in a cell, the method comprising administering to a subject lipid nanoparticles comprising a compound of formula (Ac), DSPC, cholesterol, and PEG. 2k The present invention provides a method for administering to a subject lipid nanoparticles comprising -DMG and mRNA.

[0277] In some embodiments, the disclosure provides a method for producing a polypeptide of interest in a cell, the method comprising administering to a subject 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. For example, in some embodiments, the disclosure provides a method for producing a polypeptide of interest in a cell, the method comprising administering to a subject lipid nanoparticles comprising a compound of Formula (Ac), DSPC, cholesterol, PEG-1, and mRNA. For example, in some embodiments, the disclosure provides a method of producing a polypeptide of interest in a cell, the method comprising administering to a subject lipid nanoparticles comprising a compound of Table 1, DSPC, cholesterol, PEG-1, and mRNA.

[0278] In some aspects, the disclosure provides methods of treating a disease or disorder in a mammal (e.g., a human) in need thereof. The method comprises administering a therapeutically effective amount of a disclosed loaded LNP or pharmaceutical composition to the mammal. In some embodiments, the method comprises administering to the subject 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), a phospholipid, a structured 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 a malfunction 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 diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and renal vascular diseases, and metabolic diseases.

[0279] In some embodiments, the disclosure provides a method of treating a disease or disorder in a subject, the method comprising combining 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 and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject lipid nanoparticles comprising a compound of formula (Ac), DSPC, cholesterol, and PEG. 2kand administering to a subject lipid nanoparticles comprising DMG and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to a subject lipid nanoparticles comprising a compound of Table 1, DSPC, cholesterol, and PEG. 2k - Administering to a subject lipid nanoparticles 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 of treating a disease or disorder in a subject, the method comprising administering to the subject 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 one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject lipid nanoparticles comprising a compound of Formula (Ac), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject lipid nanoparticles comprising a compound of Table 1, DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA).

[0281] In yet another aspect, the disclosure provides a method of reducing immunogenicity, comprising introducing a disclosed loaded LNP or pharmaceutical composition into a cell, wherein the loaded LNP or pharmaceutical composition reduces induction of a cellular immune response in the cell to the loaded LNP or pharmaceutical composition compared to induction of a cellular immune response in the cell induced by a reference composition. In some embodiments, the cell is in a subject, and the contacting comprises administering the cell to the subject. In some embodiments, the method includes administering to a subject 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), a phospholipid, a structured lipid, a PEG-lipid, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA), wherein the 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). Lipid nanoparticles containing a compound of formula (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 a potent potency of inducing a cellular immune response in cells induced by a reference composition, compared to that induced by a reference composition. The lipid nanoparticles containing the compound (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) induce a reduced cellular immune response in cells. For example, the cellular immune response may be an innate immune response, an adaptive immune response, or both.

[0282] In some embodiments, the disclosure provides a method of reducing immunogenicity in a subject, the method comprising combining 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 and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the disclosure provides a method of reducing immunogenicity in a subject, the method comprising administering to the subject lipid nanoparticles comprising a compound of formula (Ac), DSPC, cholesterol, and PEG. 2k and administering to a subject lipid nanoparticles comprising DMG and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the disclosure provides a method of reducing immunogenicity in a subject, the method comprising administering to a subject lipid nanoparticles comprising a compound of Table 1, DSPC, cholesterol, and PEG. 2k - Administering to a subject lipid nanoparticles 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 of reducing immunogenicity in a subject, the method comprising administering to the subject 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 one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the disclosure provides a method of reducing immunogenicity in a subject, the method comprising administering to the subject lipid nanoparticles comprising a compound of Formula (Ac), DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the disclosure provides a method of reducing immunogenicity in a subject, the method comprising administering to the subject lipid nanoparticles comprising a compound of Table 1, DSPC, cholesterol, PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA).

[0284] The disclosure also relates to methods of synthesizing 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), and methods of synthesizing 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).

[0285] Method for producing polypeptides in cells The present disclosure provides a method for producing a polypeptide of interest in mammalian cells.The method for producing a polypeptide involves contacting cells with lipid nanoparticles (for example, empty LNPs or loaded LNPs) that contain mRNA encoding the polypeptide of interest.When cells are contacted with the nanoparticle composition, the mRNA can be taken up into the cells and translated to produce the polypeptide of interest.

[0286] Generally, contacting mammalian cells with lipid nanoparticles (e.g., empty or filled LNPs) containing mRNA encoding a polypeptide of interest can be performed in vivo, ex vivo, in culture, or in vitro. The amount of lipid nanoparticles (e.g., empty or filled LNPs) contacted with cells and / or the amount of mRNA therein can depend on the type of cell or tissue to be contacted, the means of administration, the physiochemical characteristics (e.g., size, charge, and chemical composition) of the lipid nanoparticles (e.g., empty or filled LNPs) and the mRNA therein, and other factors. Generally, an effective amount of lipid nanoparticles (e.g., empty or filled LNPs) will enable efficient polypeptide production in cells. Metrics for efficiency can include polypeptide translation (indicated by polypeptide expression), mRNA degradation levels, and immune response indicators.

[0287] The step of contacting cells with lipid nanoparticles (e.g., empty or filled LNPs) containing mRNA may involve or cause transfection. Phospholipids contained in the lipid component of the lipid nanoparticles (e.g., empty or filled LNPs) may facilitate transfection and / or increase transfection efficiency, for example, by interacting with and / or fusing with cell membranes or intracellular membranes. Transfection may allow translation of the mRNA within the cell.

[0288] In some embodiments, the lipid nanoparticles described herein (e.g., empty or loaded LNPs) can be used therapeutically. For example, the mRNA contained in the lipid nanoparticles (e.g., empty or loaded LNPs) can encode a therapeutic polypeptide (e.g., in a translatable region) and produce the therapeutic polypeptide upon contact with and / or entry into cells (e.g., transfection). In other embodiments, the mRNA contained in the lipid nanoparticles (e.g., empty or loaded LNPs) can encode a polypeptide that can improve or increase a subject's immunity. For example, the mRNA can encode granulocyte colony-stimulating factor or trastuzumab.

[0289] In certain embodiments, the mRNA contained in the lipid nanoparticles (e.g., empty or filled LNPs) may encode a recombinant polypeptide that can replace one or more polypeptides that may be substantially absent in the contacted cells with the nanoparticle composition. The one or more substantially absent polypeptides may be absent due to a genetic mutation in the encoding gene or its regulatory pathway. Alternatively, the recombinant polypeptide produced by translation of the mRNA may antagonize the activity of an endogenous protein present within the cell, on the cell surface, or secreted from the cell. Antagonistic recombinant polypeptides may be desirable to counter adverse effects caused by the activity of endogenous proteins, such as altered activity or localization caused by mutations. In another alternative, the recombinant polypeptide produced by translation of the mRNA may indirectly or directly antagonize the activity of a biological moiety present within the cell, on the cell surface, or secreted from the cell. Antagonized biological moieties may include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoprotein), nucleic acids, carbohydrates, and small molecule toxins. Recombinant polypeptides produced by translation of mRNA can be engineered for localization within the cell, such as within a specific compartment such as the nucleus, or can be engineered for secretion from the cell or for translocation to the plasma membrane of the cell.

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

[0291] Methods for delivering therapeutic agents to cells and organs The present disclosure provides methods for delivering therapeutic and / or prophylactic agents to mammalian cells or organs. Delivering a therapeutic and / or prophylactic agent to a cell involves administering lipid nanoparticles (e.g., empty or loaded LNPs) containing the therapeutic and / or prophylactic agent to a subject, and administering the composition involves contacting the composition with the cell. For example, proteins, cytotoxic agents, radioactive ions, chemotherapeutic agents, or nucleic acids (e.g., mRNA) can be delivered to a cell or organ. When the therapeutic and / or prophylactic agent is mRNA, contacting the cell with the nanoparticle composition can result in translation of the translatable mRNA within the cell to produce a polypeptide of interest. However, substantially non-translatable mRNA can also be delivered to a cell. Substantially non-translatable mRNA can be useful as a vaccine and / or can sequester cellular translational components to reduce expression of other species within the cell.

[0292] In some embodiments, lipid nanoparticles (e.g., empty or loaded LNPs) can target a specific type or class of cells (e.g., cells of a specific organ or system thereof). For example, lipid nanoparticles (e.g., empty or loaded LNPs) containing a therapeutic and / or prophylactic agent of interest can be specifically delivered to the liver, kidney, spleen, or lungs of a mammal. Specific delivery to a specific class of cells, organ, or system or group thereof means that a higher percentage of lipid nanoparticles (e.g., loaded LNPs) containing a therapeutic and / or prophylactic agent are delivered to the target location (e.g., tissue) compared to other locations. In some embodiments, specific delivery of loaded LNPs containing mRNA can result in a 2-fold, 5-fold, 10-fold, 15-fold, or greater than 20-fold increase in mRNA expression in cells at the target location (e.g., tissue of interest 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 liver, kidney, lung, spleen, and tumor tissue (e.g., via intratumoral injection).

[0293] In some embodiments, specific delivery of mRNA contained in disclosed loaded LNPs (i.e., lipid nanoparticles formulated with a disclosed compound) can result in a 2-fold, 5-fold, 10-fold, 15-fold, or greater than 20-fold increase in mRNA expression compared to delivery of mRNA contained in LNPs formulated with another lipid (i.e., not including any 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] As another example of targeted or specific delivery, mRNA encoding a protein binding partner (e.g., an antibody or functional fragment thereof, a scaffolding protein, or a peptide) or a cell surface receptor can be included in the nanoparticle composition. The mRNA can additionally or alternatively be used to direct the synthesis and extracellular localization of lipids, carbohydrates, or other biological moieties. Alternatively, other therapeutic and / or prophylactic agents or components (e.g., lipids or ligands) of the lipid nanoparticles (e.g., empty or loaded LNPs) can be selected based on their affinity for a particular receptor (e.g., low-density lipoprotein receptor) so that the lipid nanoparticles (e.g., empty or loaded LNPs) can more readily interact with target cell populations containing the receptor. For example, ligands may include, but are not limited to, members of specific binding pairs, antibodies, monoclonal antibodies, Fv fragments, single-chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and fragments thereof, humanized antibodies and fragments thereof, and multivalent versions thereof; multivalent binding reagents including mono- 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 can be a surface-bound antibody, which can allow for tuning of cell targeting specificity. This is particularly useful because highly specific antibodies can be raised against epitopes of interest at the desired targeting site. In some embodiments, multiple antibodies can be expressed on the surface of a cell, each with a different specificity for a 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 biological arts based on the desired localization or function of a cell.

[0297] Target cells may include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac muscle cells, skeletal muscle cells, beta cells, pituitary cells, synovial lining 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 loaded LNPs) can target hepatocytes. Apolipoproteins such as apolipoprotein E (apoE) have been shown to associate with lipid nanoparticles (e.g., empty or loaded LNPs) containing neutral or nearly neutral lipids in the body, and are known to associate with receptors such as the low-density lipoprotein receptor (LDLR) found on the surface of hepatocytes. Therefore, lipid nanoparticles (e.g., empty or loaded LNPs) containing lipid components with a neutral or nearly neutral charge administered to a subject can acquire apoE in the subject's body and then deliver therapeutic and / or prophylactic agents (e.g., RNA) to LDLR-containing hepatocytes in a targeted manner.

[0299] Methods of Treating Diseases and Disorders Lipid nanoparticles (e.g., empty 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 defective or abnormal protein or polypeptide activity. For example, lipid nanoparticles (e.g., empty or filled LNPs) containing mRNA encoding a defective or abnormal polypeptide can be administered or delivered to cells. Subsequent translation of the mRNA can produce the polypeptide, thereby reducing or eliminating problems caused by the absence of the polypeptide or its abnormal activity. Because 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 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 for which the compositions can be administered include, but are not limited to, rare diseases, infectious diseases (both as vaccines and therapeutics), cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and renal vascular diseases, and metabolic diseases. Several diseases, disorders, and / or conditions can be characterized by deficient (or substantially reduced) protein activity such that proper protein function does not occur. Such proteins may be absent or essentially non-functional. The present disclosure provides methods for treating such diseases, disorders, and / or conditions in a subject by administering lipid nanoparticles (e.g., empty or loaded LNPs) comprising RNA and a lipid component comprising a lipid 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), a phospholipid (optionally unsaturated), a PEG-lipid, and a structural lipid, wherein the RNA can be mRNA encoding a polypeptide that antagonizes or overcomes aberrant protein activity present in the subject's cells.

[0301] The disclosure provides methods involving administering lipid nanoparticles (e.g., empty or loaded LNPs) containing one or more therapeutic and / or prophylactic agents, as well as pharmaceutical compositions containing them. The terms therapeutic agent and prophylactic agent may be used interchangeably herein with respect to features and embodiments of the present disclosure. Therapeutic compositions, or imaging, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any reasonable amount and any route of administration effective to prevent, treat, diagnose, or image 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 particular composition, the mode of administration, etc. Compositions according to the present disclosure may be formulated in unit dosage form for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment. The particular therapeutically effective, prophylactically effective, or other appropriate (e.g., for imaging) dose level for any particular patient will depend on a variety of factors, including the severity and identity of the disorder being treated (if any); the one or more therapeutic and / or prophylactic agents used; the particular composition used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and excretion rate of the particular pharmaceutical composition used; the duration of treatment; drugs used in combination with or concurrently with the particular pharmaceutical composition used; and similar factors well known in the medical arts.

[0302] Loaded LNPs can be administered by any route. In some embodiments, compositions comprising prophylactic, diagnostic, or imaging compositions comprising one or more loaded LNPs described herein are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intraarterial, subcutaneous, transdermal or intradermal, interdermal, intraperitoneal, mucosal, nasal, intratumoral, intranasal; inhalation; as an oral spray and / or powder, a nasal spray, and / or an aerosol, and / or through a portal vein catheter. In some embodiments, the compositions can be administered intravenously, intramuscularly, intradermal, intraarterial, intratumoral, subcutaneous, or any other parenteral administration route, or by inhalation. However, the present disclosure encompasses delivery or administration of the compositions described herein by any appropriate route, taking into account potential advances in the science of drug delivery. Generally, the most appropriate administration route will depend on various factors, including the properties of the loaded LNPs comprising one or more therapeutic and / or prophylactic agents (e.g., their stability in various bodily environments, such as the bloodstream and gastrointestinal tract), the condition of the patient (e.g., whether the patient can tolerate a particular administration route), etc.

[0303] In certain embodiments, compositions according to the present disclosure provide a therapeutically effective amount of steroids at a given dose, such as from about 0.0001 mg / kg to about 10 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, or from about 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 LNPs may be administered at dosage levels sufficient to deliver 0.25 mg / kg, about 0.001 mg / kg to about 0.25 mg / kg, about 0.005 mg / kg to about 0.25 mg / kg, about 0.01 mg / kg to about 0.25 mg / kg, about 0.05 mg / kg to about 0.25 mg / kg, or about 0.1 mg / kg to about 0.25 mg / kg of a therapeutic and / or prophylactic agent (e.g., mRNA), with a 1 mg / kg (mpk) dose providing 1 mg of therapeutic and / or prophylactic agent per kg of subject body weight. In some embodiments, a dose of about 0.001 mg / kg to about 10 mg / kg of loaded LNP may be administered. In other embodiments, a dose of about 0.005 mg / kg to about 2.5 mg / kg of therapeutic and / or prophylactic agent may be administered.In certain embodiments, a dose of about 0.1 mg / kg to about 1 mg / kg may be administered. In other embodiments, a dose of about 0.05 mg / kg to about 0.25 mg / kg may be administered. Doses may be administered one or more times daily in the same or different amounts to achieve the desired level of mRNA expression and / or therapeutic, diagnostic, preventative, or imaging effect. The desired dosage may be delivered, for example, three times a day, twice a day, once a day, every other day, every three days, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations). 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 or loaded 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. "Combined 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 or loaded 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 agents or medical procedures. Generally, each agent is administered at a dose and / or time schedule determined for that agent. In some embodiments, the present disclosure encompasses delivery of compositions, or imaging, diagnostic, or prophylactic compositions thereof, 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 agents utilized in combination can be administered together in a single composition or administered separately in different compositions. In general, it is expected that agents utilized in combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination may be lower than the levels utilized individually.

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

[0307] Lipid nanoparticles (e.g., empty or loaded LNPs) can be used in combination with drugs to increase the efficacy and / or therapeutic time frame of the composition. Such drugs can 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 or loaded LNPs) can be used in combination with dexamethasone, methotrexate, acetaminophen, H1 receptor blockers, or H2 receptor blockers. In some embodiments, methods of treating a subject in need of treatment for a disease or disorder or delivering therapeutic and / or prophylactic agents to a subject (e.g., a mammal) can involve pre-treating the subject with one or more drugs prior to administering the nanoparticle composition. For example, a subject may be pretreated with a useful amount (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 amount) of dexamethasone, methotrexate, acetaminophen, an H1 receptor blocker, or an H2 receptor blocker. The pretreatment may be administered 24 hours or less (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) prior to administration of the lipid nanoparticles (e.g., empty or loaded LNPs), and may be administered once, twice, or more times, e.g., in increasing dosages.

[0308] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above detailed description, but rather is intended to be set forth in the appended claims.

[0309] In the claims, articles such as "a," "an," and "the" may mean one or more unless indicated to the contrary or clear from context. A claim or description including "or" between one or more members of a group is deemed to be satisfied when one, more than one, or all of the members of the group are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or clear from context. The disclosure includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. As used herein, phrases such as "one or more of A, B, or C," "one or more of A, B, or C," "one or more of A, B, and C," "one or more of A, B, and C," "selected from A, B, and C," "selected from the group consisting of A, B, and C," and the like are used interchangeably and all refer to selection from the group consisting of A, B, and / or C, i.e., one or more A, one or more B, one or more C, or any combination thereof, unless otherwise specified.

[0310] It should also be noted that the term "comprising" is intended to be open-ended, permitting but not requiring the inclusion of additional elements or steps. Thus, when the term "comprising" is used herein, the terms "consisting essentially of" and "consisting of" are also encompassed and disclosed. Throughout this specification, when a composition is described as having, including, or comprising particular components, it is contemplated that the composition also consists essentially of or consists of the recited components. Similarly, when a method or process is described as having, including, or comprising particular process steps, the process also consists essentially of or consists of the recited processing steps. Furthermore, it should be understood that the order or sequence of steps for performing a particular act is immaterial so long as the invention remains operable. Moreover, two or more steps or acts can be performed simultaneously.

[0311] When ranges are specified, the endpoints are included. Furthermore, unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges should be understood to contemplate any particular value or subrange within the range set forth in different embodiments of the disclosure to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0312] The disclosed synthetic processes can tolerate a wide variety of functional groups, and therefore various substituted starting materials can be used. Although the processes generally provide the desired final compound at or near the completion of the entire process, in certain cases it may be desirable to further convert the compound to its pharmaceutically acceptable salt.

[0313] The compounds of the present disclosure can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates by using standard synthetic methods and procedures known to those skilled in the art or that will 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 the relevant scientific literature or standard textbooks in the field. Examples include, but are not limited to, any one or several sources, such as Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5, incorporated herein by reference. th edition, John Wiley&Sons: New York, 2001, Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3 rd edition, 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 textbooks of organic synthesis known to those skilled in the art. The following description of synthetic methods is designed to illustrate, but not limit, the general procedures for the preparation of compounds of the present disclosure.

[0314] Compounds of the present disclosure having any of the formulas described herein can be prepared according to the procedures shown in Schemes 1, 2, and 3 below from starting materials that are commercially available or can be prepared using literature procedures. 1 , R 2 , and R 3 etc. are as defined herein.) Those skilled in the art will note that the order of certain steps, such as the introduction and removal of protecting groups, may be varied in the reaction sequences and synthetic schemes described herein.

[0315] Those skilled in the art will recognize that certain groups may require protection from the reaction conditions through the use of protecting groups. Protecting groups can also be used to modify similar functional groups within a molecule. Lists 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 edition, John Wiley & Sons: New York, 1999.

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

[0317] The hydroxyl moiety includes 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] For diols, Ac(x2)TBS(x2) or when combined with acetonide.

[0321] For thiols, AC.

[0322] Benzimidazoles include SEM, benzil, PMB, and DMB.

[0323] For aldehydes, di-alkyl acetals such as dimethoxy acetal or diethylacetyl.

[0324] In the reaction scheme described herein, multiple stereoisomers can be produced.When no specific stereoisomer is indicated, it is understood that it means all possible stereoisomers that can be produced from the reaction.Those skilled in the art will recognize that the reaction can be optimized to give one isomer preferentially, or new schemes can be devised to produce a single isomer.When a mixture is produced, techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC or preparative SFC can be used to separate isomers.

[0325] [ka] As shown in Scheme 1 above, 8-bromooctanoic acid is reacted with an alcohol a1 (e.g., heptadecan-9-ol) to give an ester b1 (e.g., heptadecan-9-yl 8-bromooctanoate). Step 1 can be carried out, for example, in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine, and DMAP in an organic solvent (e.g., dichloromethane). Step 1 can be carried out at room temperature for 18 hours. Next, ester b1 is reacted with 2-aminoethan-1-ol to give amine c1 (e.g., heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate). Step 2 can be carried out, for example, in ethanol at a temperature of about 60°C. Amine c1 is then reacted with a bromoalkyl group R 1-Br (e.g., 1-bromotetradecane) to give 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, e.g., 8-bromooctanoic acid) is reacted with alcohol b2 (e.g., nonan-1-ol) to give ester c2 (e.g., nonyl-8-bromooctanoate). Step 1 can be carried out, for example, in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine, and DMAP in an organic solvent (e.g., dichloromethane). Alcohol e2 (e.g., heptadecan-9-ol) can be converted to aldehyde d2 (e.g., nonanol) via step 2 using a Grignard reagent R. 3 -MgX (e.g., n-CH 17MgBr). Next, 8-bromooctanoic acid is reacted with alcohol e2 (e.g., heptadecan-9-ol) to give ester f2 (e.g., heptadecan-9-yl 8-bromooctanoate). Step 3 can be carried out, for example, in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine, and DMAP in an organic solvent (e.g., dichloromethane). Next, ester f2 is reacted with 2-aminoethan-1-ol to give 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. Amine g2 is then reacted with ester c2 (e.g., nonyl-8-bromooctanoate) to give 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 elevated temperatures (e.g., about 70-90°C, e.g., about 80°C) in the presence of a base (e.g., an 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) in an organic solvent (e.g., a mixture of CPME and MeCN).

[0327] [ka] As shown in Scheme 3 above, haloalkanols (x 3 is an integer from 1 to 12, for example, 6-bromohexan-1-ol) is used as the starting material a3 (x 2is an integer from 1 to 6, for example, 4-(hexyloxy)-4-oxobutanoic acid, to obtain a halogenated diester b3 (e.g., 6-bromohexylhexyl succinate). Compound a3 can be obtained by reacting 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, for example, in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine, and DMAP in an organic solvent (e.g., dichloromethane). Next, halogenated diester b3 is reacted with amine c3(x 4 is an integer between 5 and 13, and x 5 is an integer from 1 to 5, for example, heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate) to give product d3. Step 2 can be carried out at elevated temperature (e.g., about 90°C) in an organic solvent (e.g., a mixture of CPME and MeCN) in the presence of a base (e.g., an inorganic base (e.g., KCO), and a catalyst (e.g., an iodide such as KI), and an ethereal solvent (e.g., cyclopentyl methyl ether).

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

[0329] In certain aspects, the disclosure also includes methods of synthesizing compounds of any 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), as well as intermediate(s) for synthesizing the compounds.

[0330] In some embodiments, the method of synthesizing the disclosed compounds comprises providing a compound of formula (X2): [ka] The compound R 1 -Br to obtain the disclosed compounds, wherein each variable 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 is H. For example, M is -C(O)O- or -OC(O)-. For example, R 4 is the unsubstituted C 1-3 Alkyl or -(CH2) n Q, 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, a compound of formula (X2) and R 1 The reaction with -Br is carried out in the presence of a base (such as an inorganic base (e.g., K2CO3) or a non-nucleophilic organic base (e.g., i-Pr2EtN)). For example, the reaction is carried out 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 is carried out at an elevated temperature, for example, about 50-100°C, 70-90°C, or about 80°C.

[0331] The method also includes the step of: [ka] The compound R 4 with NH2 to obtain a compound of formula (X2), wherein each variable is as defined herein.

[0332] In some embodiments, the intermediate(s) have formulas (X1) and (X2): [ka] wherein each variable is as defined herein. For example, intermediates include heptadecan-9-yl 8-bromooctanoate and heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate, and structural forms (e.g., crystalline forms) thereof.

[0333] In addition, it should be understood that any particular embodiment of the present disclosure that falls within the prior art may be expressly excluded from any one or more of the claims. Such embodiments may be excluded even if the exclusion is not expressly set forth herein because they are deemed to be known to those of ordinary skill in the art.

[0334] All cited documents, e.g., references, publications, databases, database entries, and arts cited herein, are incorporated by reference into this application, even if not explicitly stated in the cited document. In the event of a conflict between the statements in the cited document and this application, the statements in this application shall control. [Example]

[0335] Example 1: Synthesis of compounds in Table 1 A. General considerations All solvents and reagents used were commercially available and used as received unless otherwise stated. 1 H NMR spectra were recorded in CDCl3 using a Bruker Ultrashield 300 MHz instrument at 300 K. Chemical shifts are 1Values ​​are reported as parts per million (ppm) relative to TMS (0.00) in H. Silica gel chromatography was performed on an ISCO CombiFlash Rf+Lumen Instruments using ISCO RediSep Rf Gold Flash Cartridges (particle size: 20-40 microns). Reverse-phase chromatography was performed on an ISCO CombiFlash Rf+Lumen Instruments using RediSep Rf Gold C18 High Performance columns. All final compounds were determined to be >85% pure via analysis by reverse-phase UPLC-MS (retention time, RT, in minutes) using a Waters Acquity UPLC instrument equipped with a DAD and ELSD and a ZORBAX Rapid Resolution High Definition (RRHD) SB-C18 LC column, 2.1 mm, 50 mm, 1.8 μm, and a gradient of 65% to 100% acetonitrile in water containing 0.1% TFA over 5 minutes at 1.2 mL / min. The injection volume was 5 μL and the column temperature was 80° C. Detection was based on electrospray ionization (ESI) in positive mode 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 3.5 μm, 100 Å, 4.6 x 50 mm 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, 100% B to 70% B in 1 minute, then stop. Column temperature: ambient temperature Detector: ELSD

[0336] The procedures described below are useful for the synthesis of the compounds in Table 1.

[0337] The following abbreviations are used herein: 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] Representative Procedure A: 1,4-Addition of Grignard Reagent RMgX to Methyl (E)-non-2-enoate, 1 A1. Compound 2a: Methyl 3-butylnonanoate [ka] To an oven-dried 100 mL round-bottom flask were added copper(I) bromide (421.3 mg, 2.93 mmol) and lithium chloride (249 mg, 5.87 mmol), followed by anhydrous THF (15 mL). The mixture was stirred for 10 min, during which time the solids dissolved. The flask was placed in an ice bath, and methyl (E)-non-2-enoate 1 (5 g, 29.37 mmol) was added, followed by TMSCl (4 mL, 32.31 mmol). The reaction was stirred for 15 min. A THF solution of butylmagnesium bromide (17.6 mL, 35.2 mmol, 2.0 M in THF) was slowly added, and the reaction was stirred for 2 h. The reaction was quenched with saturated NH4Cl (10 mL), extracted with diethyl ether (100 mL), and dried over anhydrous sodium sulfate. After removal of the solvent, the crude was purified by flash chromatography (SiO2: ethyl acetate / hexane 0-100%) to give a colorless oil product 2a (3 g, 45%). 1 H NMR (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-isopropylnonanoate [ka] Same as procedure A1 but using isopropylmagnesium bromide. Yield = 2.4 g (38%). 1 H NMR (300 MHz, CDCl3): δ ppm 3.65 (s, 3H);2.25 (dd, 1H, J = 15.1, 6.1 Hz);2.14 (dd, 1H, J = 15.1, 7.1 Hz);1.23-1.20 (m, 12H);0.87-0.84 (m, 9H).

[0340] A3. Compound 2c: Methyl 3-propylnonanoate [ka] Same as procedure A1 but using n-propylmagnesium bromide. Yield = 2.2 g (35%). 1 H NMR (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] Representative 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 LiAlH (0.73 g, 19.27 mmol) in THF (10 mL) under N. The mixture was heated under reflux for 5 h. The reaction was cooled to room temperature. Under an ice-water bath at 0 °C, 0.7 mL of HO, 0.7 mL of 15% NaOH, and 2.1 mL of HO were added sequentially. The white precipitate was filtered, and the filtrate was concentrated. The crude product was purified by flash chromatography (SiO: ethyl acetate / hexane 0–100%) to give the colorless oil product 3a (980 mg, 51%). 1 H NMR (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] Same as procedure B1 but using methyl 3-isopropylnonanoate, 2b. Yield = 1.7 g (81%). 1 H NMR (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] Same as procedure B1 but using methyl 3-propylnonanoate, 2c. Yield = 1.28 g (67%). 1 H NMR (300 MHz, CDCl3): δ ppm 3.64 (t, 2H, J = 6.6 Hz);1.52 (q, 2H, J = 6.3 Hz);1.23-1.20 (m, 16H);0.88-0.84 (m, 6H).

[0344] Representative 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 at room temperature overnight. TLC showed the reaction was complete. The reaction mixture was cooled to 0 °C, and 1 N hydrochloric acid (3 mL) was slowly added. The mixture was then extracted with diethyl ether (100 mL), and the layers were separated. 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 give a colorless oil product 5a (680 mg, 73%). 1 H NMR (300 MHz, CDCl3): δ ppm 4.07 (t, 2H, J = 6.8 Hz);3.39 (t, 2H, J = 6.8 Hz);2.28 (t, 2H, J = 7.6 Hz);1.88-1.79 (m, 2H);1.70-1.42 (m, 6H);1.38-1.17 (m, 21H);0.88-0.82 (m, 6H).

[0345] C2. Compound 5b: 3-Isopropylnonyl 8-bromooctanoate [ka] Same as procedure C1 but using 3-isopropylnonan-1-ol, 3b. Yield = 297 mg (71%). 1 H NMR (300 MHz, CDCl3): δ ppm 4.05 (dd, 2H, J = 14.3, 6.6 Hz);3.39 (t, 2H, J = 6.8 Hz);2.28 (t, 2H, J = 7.7 Hz);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] Same as procedure C1 but using 3-propylnonan-1-ol, 3c. Yield = 430 mg (68%). 1 H NMR (300 MHz, CDCl3): δ ppm 3.96 (d, 2H, J = 5.8 Hz);3.38 (t, 2H, J = 5.5 Hz);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] Representative Procedure D: N-Alkylation of Heptadecan-9-yl 8-((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 equipped with 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 h. MS showed clean conversion, and the mixture was cooled to room temperature and diluted with hexane. The mixture was filtered through a pad of Celite. After washing with hexane, the filtrate was concentrated to give a brown oil, which was purified by flash chromatography (SiO: hexane / diethyl ether 0–100%) to give 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 for NO5. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.85 (quintet, 1H, J = 6.1 Hz);4.07 (t, 2H, J = 6.9 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 Hz);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] Same as procedure D1, but using 3-isopropylnonyl 8-bromooctanoate, 5b. Yield = 258 mg (50%). HPLC / ELSD: RT = 6.98 min, MS (CI): m / z (MH + )C 47 H 93 752.6 for NO5. 1 H NMR (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 Hz);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] Same as procedure D1, but using 3-propylnonyl 8-bromooctaato, 5c. Yield = 510 mg (68%). HPLC / ELSD: RT = 7.01 min, MS (CI): m / z (MH + )C 47 H 93 752.6 for NO5. 1H NMR (300 MHz, 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 Hz);1.65-1.48 (m, 17H);1.26 (br. m, 48H);0.88-0.84 (m, 12H).

[0350] Synthetic scheme for the preparation of compound 8 [ka] C4. Compound 5d: 2-Propylnonyl 8-bromooctanoate [ka] Same as procedure C1 but using 2-propylnonan-1-ol 15a. Yield = 1.67 g (79%). 1 H NMR (300 MHz, CDCl3): δ ppm 3.96 (d, 2H, J = 5.8 Hz);3.38 (t, 2H, J = 5.5 Hz);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] Same as procedure D1, but using 2-propylnonyl 8-bromooctanoate, 5d. Yield = 355 mg (68%). HPLC / ELSD: RT = 7.0 min, MS (CI): m / z (MH + )C 47 H 93 752.6 for NO5. 1 H NMR (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 = 13.9, 6.6 Hz);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-propylhex-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 min, and the mixture was stirred at room temperature until gas evolution ceased (approximately 30 min). The reaction mixture was cooled to 0 °C, and 4-heptanone (6.12 mL, 43.8 mmol) was added portionwise. The reaction was gradually warmed to room temperature and stirred under reflux for 24 h. After cooling to room temperature, the reaction was quenched with saturated aqueous sodium bicarbonate. The aqueous phase was extracted with diethyl ether, and the organic extract was washed with brine, dried (MgSO4), and concentrated. The crude material was purified by silica gel chromatography (0–20% EtOAc:hexanes) to afford ethyl 3-propylhex-2-enoate (8.07 g, 43.8 mmol, 100%) as a clear oil and as a mixture of regioisomers. 1H NMR (300 MHz, CDCl3) δ as a mixture of regioisomers: 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);1.57-1.35 (m, 6.10H);1.34-1.21 (m, 7.59H);1.01-0.82 (m, 12.9H).

[0353] Intermediate AB: Ethyl 3-propylhexanoate [ka] A steel Parr reactor equipped with a stir bar was charged with ethyl 3-propylhex-2-enoate (8.07 g, 43.8 mmol) in ethanol (44 mL). Palladium hydroxide on carbon (922 mg, 6.57 mmol) was added, the vessel was sealed, evacuated, and refilled with H gas (3x), and the pressure was set to 200 psi. The reaction was stirred at room temperature for 2 hours at 500 rpm under 200 psi H gas. The vessel was then evacuated, refilled with N gas, and opened. The crude reaction mixture was filtered through a Celite pad. The Celite pad was washed with EtOH, and the crude material was concentrated to give ethyl 3-propylhexanoate (6.55 g, 35.2 mmol, 80%) as a clear oil. The compound was carried on to the next step without further purification. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.12 (q, 2H, J = 6.0 Hz);2.22 (d, 2H, J = 9.0 Hz);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-propylhexan-1-ol [ka] Ethyl 3-propylhexanoate (6.55 g, 35.2 mmol) in anhydrous ether (28 mL) was added dropwise to a mixture of lithium aluminum hydride (1.60 g, 42.2 mmol) in anhydrous ether (42 mL) at 0 °C under N2. The mixture was stirred at room temperature for 2.5 h 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 material was purified by silica gel chromatography (0–40% EtOAc:hexanes) to give 3-propylhexan-1-ol (4.82 g, 33.4 mmol, 95%) as a clear oil. 1 H NMR (300 MHz, CDCl3) δ: ppm 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-butylhept-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 min, and the mixture was stirred at room temperature until gas evolution ceased (approximately 30 min). The reaction mixture was cooled to 0 °C, and 5-nonanone (6.05 mL, 35.2 mmol) was added portionwise. The reaction was gradually warmed to room temperature and stirred under reflux for 24 h. After cooling to room temperature, the reaction was quenched with saturated aqueous sodium bicarbonate. The aqueous phase was extracted with diethyl ether, and the organic extract was washed with brine, dried (MgSO4), and concentrated. The crude material was purified by silica gel chromatography (0–20% EtOAc:hexanes) to afford ethyl 3-butylhept-2-enoate (5.27 g, 24.8 mmol, 71%) as a clear oil. 1 H NMR (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 stir bar was charged with ethyl 3-butylhept-2-enoate (10.5 g, 49.5 mmol) in ethanol (50 mL). Palladium hydroxide on carbon (1.04 g, 7.42 mmol) was added, the vessel was sealed, evacuated, and refilled with H gas (3x), and the pressure was set to 200 psi. The reaction was stirred at room temperature for 2 hours at 500 rpm under 200 psi H gas. The vessel was then evacuated, refilled with N gas, and opened. The crude reaction mixture was filtered through a Celite pad. The Celite pad was washed with EtOH, and the crude material was concentrated to give ethyl 3-butylheptanoate (9.69 g, 45.2 mmol, 91%) as a clear oil. The compound was carried on to the next step without further purification.1 H NMR (300 MHz, CDCl3) δ: ppm 4.12 (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] 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) at 0 °C under N2. The mixture was stirred at room temperature for 2.5 h 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 material was purified by silica gel chromatography (0–40% EtOAc:hexanes) to give 3-butylheptan-1-ol (3.19 g, 18.5 mmol, 99%) as a clear oil. 1 H NMR (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-pentyloct-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 min, and the mixture was stirred at room temperature until gas evolution ceased (approximately 30 min). The reaction mixture was cooled to 0 °C, and 6-undecanone (8.42 mL, 41.1 mmol) was added portionwise. The reaction was gradually warmed to room temperature and stirred under reflux for 60 h. After cooling to room temperature, the reaction was quenched with saturated aqueous sodium bicarbonate. The aqueous phase was extracted with diethyl ether, and the organic extract was washed with brine, dried (MgSO4), and concentrated. The crude material was purified by silica gel chromatography (0–20% EtOAc:hexanes) to give ethyl 3-pentyloct-2-enoate (8.76 g, 36.5 mmol, 89%) as a clear oil. 1 H NMR (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, 3.0 Hz);1.52-1.38 (m, 3H);1.38-1.23 (m, 12H);0.93-0.86 (m, 6H).

[0359] Intermediate AH: Ethyl 3-pentyl octanoate [ka] A steel Parr reactor equipped with a stir bar was charged with ethyl 3-pentyl oct-2-enoate (8.76 g, 36.5 mmol) in ethanol (37 mL). Palladium hydroxide on carbon (768 mg, 5.47 mmol) was added, the vessel was sealed, evacuated, and refilled with H gas (3x), and the pressure was set to 200 psi. The reaction was stirred at room temperature for 2 hours at 500 rpm under 200 psi H gas. The vessel was then evacuated, refilled with N gas, and opened. The crude reaction mixture was filtered through a Celite pad. The Celite pad was washed with EtOH, and the crude material was concentrated to give ethyl 3-pentyl octanoate (8.45 g, 34.9 mmol, 96%) as a clear oil. The compound was carried on to the next step without further purification. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.12 (q, 2H, J = 6.0 Hz);2.22 (d, 2H, J = 6.0 Hz);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-pentyloctan-1-ol [ka] Ethyl 3-pentyloctanoate (8.45 g, 34.9 mmol) in anhydrous ether (28 mL) was added dropwise to a mixture of lithium aluminum hydride (1.59 g, 41.8 mmol) in anhydrous ether (42 mL) at 0 °C under N2. The mixture was stirred at room temperature for 2.5 h 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 material was purified by silica gel chromatography (0–40% EtOAc:hexanes) to give 3-pentyloctan-1-ol (6.98 g, 34.9 mmol, 100%) as a clear oil. 1 H NMR (300 MHz, CDCl3) δ: ppm 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-pentyl octanal [ka] To a stirred suspension of pyridinium chlorochromate (9.02 g, 41.8 mmol) and silica gel (9.02 g per g of pyridinium chlorochromate) in dichloromethane (90 mL) under a N atmosphere was added 3-pentyloctan-1-ol (6.98 g, 34.9 mmol). The suspension was stirred at room temperature for 1 h. The reaction was then filtered through a Celite pad, the Celite pad was washed with dichloromethane, and the filtrate was concentrated. The crude material was purified by silica gel chromatography (0–20% EtOAc:hexanes) to give 3-pentyloctanal (4.66 g, 23.5 mmol, 67%) as a clear oil. 1H NMR (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-allylundecane [ka] To a suspension of methyltriphenylphosphonium bromide (4.68 g, 13.1 mmol) in anhydrous ether (190 mL) under N2, potassium tert-butoxide (1.47 g, 13.1 mmol) was added in one portion. The mixture was stirred at room temperature for 15 minutes, after which 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 organics were dried (MgSO4), filtered, and concentrated. The crude material was purified by silica gel chromatography (0–10% EtOAc:hexanes) to give 6-allylundecane (1.65 g, 8.38 mmol, 83%) as a clear oil. 1 H NMR (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-pentylnonan-1-ol [ka] To a stirred solution of sodium borohydride (131 mg, 3.46 mmol) in anhydrous diglyme (3.6 mL) under a N atmosphere was added a solution of 6-allylundecane (2.26 g, 11.5 mmol) in anhydrous diglyme (2.3 mL). Next, a solution of boron trifluoride ethoxylate (569 μL, 4.61 mmol) in 1.2 mL of anhydrous diglyme at room temperature was added over 15 min. The resulting mixture was stirred for 1 h, after which water (1.2 mL) was added dropwise. When gas evolution ceased, 2.3 mL of 3 M NaOH at room temperature was added, followed by the dropwise addition of 2.3 mL of 30% HO at 40 °C. After stirring for 1 h at 40 °C, the reaction 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 extracts were washed with water (×5). The ethereal extract was dried (MgSO), filtered, and concentrated. The crude residue was purified by silica gel chromatography (0-40% EtOAc:hexanes) to give 4-pentylnonan-1-ol (1.88 g, 8.77 mmol, 76%) as a clear oil. 1 H NMR (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-propylhexan-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 at room temperature overnight. The reaction mixture was then cooled to 0 °C, and a solution of 10% hydrochloric acid (180 mL) was added slowly over 20 minutes. The layers were separated, and the organic layer was concentrated in vacuo to give a 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 (MgSO4), and filtered. The solvent was removed in vacuo to give 3-propylhexyl 8-bromooctanoate (10.9 g, 31.2 mmol, 93%) as a clear oil. The compound was carried on to the next step without further purification. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.08 (t, 2H, J = 6.0 Hz);3.40 (t, 2H, J = 6.0 Hz);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);0.88 (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 at room temperature overnight. The reaction mixture was then cooled to 0 °C, and a solution of 10% hydrochloric acid (150 mL) was added slowly over 20 minutes. The layers were separated, and the organic layer was concentrated in vacuo to give a 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 (MgSO4), and filtered. The solvent was removed in vacuo to give 3-butylheptyl 8-bromooctanoate (6.90 g, 18.3 mmol, 99%) as a clear oil. The compound was carried on to the next step without further purification. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.08 (t, 2H, J = 6.0 Hz);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 at room temperature overnight. The reaction mixture was then cooled to 0 °C, and a solution of 10% hydrochloric acid (70 mL) was added slowly over 20 minutes. The layers were separated, and the organic layer was concentrated in vacuo to give a 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 (MgSO4), and filtered. The solvent was removed in vacuo to give 3-pentyloctyl 8-bromooctanoate (3.94 g, 9.72 mmol, 97%) as a clear oil. The compound was carried on to the next step without further purification. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.08 (t, 2H, J = 6.0 Hz);3.40 (t, 2H, J = 6.0 Hz);3.29 (t, 2H, J = 6.0 Hz);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 at room temperature overnight. The reaction mixture was then cooled to 0 °C, and a solution of 10% hydrochloric acid (60 mL) was added slowly over 20 minutes. The layers were separated, and the organic layer was concentrated in vacuo to give a 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 (MgSO4), and filtered. The solvent was removed in vacuo to give 4-pentylnonyl-8-bromooctanoate (3.68 g, 8.77 mmol, 100%) as a clear oil. The compound was carried on to the next step without further purification. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.04 (t, 2H, J = 6.0 Hz);3.40 (t, 2H, J = 6.0 Hz);2.29 (t, 2H, J = 6.0 Hz);1.85 (quintet, 2H, J = 6.0 Hz);1.70-1.52 (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) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.13 g, 11.1 mmol), 4-(dimethylamino)pyridine (0.217 g, 1.77 mmol), and pentadecan-8-ol (2.03 g, 8.87 mmol). The reaction was stirred at room temperature for 18 hours. The reaction mixture was cooled to 0 °C, and a solution of 10% hydrochloric acid was added slowly. 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 (aq). 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 hexanes) to give a ∼13.2:1 mixture of pentadecane-8-yl 8-bromooctanoate and pentadecane-8-yl 8-chlorooctanoate (3.19 g, 83.1%) as a colorless liquid. 1 H NMR (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: Tridecan-7-yl 8-bromooctanoate [ka] To a solution of 8-bromooctanoic acid (1.96 g, 8.76 mmol) in dichloromethane (30 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.10 g, 10.9 mmol), 4-(dimethylamino)pyridine (0.234 g, 1.92 mmol), and tridecan-7-ol (1.75 g, 8.73 mmol). The reaction was stirred at room temperature for 18 hours. The reaction was diluted with dichloromethane and extracted with saturated NaHCO3 (aq). The organic layer was separated, washed with brine, dried over MgSO4, then filtered and evaporated in vacuo. The residue was purified by silica gel chromatography (0-10% ethyl acetate in hexanes) to give a ∼12.3:1 mixture of tridecan-7-yl 8-bromooctanoate and tridecan-7-yl 8-chlorooctanoate (2.10 g, 59.4%) as a colorless liquid. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.89 (p, 1H);3.55 (t, 0.15H);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: Undecan-6-yl 8-bromooctanoate [ka] To a solution of 8-bromooctanoic acid (4.00 g, 17.9 mmol) in dichloromethane (60 mL) was added 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). The reaction was stirred at room temperature for 18 hours. The reaction mixture was cooled to 0 °C, and a solution of 10% hydrochloric acid was slowly added. The organic layer was separated and evaporated in vacuo. The residue was dissolved in hexane and washed with a 1:1 mixture of acetonitrile and saturated NaHCO3 (aq). The hexane layer was separated, dried over MgSO4, filtered, and evaporated in vacuo. The residue was purified by silica gel chromatography (0-10% ethyl acetate in hexanes) to give a ∼19:1 mixture of undecane-6-yl 8-bromooctanoate and undecane-6-yl 8-chlorooctanoate (4.33 g, 64.01%) as a colorless liquid. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.89 (p, 1H);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) was added 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). The reaction was stirred at room temperature for 18 hours. The reaction mixture was cooled to 0 °C, and a solution of 10% hydrochloric acid was added slowly. The organic layer was separated and evaporated in vacuo. The residue was dissolved in hexane and washed with a 1:1 mixture of acetonitrile and saturated NaHCO3 (aq). The hexane layer was separated, dried over MgSO4, then filtered and evaporated in vacuo. The residue was purified by silica gel chromatography (0-10% ethyl acetate in hexanes) to give a ca. 7:1 mixture of nonan-5-yl 8-bromooctanoate and nonan-5-yl 8-chlorooctanoate (5.23 g, 83.5%) as a colorless liquid. 1 H NMR (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) was added 3-propylhexyl 8-bromooctanoate (8.00 g, 22.3 mmol) in EtOH (55 mL) over 20 minutes. The reaction was heated to 60 °C and stirred at this temperature for 16 hours. Upon cooling, the solvent was evaporated, and the residue was diluted with ethyl acetate and washed with saturated aqueous NaHCO and brine (5x) until no white precipitate was observed in the aqueous layer. The organic layer was separated, washed with brine, dried (MgSO), filtered, and concentrated. The residue was purified by flash chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH, 20% MeOH in dichloromethane)) to give 3-propylhexyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate (5.81 g, 13.1 mmol, 57%) as a clear oil. 1 H NMR (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] To a round-bottom flask equipped with a stir bar was added 3-propylhexyl 8-bromooctanoate (2.82 g, 8.06 mmol), ethanolamine (14.6 mL, 242 mmol), and ethyl alcohol (6 mL). The resulting mixture was stirred at 40 °C for 16 hours. The reaction was diluted with dichloromethane, washed with water (2x), and the layers were separated. The organic layer was dried (MgSO), filtered, and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (mixture of 1% NHOH, 20% MeOH in dichloromethane)) to give 3-propylhexyl 8-((2-hydroxyethyl)amino)octanoate (876 mg, 2.66 mmol, 33%) as a clear oil. 1 H NMR (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)amino)propyl)amino)octanoate [ka] To a solution of tert-butyl N-(3-aminopropyl)carbamate (15.5 g, 88.8 mmol) in EtOH (38 mL) was added 3-pentyloctyl 8-bromooctanoate (6.00 g, 14.8 mmol) in EtOH (36 mL) over 20 minutes. The reaction was heated to 60 °C and stirred at this temperature for 16 hours. Upon cooling, the solvent was evaporated, and the residue was diluted with ethyl acetate and washed with saturated aqueous NaHCO and brine (5x) until no white precipitate was observed in the aqueous layer. The organic layer was separated, washed with brine, dried (MgSO), filtered, and concentrated. The residue was purified by flash chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH, 20% MeOH in dichloromethane)) to give 3-pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate (4.23 g, 8.49 mmol, 57%) as a clear oil. 1 H NMR (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] To a round-bottom flask equipped with a stir bar was added 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 (2x), and the layers were separated. The organic layer was dried (MgSO), filtered, and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (mixture of 1% NHOH, 20% MeOH in dichloromethane)) to give 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 49 400.31 for NO3. 1 H NMR (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, 6H);1.32-1.10 (m, 25H);0.81 (t, 6H, J = 6.0 Hz).

[0376] Intermediate AY: 3-pentyloctyl 8-((3-hydroxypropyl)amino)octanoate [ka] To a round-bottom flask equipped with a stir bar was added 3-pentyloctyl 8-bromooctanoate (1.00 g, 2.47 mmol), propanolamine (5.66 mL, 74.0 mmol), and ethyl alcohol (2 mL). The resulting mixture was stirred at 40 °C for 16 h. The reaction was diluted with dichloromethane, washed with water (2x), and the layers were separated. The organic layer was dried (MgSO), filtered, and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (mixture of 1% NHOH, 20% MeOH in dichloromethane)) to give 3-pentyloctyl 8-((3-hydroxypropyl)amino)octanoate (374 mg, 0.94 mmol, 38%) as a clear oil. UPLC / ELSD: RT = 1.64 min, MS (ES): m / z (MH + )C 24 H 49 400.18 for NO3.

[0377] Intermediate AZ: Heptadecan-9-yl 8-((3-hydroxypropyl)amino)octanoate [ka] To a round-bottom flask equipped with a stir bar was added heptadecan-9-yl 8-bromooctanoate (1.00 g, 2.17 mmol), propanolamine (4.97 mL, 65.0 mmol), and ethyl alcohol (2 mL). The resulting mixture was stirred at 40 °C for 16 hours. The reaction was diluted with dichloromethane, washed with water (2x), and the layers were separated. The organic layer was dried (MgSO), filtered, and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (mixture of 1% NHOH, 20% MeOH in dichloromethane)) to afford 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 for NO3.

[0378] Intermediate BA: Heptadecan-9-yl 8-((4-hydroxybutyl)amino)octanoate [ka] To a round-bottom flask equipped with a stir bar was added heptadecan-9-yl 8-bromooctanoate (1.00 g, 2.17 mmol), 4-aminobutan-1-ol (5.99 mL, 65.0 mmol), and ethyl alcohol (2 mL). The resulting mixture was stirred at 40 °C for 16 hours. The reaction was diluted with dichloromethane, washed with water (2x), and the layers were separated. The organic layer was dried (MgSO), filtered, and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (mixture of 1% NHOH, 20% MeOH in dichloromethane)) to afford heptadecan-9-yl 8-((4-hydroxybutyl)amino)octanoate (773 mg, 1.65 mmol, 76%) as a clear oil. UPLC / ELSD: RT=2.02 min, MS(ES): m / z(MH + )C 29 H 59 470.23 for NO3.

[0379] Intermediate BB: 3-pentyloctyl 8-((4-hydroxybutyl)amino)octanoate [ka] To a round-bottom flask equipped with a stir bar was added 3-pentyloctyl 8-bromooctanoate (1.00 g, 2.47 mmol), 4-aminobutan-1-ol (6.82 mL, 74.0 mmol), and ethyl alcohol (2 mL). The resulting mixture was stirred at 40 °C for 16 hours. The reaction was diluted with dichloromethane, washed with water (2x), and the layers were separated. The organic layer was dried (MgSO), filtered, and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (mixture of 1% NHOH, 20% MeOH in dichloromethane)) to give 3-pentyloctyl 8-((4-hydroxybutyl)amino)octanoate (501 mg, 1.21 mmol, 49%) as a clear oil. UPLC / ELSD: RT=1.67 min, MS(ES): m / z(MH + )C 25 H 51 414.24 for NO3.

[0380] Intermediate BC: 3-pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate [ka] 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) was added potassium carbonate (1.66 g, 12.0 mmol) and potassium iodide (366 mg, 2.21 mmol). The reaction was stirred at 80° C. for 16 h. Upon cooling, the volatiles were evaporated in vacuo. The residue was diluted with dichloromethane and washed with water. The organic layer was separated, washed with brine, dried (MgSO), 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, 20% MeOH in dichloromethane)) to give 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 for N2O6.

[0381] Intermediate BD: 3-butylheptyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate [ka] 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) was added potassium carbonate (1.87 g, 13.6 mmol) and potassium iodide (412 mg, 2.49 mmol). The reaction was stirred at 80° C. for 16 h. Upon cooling, the volatiles were evaporated in vacuo. The residue was diluted with dichloromethane and washed with water. The organic layer was separated, washed with brine, dried (MgSO), 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, 20% MeOH in dichloromethane)) to give 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 for N2O6.

[0382] Intermediate BE: Bis(3-propylhexyl) 8,8'-((3-((tert-butoxycarbonyl)amino)propyl)azanediyl)dioctanoate [ka] 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) was added potassium carbonate (1.87 g, 13.6 mmol) and potassium iodide (412 mg, 2.49 mmol). The reaction was stirred at 80° C. for 16 h. Upon cooling, the volatiles were evaporated in vacuo. The residue was diluted with dichloromethane and washed with water. The organic layer was separated, washed with brine, dried (MgSO), 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, 20% MeOH in dichloromethane)) to give bis(3-propylhexyl) 8,8'-((3-((tert-butoxycarbonyl)amino)propyl)azanediyl) 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 for N2O6.

[0383] Intermediate BF: 3-butylheptyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate [ka] 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) was added potassium carbonate (1.66 g, 12.0 mmol) and potassium iodide (366 mg, 2.21 mmol). The reaction was stirred at 80° C. for 16 h. Upon cooling, the volatiles were evaporated in vacuo. The residue was diluted with dichloromethane and washed with water. The organic layer was separated, washed with brine, dried (MgSO), 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, 20% MeOH in dichloromethane)) to give 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 for N2O6.

[0384] Intermediate BG: 3-pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-(undecan-6-yloxy)octyl)amino)octanoate [ka] UPLC / ELSD: RT=2.93 minutes. MS(ESI):m / z C 48 H 95 N2O6 +Calculated for (M+H) 795.288; found 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, 12H).

[0385] Intermediate BH: Nonan-5-yl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate [ka] 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) was added potassium carbonate (2.00 g, 14.4 mmol) and potassium iodide (439 mg, 2.65 mmol). The reaction was stirred at 80° C. for 16 h. Upon cooling, the volatiles were evaporated in vacuo. The residue was diluted with dichloromethane and washed with water. The organic layer was separated, washed with brine, dried (MgSO), 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, 20% MeOH in dichloromethane)) to give 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. 1H NMR (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: Pentadecan-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 for (M+H) 795.288; found 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-(tridecan-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) was added potassium iodide (0.588 g, 3.54 mmol), potassium carbonate (1.73 g, 12.5 mmol), and a solution of tridecan-7-yl 8-bromooctanoate (1.26 g, 3.11 mmol) in CMPE (9 mL). The reaction was stirred at 77 °C for 18 hours. The reaction was cooled to room temperature, filtered, and the filtrate was evaporated in vacuo. The residue was purified by silica gel chromatography [0-70% in dichloromethane (1% NH4OH, 20% MeOH mixture in dichloromethane)] to give 3-propylhexyl 8-({3-[(tert-butoxycarbonyl)amino]propyl}[8-oxo-8-(tridecan-7-yloxy)octyl]amino)octanoate (1.32 g, 55.1%) as a yellowish oil. UPLC / ELSD: RT = 2.70 min, found 767.34. 1 H NMR (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, 14H);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-(undecan-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) was added potassium iodide (0.619 g, 3.73 mmol), potassium carbonate (1.87 g, 13.6 mmol), and a solution of undecan-6-yl 8-bromooctanoate (1.28 g, 3.39 mmol) in CPME (10 mL). The reaction was stirred at 77 °C for 18 hours. The reaction was cooled to room temperature, filtered, and the filtrate was evaporated in vacuo. The residue was purified by silica gel chromatography [0-70% in dichloromethane (a mixture of 1% NH4OH, 20% MeOH in dichloromethane)] to give 3-propylhexyl 8-({3-[(tert-butoxycarbonyl)amino]propyl}[8-oxo-8-(undecan-6-yloxy)octyl]amino)octanoate (1.53 g, 61.2%) as a yellowish oil. UPLC / ELSD: RT = 2.56 min, found 739.46. 1 H NMR (300 MHz, CDCl3) 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);2.44-2.35 (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) was added 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). The reaction was stirred at 77 °C for 18 h. The reaction was cooled to room temperature, filtered, and the filtrate was evaporated in vacuo. The residue was purified by silica gel chromatography [0-70% in dichloromethane (mixture of 1% NH4OH, 20% MeOH in dichloromethane)] to give 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, found 711.46. 1 H NMR (300 MHz, CDCl3) 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] To 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 added trifluoroacetic acid (1.39 mL, 18.2 mmol). The reaction was stirred at room temperature for 4 hours. The reaction was quenched with saturated aqueous NaHCO3 and extracted with dichloromethane. The organic layer was separated, washed with brine, dried (MgSO4), filtered, and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NHOH, 20% MeOH in dichloromethane)) to give 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 for 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) was added trifluoroacetic acid (2.01 mL, 26.3 mmol). The reaction was stirred at room temperature for 4 hours. The reaction was quenched with saturated aqueous NaHCO3 and extracted with dichloromethane. The organic layer was separated, washed with brine, dried (MgSO4), filtered, and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NHOH, 20% MeOH in dichloromethane)) to give 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 for N2O4.

[0392] Intermediate BO: Bis(3-propylhexyl) 8,8'-((3-aminopropyl)azanediyl) dioctanoate [ka] To a solution of bis(3-propylhexyl) 8,8'-((3-((tert-butoxycarbonyl)amino)propyl)azanediyl)dioctanoate (730 mg, 1.03 mmol) in methylene chloride (21 mL) was added trifluoroacetic acid (1.57 mL, 20.5 mmol). The reaction was stirred at room temperature for 4 hours. The reaction was quenched with saturated aqueous NaHCO3 and extracted with dichloromethane. The organic layer was separated, washed with brine, dried (MgSO4), filtered, and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NH4OH, 20% MeOH in dichloromethane)) to give bis(3-propylhexyl) 8,8'-((3-aminopropyl)azanediyl) 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) was added trifluoroacetic acid (1.72 mL, 22.5 mmol). The reaction was stirred at room temperature for 4 hours. The reaction was quenched with saturated aqueous NaHCO3 and extracted with dichloromethane. The organic layer was separated, washed with brine, dried (MgSO4), filtered, and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NHOH, 20% MeOH in dichloromethane)) to give 3-butylheptyl 8-((3-aminopropyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate (632 mg, 0.91 mmol, 81%) as a clear oil. UPLC / ELSD: RT=2.47 min, MS(ES): m / z (MH + )C 43 H 86 695.68 for N2O4

[0394] Intermediate BQ: 3-pentyloctyl 8-((4-aminobutyl)(8-oxo-8-(undecan-6-yloxy)octyl)amino)octanoate [ka] UPLC / ELSD: RT=2.49 minutes. MS(ESI):m / z C 44 H 89 N2O6 + Calculated for (M+H) 709.198; Found 695.43. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.88 (p, 1H);4.09 (t, 2H);2.74 (t, 2H);2.47 (t, 2H);2.39 (t, 4H);2.29 (t, 4H);1.69-1.38 (m, 25H);1.29 (br. m, 38H);0.90 (t, 12H).

[0395] Intermediate BR: Nonan-5-yl 8-((3-aminopropyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate [ka] To a solution of nonan-5-yl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate (1.06 g, 1.39 mmol) in methylene chloride (28 mL) was added trifluoroacetic acid (2.12 mL, 27.7 mmol). The reaction was stirred at room temperature for 4 hours. The reaction was quenched with saturated aqueous NaHCO3 and extracted with dichloromethane. The organic layer was separated, washed with brine, dried (MgSO4), filtered, and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (a mixture of 1% NHOH, 20% MeOH in dichloromethane)) to give nonan-5-yl 8-((3-aminopropyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate (718 mg, 1.08 mmol, 78%) as a clear oil. UPLC / ELSD: RT=2.31 min, MS(ES): m / z (MH + )C 41 H 82 667.43 for N2O4

[0396] Intermediate BS: 3-pentyloctyl 8-((3-aminopropyl)(8-oxo-8-(undecan-6-yloxy)octyl)amino)octanoate [ka] UPLC / ELSD: RT=2.34 minutes. MS(ESI):m / z C 43 H 87 N3O6 +Calculated value for (M+H) 695.171; Actual value 695.430.1H NMR (300 MHz, CDCl3) δ: ppm 4.88 (p, 1H);4.10 (t, 2H);4.10 (t, 2H);2.92 (t, 2H);2.61 (t, 2H);2.46 (t, 4H);2.30 (t, 4H);1.73-1.41 (m, 18H);1.28 (br. m, 40H);0.90 (t, 12H).

[0397] Intermediate BT: 3-Propylhexyl 8-((3-aminopropyl)(8-oxo-8-(tridecan-7-yloxy)octyl)amino)octanoate [ka] To a solution of 3-propylhexyl 8-({3-[(tert-butoxycarbonyl)amino]propyl}[8-oxo-8-(tridecan-7-yloxy)octyl]amino)octanoate (1.32 g, 1.72 mmol) in dichloromethane (33 mL) was added trifluoroacetic acid (6.63 mL, 34.3 mmol). The reaction was stirred at room temperature for 4 hours. Saturated NaHCO3 (aq) was added and the reaction was diluted with dichloromethane. The organic layer was separated and washed two more times with saturated NaHCO3 (aq) and then with brine. The organic layer was dried over MgSO4, then filtered and evaporated in vacuo. The residue was purified by silica gel chromatography [0-70% in dichloromethane (1% NH4OH, 20% MeOH in dichloromethane)] to give 3-propylhexyl 8-[(3-aminopropyl)[8-oxo-8-(tridecan-7-yloxy)octyl]amino]octanoate (0.774 g, 67.6%) as a yellowish oil. UPLC / ELSD: RT = 2.03 min, found 667.31. 1H NMR (300 MHz, CDCl3) δ: ppm 4.88 (p, 1H);4.10 (t, 2H);2.79 (t, 2H);2.51 (t, 2H);2.42 (t, 4H);2.30 (t, 4H);1.79-1.39 (m, 17H);1.39-1.18 (m, 36H);0.90 (t, 12H).

[0398] Intermediate BU: 3-propylhexyl 8-((3-aminopropyl)(8-oxo-8-(undecan-6-yloxy)octyl)amino)octanoate [ka] To a solution of 3-propylhexyl 8-({3-[(tert-butoxycarbonyl)amino]propyl}[8-oxo-8-(undecan-6-yloxy)octyl]amino)octanoate (1.53 g, 2.07 mmol) in dichloromethane (40 mL) was added trifluoroacetic acid (3.05 mL, 39.9 mmol). The reaction was stirred at room temperature for 4 hours. Saturated NaHCO3 (aq) was added and the reaction was diluted with dichloromethane. The organic layer was separated and washed two more times with saturated NaHCO3 (aq) and then with brine. The organic layer was dried over MgSO4, then filtered and evaporated in vacuo. The residue was purified by silica gel chromatography [0-70% in dichloromethane (1% NH4OH, 20% MeOH in dichloromethane)] to give 3-propylhexyl 8-[(3-aminopropyl)[8-oxo-8-(undecan-6-yloxy)octyl]amino]octanoate (0.682 g, 51.5%) as a yellowish oil. UPLC / ELSD: RT = 1.85 min, found 639.19. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.89 (p, 1H);4.10 (t, 2H);2.82 (t, 2H);2.53 (t, 2H);2.44 (t, 4H);2.30 (t, 4H);1.75-1.40 (m, 17H);1.40-1.18 (m, 32H);0.91 (t, 12H). Intermediate BV: Nonan-5-yl 8-((3-aminopropyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate [ka]

[0399] To a solution of nonan-5-yl 8-({3-[(tert-butoxycarbonyl)amino]propyl}({8-oxo-8-[(3-propylhexyl)oxy]octyl})amino)octanoate (0.483 g, 0.679 mmol) in dichloromethane (13 mL) was added trifluoroacetic acid (1.00 mL, 13.1 mmol). The reaction was stirred at room temperature for 4 hours. Saturated NaHCO3 (aq) was added and the reaction was diluted with dichloromethane. The organic layer was separated and washed two more times with saturated NaHCO3 (aq) and then with brine. The organic layer was dried over MgSO4, then filtered and evaporated in vacuo. The residue was purified by silica gel chromatography [0-70% in dichloromethane (1% NH4OH, 20% MeOH mixture in dichloromethane)] to give nonan-5-yl 8-[(3-aminopropyl)({8-oxo-8-[(3-propylhexyl)oxy]octyl})amino]octanoate (0.202 g, 48.7%) as a yellowish oil. UPLC / ELSD: RT = 1.85 min, found 611.44. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.89 (p, 1H);4.10 (t, 2H);2.80 (t, 2H);2.52 (t, 2H);2.42 (t, 4H);2.30 (t, 4H);1.74-1.39 (m, 17H);1.39-1.20 (m, 28H);0.91 (t, 12H).

[0400] Synthesis of the final compound: AA. Compound 3: Heptadecan-9-yl 8-((2-hydroxyethyl)(6-((((3-pentyloctyl)oxy)carbonyl)oxy)hexyl)amino)octanoate [ka] UPLC / ELSD: RT=3.04 min, MS(ES): m / z(MH + )C 47 H 93 769.313 for NO6. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.89 (p, 1H);4.14 (m, 4H);3.54 (bm, 2H);2.66-2.37 (m, 6H);2.30 (m, 2H);1.77-1.17 (m, 66H);0.91 (m, 12H).

[0401] AB. Compound 4: Heptadecan-9-yl 8-((6-((((3-hexylnonyl)oxy)carbonyl)oxy)hexyl)(3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)amino)octanoate Ethyl 3-hexylnon-2-enoate [ka] To a suspension of sodium hydride ...

Claims

1. Formula (A): 【Chemistry 1】 or a salt thereof, In the formula, R' ａ R' is branched; R' 分岐状 but, 【Chemistry 2】 and 【Chemistry 3】 indicates the attachment point, R ａα is H, R aβ and R ａδ is independently H and unsubstituted C ２－１２ alkyl, and R aγ is selected from unsubstituted C ２－１２ Alkyl or unsubstituted C ２－１２ alkenyl, R ２ and R ３ each independently represents an unsubstituted C １－１４ is alkyl, R ４ But -(CH ２ ) ２ OH, -(CH ２ ) ３ OH, -(CH ２ ) ４ OH, -(CH ２ ) ５ OH, and 【Chemistry 4】 R １０ But N(R) ２ and each R is independently an unsubstituted C １－６ Alkyl, unsubstituted C ２－３ 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 is independently an unsubstituted C １－３ Alkyl, unsubstituted C ２－３ alkenyl, and H; Each R ６ is independently an unsubstituted C １～３ Alkyl, unsubstituted C ２－３ alkenyl, and H; R ７ is H, M and M' are independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is unsubstituted C １－１２ Alkyl or unsubstituted C ２－１２ alkenyl, R*"a is unsubstituted C １－１５ Alkyl and unsubstituted C ２－１５ alkenyl, l is selected from the group consisting of 1, 2, 3, 4, and 5; and A compound or a salt thereof, wherein m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

2. Formula (1-1): 【Chemistry 5】 or a salt thereof, In the formula, R' ａ R' is branched; R' 分岐状 but, 【Chemistry 6】 and R′ b is 【Chemistry 7】 and 【Chemistry 8】 indicates the attachment point, R ａγ and R ｂγ each independently represents an unsubstituted C ２－１２ Alkyl or unsubstituted C ２－１２ alkenyl, R ２ and R ３ each independently represents an unsubstituted C １－１４ Alkyl and unsubstituted C ２－１４ alkenyl, R ４ But -(CH ２ ) ２ OH, and Each R' is independently an unsubstituted C １－１２ Alkyl or unsubstituted C ２－１２ The compound or a salt thereof, wherein:

3. The compound has formula (I-a), (A-c) or (B-c): 【Chemistry 9】 3. The compound according to claim 1 or 2, or a salt thereof.

4. R ａγ is unsubstituted C ２－６ The compound according to any one of claims 1 to 3, which is alkyl.

5. R ｂγ is unsubstituted C ２－６ 4. The compound of claim 2 or 3, which is alkyl.

6. R ａγ and R ｂγ each independently represents an unsubstituted C ２－６ 4. The compound of claim 2 or 3, which is alkyl.

7. R ｂγ is unsubstituted C ４－６ 4. The compound of claim 2 or 3, which is alkyl.

8. R ２ and R ３ Each of the unsubstituted C ８ The compound according to any one of claims 1 to 3, which is alkyl.

9. Each R ’ is independently an unsubstituted C ２－５ The compound according to any one of claims 1 to 3, which is alkyl.

10. A compound, or a pharma- ceutically acceptable salt thereof, selected from: 【Table 1】

11. A compound, or a pharma- ceutically acceptable salt thereof, selected from: 【Table 2】

12. 12. An empty lipid nanoparticle (empty LNP) comprising a compound according to claim 1 or 11, a phospholipid, a structured lipid, and a PEG lipid.

13. (a) about 40 mol% to about 60 mol% of the compound, about 0 mol% to about 20 mol% of a phospholipid, about 30 mol% to about 50 mol% of a structural lipid, and about 0 mol% to about 5 mol% of a PEG lipid; (b) the phospholipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-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), Sphingomyelins and their mixtures is selected from the group consisting of (c) the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, and mixtures thereof; (d) 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; and / or (e) the PEG lipid is ２ｋ -DMG and PEG-1: 【Chemistry 10】 and mixtures thereof; The empty LNP of claim 12.

14. A loaded lipid nanoparticle (loaded LNP) comprising the empty LNP of claim 13 and one or more therapeutic and / or prophylactic agents.

15. The loaded LNP of claim 14 , wherein the one or more therapeutic and / or prophylactic agents are nucleic acids.

16. 16. The loaded LNP of claim 15, 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), antagomir, antisense RNA, ribozymes, dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

17. The loaded LNP of claim 16, wherein the RNA is mRNA.

18. A pharmaceutical composition comprising the loaded LNP of any one of claims 14 to 17 and a pharma- ceutically acceptable carrier.

19. A pharmaceutical composition for delivering a therapeutic and / or prophylactic agent to cells in a subject, comprising the loaded LNPs of any one of claims 14 to 17.

20. A pharmaceutical composition for specifically delivering a therapeutic and / or prophylactic agent to an organ of a target, comprising the loaded LNPs of any one of claims 14 to 17.

21. A pharmaceutical composition for producing a polypeptide of interest in cells in a subject, comprising the loaded LNPs of any one of claims 14 to 17.

22. A pharmaceutical composition for treating a disease or disorder in a subject in need thereof, comprising the loaded LNPs of any one of claims 14 to 17.

23. 21. The pharmaceutical composition of claim 20, wherein the organ is selected from the group consisting of liver, kidney, lung, and spleen.

24. 24. The pharmaceutical composition of claim 22 or 23, wherein the pharmaceutical composition is administered to the subject parenterally, intramuscularly, intradermally, subcutaneously, and / or intravenously.

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