Branched tail end lipid compounds and compositions for intracellular delivery of therapeutic agents

Abstract

The present application relates to branched tail end lipid compounds and compositions for intracellular delivery of therapeutic agents. The present application relates to lipids of formula (A), formula (B) and formula (1-1), and to lipid nanoparticles (empty LNP or loaded LNP) comprising such lipids and additional lipids, such as phospholipids, structural lipids and PEG lipids. Lipid nanoparticles further comprising therapeutic and / or prophylactic agents such as RNA may be used to deliver therapeutic and / or prophylactic agents to mammalian cells or organs to, for example, modulate polypeptide, protein or gene expression. # imgabs0 #

Description

[0001] Related applications

[0002] 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. Technical Field

[0003] 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 mammalian cells or organs. In addition to novel lipids, the lipid nanoparticle compositions of the present disclosure may also include a specific fraction of one or more cationic and / or ionizable amino lipids, phospholipids including polyunsaturated lipids, PEG lipids, structured lipids, and / or therapeutic and / or prophylactic agents. Background of the Invention

[0004] Effective targeted delivery of biologically active substances, such as small molecule drugs, proteins, and nucleic acids, represents an ongoing medical challenge. In particular, delivery of nucleic acids to cells is difficult due to the relative instability and low cell permeability of such substances. Therefore, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and / or prophylactic agents, such as nucleic acids, to cells.

[0005] Lipid-containing nanoparticle compositions, liposomes, and liposome complexes have been shown to be effective as transport vehicles for bioactive substances such as small molecule drugs, proteins, and nucleic acids into cells and / or intracellular compartments. Such compositions typically include one or more "cationic" and / or amino (ionizable) lipids, phospholipids including polyunsaturated lipids, structured lipids (e.g., sterols), and / or lipids containing polyethylene glycol (PEG lipids). Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be readily protonated. Although a variety of such lipid-containing nanoparticle compositions have been demonstrated, improvements in safety, efficacy, and specificity are still lacking. Summary of the Invention

[0006] The present disclosure provides novel compounds and compositions and methods involving the same.

[0007] In some aspects, the present disclosure relates to a compound of formula (1-1):

[0008] or an N-oxide thereof, or a salt or isomer thereof,

[0009] where R'a is R' 支链 or R' 环状 ;in

[0010] R'支链 yes: And R' 环状 yes: and

[0011] R' b yes:

[0012] in Indicates a connection point;

[0013] where R aγ and R bγ Each is independently C 2-12 Alkyl or C 2-12 alkenyl;

[0014] R 2 and R 3 Each independently selected from C 1-14 Alkyl and C 2-14 a group consisting of alkenyl groups;

[0015] R 4 is -(CH2)2OH;

[0016] Each R' is independently C 1-12 Alkyl or C 2-12 alkenyl;

[0017] Y a It is C 3-6 carbon ring;

[0018] R*” a Choose from C 1-15 Alkyl and C 2-15 and

[0019] s is 2 or 3.

[0020] In some aspects, the present disclosure relates to a compound of formula (2-1):

[0021] or an N-oxide thereof, or a salt or isomer thereof,

[0022] where R' a It's R' 支链 or R' 环状 ;in

[0023] R' 支链 yes: And R' 环状 yes: and

[0024] R' b yes:

[0025] in Indicates a connection point;

[0026] where R aγ and R bγ Each is independently C 2-12 Alkyl or C 2-12 alkenyl;

[0027] R 2 and R 3 Each independently selected from C 1-14 Alkyl and C 2-14 a group consisting of alkenyl groups;

[0028] R 4 yes in Indicates the connection point; R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0029] Each R' is independently C 1-12 Alkyl or C 2-12 alkenyl;

[0030] Y a It is C 3-6 carbon ring;

[0031] R*"a is selected from C 1-15 Alkyl and C 2-15 alkenyl; and

[0032] s is 2 or 3.

[0033] In some aspects, the present disclosure relates to a compound of formula (A):

[0034] or an N-oxide thereof, or a salt or isomer thereof,

[0035] where R' a It's R' 支链 or R' 环状 ;in

[0036] R' 支链 yes: R' 环状 yes: in Indicates a connection point;

[0037] where R aα is H, and R aβ 、R aγand R aδ Each independently selected from H, C 2-12 Alkyl and C 2-12 A group consisting of alkenyl groups, wherein R aβ 、R aγ and R aδ At least one of which is selected from C 2-12 Alkyl and C 2-12 a group consisting of alkenyl groups;

[0038] R 2 and R 3 Each is C 1-14 alkyl;

[0039] R 4 Selected from -(CH2)2OH, -(CH2)3OH, -(CH2)4OH, -(CH2)5OH and The group composed of

[0040] in Indicates a connection point;

[0041] R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0042] Each R 5 Independently selected from OH, C 1-3 Alkyl, C 2-3 A group consisting of an alkenyl group and H;

[0043] Each R 6 Independently selected from OH, C 1-3 Alkyl, C 2-3 A group consisting of an alkenyl group and H;

[0044] R 7 It is H;

[0045] M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-;

[0046] R' is C 1-12 Alkyl or C 2-12 alkenyl;

[0047] Y a It is C 3-6 carbon ring;

[0048] R*” a Choose from C 1-15 Alkyl and C 2-15 a group consisting of alkenyl groups;

[0049] l is selected from the group consisting of 1, 2, 3, 4 and 5;

[0050] s is 2 or 3; and

[0051] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

[0052] In some aspects, the present disclosure relates to a compound of formula (B):

[0053] or an N-oxide thereof, or a salt or isomer thereof,

[0054] where R' a It's R' 支链 or R' 环状 ;in

[0055] R' 支链 yes: R' 环状 yes: in Indicates a connection point;

[0056] where R aα and R aβ Each is H, and R aγ and R aδ Each independently selected from H, C 2-12 Alkyl and C 2-12 A group consisting of alkenyl groups, wherein R aγ and R aδ At least one of which is selected from C 2-12 Alkyl and C 2-12 a group consisting of alkenyl groups;

[0057] R bα 、R bβ 、R bγ and R bδ Each independently selected from H, C 2-30 Alkyl and C 5-20 A group consisting of alkenyl groups, wherein R bα 、R bβ 、R bγ and R bδ At least one of which is selected from C 2-30 Alkyl and C 5-20 a group consisting of alkenyl groups;

[0058] R 4 Selected from -(CH2)2OH, -(CH2)3OH, -(CH2)4OH, -(CH2)5OH and The group composed of

[0059] in Indicates a connection point;

[0060] R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0061] Each R 5 Independently selected from OH, C 1-3 Alkyl, C 2-3 A group consisting of an alkenyl group and H;

[0062] Each R 6 Independently selected from OH, C 1-3 Alkyl, C 2-3 A group consisting of an alkenyl group and H;

[0063] M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-;

[0064] R' is C 1-12 Alkyl or C 2-12 alkenyl;

[0065] Y a It is C 3-6 carbon ring;

[0066] R*"a is selected from C 1-15 Alkyl and C 2-15 a group consisting of alkenyl groups;

[0067] l is selected from the group consisting of 1, 2, 3, 4 and 5;

[0068] s is 2 or 3; and

[0069] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

[0070] In some aspects, the present disclosure relates to a compound of formula (Aa):

[0071] or an N-oxide thereof, or a salt or isomer thereof,

[0072] where R aβ 、R aγ and R aδ Each independently selected from H, C 2-12 Alkyl and C 2-12 A group consisting of alkenyl groups, wherein R aβ 、R aγ and R aδAt least one of which is selected from C 2-12 Alkyl and C 2-12 a group consisting of alkenyl groups;

[0073] R 4 Selected from the group consisting of -(CH2)2OH, -(CH2)3OH, -(CH2)4OH and -(CH2)5OH;

[0074] M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; and

[0075] R' is C 1-12 Alkyl or C 2-12 Alkenyl.

[0076] In some aspects, the present disclosure relates to a compound of formula (Ab):

[0077] or an N-oxide thereof, or a salt or isomer thereof,

[0078] where R aβ 、R aγ and R aδ Each independently selected from H, C 2-12 Alkyl and C 2-12 A group consisting of alkenyl groups, wherein R aβ 、R aγ and R aδ At least one of which is selected from C 2-12 Alkyl and C 2-12 a group consisting of alkenyl groups;

[0079] R 4 is selected from the group consisting of -(CH2)2OH, -(CH2)3OH, -(CH2)4OH, and -(CH2)5OH; and

[0080] R' is C 1-12 Alkyl or C 2-12 Alkenyl. DETAILED DESCRIPTION

[0081] The disclosure relates to novel lipids and lipid nanoparticles (such as empty LNP or loaded LNP) comprising novel lipids. The disclosure also provides therapeutic and / or prophylactic delivery to mammalian cells, specifically to mammalian organs, producing target polypeptide in mammalian cells, improving the level of the protein produced in mammalian cells compared with the LNP comprising other lipids, and treating a mammalian disease or the method for illness in need. For example, a method of producing target polypeptide in cells relates to contacting the nanoparticle comprising mRNA with mammalian cells, wherein the mRNA can be translated to produce target polypeptide. A method of delivering therapeutic and / or prophylactic to mammalian cells or organs can relate to the nanoparticle composition comprising the therapeutic and / or prophylactic being administered to a subject, wherein the administration relates to contacting the cell or organ with the composition, wherein the therapeutic and / or prophylactic is delivered to the cell or organ. This type of delivery method can be in vitro or in vivo.

[0082] The present disclosure provides the lipid that comprises central amine moiety and at least one biodegradable group.Lipid as herein described can be advantageously used for lipid nanoparticle (for example empty LNP or load LNP) so that therapeutic agent and / or preventive agent are delivered to mammalian cell or organ.For example, lipid as herein described hardly has or does not have immunogenicity.For example, compared with reference lipid (for example MC3, KC2 or DLinDMA), the lipid compound of formula (I-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) has lower immunogenicity. 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 (eg, MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.

[0083] In some aspects, the present disclosure relates to a compound of formula (A-1):

[0084] or an N-oxide thereof, or a salt or isomer thereof,

[0085] where R' a It's R' 支链 or R' 环状 ;in

[0086] R' 支链 yes: R' 环状 yes: in Indicates a connection point;

[0087] where R aα 、R aγ and R aδ Each independently selected from H, C 2-12 Alkyl and C 2-12 alkenyl; and R aβ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0088] R 2 and R 3 Each is C 1-14 alkyl;

[0089] R 4 is -(CH2)2OH or

[0090] in Indicates a connection point;

[0091] R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0092] R 5 and R 6 Each is H;

[0093] R 7 It is H;

[0094] M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-;

[0095] R' is C 1-12 Alkyl or C 2-12 alkenyl;

[0096] Y a It is C 3-6 carbon ring;

[0097] R*” a Choose from C 1-15 Alkyl and C 2-15 a group consisting of alkenyl groups;

[0098] l is selected from the group consisting of 1, 2, 3, 4 and 5;

[0099] s is 2 or 3; and

[0100] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

[0101] In some aspects, the present disclosure relates to a compound of formula (A-2):

[0102] or an N-oxide thereof, or a salt or isomer thereof,

[0103] where R' a It's R' 支链 or R' 环状 ;in

[0104] R' 支链 yes: R' 环状 yes: in Indicates a connection point;

[0105] where R aα 、R aβ and R aδ Each independently selected from H, C 2-12 Alkyl and C 2-12 alkenyl; and R aγ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0106] R 2 and R 3 Each is C 1-14 alkyl;

[0107] R 4 is -(CH2)2OH or

[0108] in Indicates a connection point;

[0109] R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0110] R 5 and R 6 Each is H;

[0111] R 7 It is H;

[0112] M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-;

[0113] R' is C 1-12Alkyl or C 2-12 alkenyl;

[0114] Y a It is C 3-6 carbon ring;

[0115] R*” a Choose from C 1-15 Alkyl and C 2-15 a group consisting of alkenyl groups;

[0116] l is selected from the group consisting of 1, 2, 3, 4 and 5;

[0117] s is 2 or 3; and

[0118] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

[0119] In some aspects, the present disclosure relates to a compound of formula (A-3):

[0120] or an N-oxide thereof, or a salt or isomer thereof,

[0121] where R' a It's R' 支链 or R' 环状 ;in

[0122] R' 支链 yes: R' 环状 yes: in Indicates a connection point;

[0123] where R aα 、R aγ and R aβ Each independently selected from H, C 2-12 Alkyl and C 2-12 alkenyl; and R aδ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0124] R 2 and R 3 Each is C 1-14 alkyl;

[0125] R 4 is -(CH2)2OH or

[0126] in Indicates a connection point;

[0127] R 10 is N(R)2; each R is independently selected from C 1-6Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0128] R 5 and R 6 Each is H;

[0129] R 7 It is H;

[0130] M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-;

[0131] R' is C 1-12 Alkyl or C 2-12 alkenyl;

[0132] Y a It is C 3-6 carbon ring;

[0133] R*” a Choose from C 1-15 Alkyl and C 2-15 a group consisting of alkenyl groups;

[0134] l is selected from the group consisting of 1, 2, 3, 4 and 5;

[0135] s is 2 or 3; and

[0136] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

[0137] In some embodiments, compounds of the present disclosure have one of the following structures:

[0138]

[0139] In some aspects, the present disclosure relates to a compound of formula (B-1):

[0140] or an N-oxide thereof, or a salt or isomer thereof,

[0141] where R' a It's R' 支链 or R' 环状 ;in

[0142] R' 支链 yes: R' 环状 yes: in Indicates a connection point;

[0143] where R aα 、R aγ and Raδ Each independently selected from H, C 2-12 Alkyl and C 2-12 alkenyl; and R aβ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0144] R bα 、R bγ and R bδ Each independently selected from H, C 2-30 Alkyl and C 5-20 alkenyl; and R bβ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0145] R 4 yes

[0146] in Indicates a connection point;

[0147] where R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0148] R 5 and R 6 Each is H;

[0149] M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-;

[0150] R' is C 1-12 Alkyl or C 2-12 alkenyl;

[0151] Y a It is C 3-6 carbon ring;

[0152] R*” a Choose from C 1-15 Alkyl and C 2-15 a group consisting of alkenyl groups;

[0153] l is selected from the group consisting of 1, 2, 3, 4 and 5;

[0154] s is 2 or 3; and

[0155] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

[0156] In some aspects, the present disclosure relates to a compound of formula (B-2):

[0157] or an N-oxide thereof, or a salt or isomer thereof,

[0158] where R' a It's R' 支链 or R' 环状 ;in

[0159] R' 支链 yes: R' 环状 yes: in Indicates a connection point;

[0160] where R aα 、R aβ and R aδ Each independently selected from H, C 2-12 Alkyl and C 2-12 alkenyl; and R aγ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0161] R bα 、R bβ and R bδ Each independently selected from H, C 2-30 Alkyl and C 5-20 alkenyl; and R bγ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0162] R 4 is -(CH2)2OH or

[0163] in Indicates a connection point;

[0164] R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0165] R 5 and R 6 Each is H;

[0166] M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-;

[0167] R' is C 1-12 Alkyl or C 2-12 alkenyl;

[0168] Y a It is C 3-6 carbon ring;

[0169] R*"a is selected from C 1-15 Alkyl and C 2-15 a group consisting of alkenyl groups;

[0170] l is selected from the group consisting of 1, 2, 3, 4 and 5;

[0171] s is 2 or 3; and

[0172] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

[0173] In some aspects, the present disclosure relates to a compound of formula (B-3):

[0174] or an N-oxide thereof, or a salt or isomer thereof,

[0175] where R' a It's R' 支链 or R' 环状 ;in

[0176] R' 支链 yes: R' 环状 yes: in Indicates a connection point;

[0177] where R aα 、R aβ and R aγ Each independently selected from H, C 2-12 Alkyl and C 2-12 alkenyl; and R aδ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0178] R bα 、R bβ and R bγ Each independently selected from H, C 2-30 Alkyl and C 5-20 alkenyl; and R bδ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0179] R 4 is -(CH2)2OH or

[0180] in Indicates a connection point;

[0181] R10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0182] R 5 and R 6 Each is H;

[0183] M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-;

[0184] R' is C 1-12 Alkyl or C 2-12 alkenyl;

[0185] Y a It is C 3-6 carbon ring;

[0186] R*” a Choose from C 1-15 Alkyl and C 2-15 a group consisting of alkenyl groups;

[0187] l is selected from the group consisting of 1, 2, 3, 4 and 5;

[0188] s is 2 or 3; and

[0189] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

[0190] In some aspects, the present disclosure relates to a compound of formula (A-a1):

[0191] or an N-oxide thereof, or a salt or isomer thereof,

[0192] where R aγ and R aδ Each independently selected from H, C 2-12 Alkyl and C 2-12 alkenyl; and R aβ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0193] R 4 is -(CH2)2OH or

[0194] in Indicates a connection point;

[0195] R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0196] M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; and

[0197] R' is C 1-12 Alkyl or C 2-12 Alkenyl.

[0198] In some aspects, the present disclosure relates to a compound of formula (A-a2):

[0199] or an N-oxide thereof, or a salt or isomer thereof,

[0200] where R aβ and R aδ Each independently selected from H, C 2-12 Alkyl and C 2-12 alkenyl; and R aγ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0201] R 4 is -(CH2)2OH or

[0202] in Indicates a connection point;

[0203] R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0204] M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; and

[0205] R' is C 1-12 Alkyl or C 2-12 Alkenyl.

[0206] In some aspects, the present disclosure relates to a compound of formula (A-a3):

[0207] or an N-oxide thereof, or a salt or isomer thereof,

[0208] where R aβ and R aγ Each independently selected from H, C 2-12 Alkyl and C 2-12alkenyl; and R aδ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0209] R 4 is -(CH2)2OH or

[0210] in Indicates a connection point;

[0211] R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0212] M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; and

[0213] R' is C 1-12 Alkyl or C 2-12 Alkenyl.

[0214] In some aspects, the present disclosure relates to a compound of formula (A-b1):

[0215] or an N-oxide thereof, or a salt or isomer thereof,

[0216] where R aγ and R aδ Each independently selected from H, C 2-12 Alkyl and C 2-12 alkenyl; and R aβ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0217] R 4 is -(CH2)2OH or

[0218] in Indicates a connection point;

[0219] R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; and

[0220] R' is C 1-12 Alkyl or C 2-12 Alkenyl.

[0221] In some aspects, the present disclosure relates to a compound of formula (A-b2):

[0222] or an N-oxide thereof, or a salt or isomer thereof,

[0223] where R aβ and R aδ Each independently selected from H, C 2-12 Alkyl and C 2-12 alkenyl; and R aγ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0224] R 4 is -(CH2)2OH or

[0225] in Indicates the connection point; R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; and

[0226] R' is C 1-12 Alkyl or C 2-12 Alkenyl.

[0227] In some aspects, the present disclosure relates to a compound of formula (A-b3):

[0228] or an N-oxide thereof, or a salt or isomer thereof,

[0229] where R aβ and R aγ Each independently selected from H, C 2-12 Alkyl and C 2-12 alkenyl; and R aδ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0230] R 4 is -(CH2)2OH or

[0231] in Indicates a connection point;

[0232] R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; and

[0233] R' is C 1-12 Alkyl or C 2-12 Alkenyl.

[0234] In some aspects, the present disclosure relates to a compound of formula (Ac):

[0235] or an N-oxide thereof, or a salt or isomer thereof,

[0236] where R aγ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0237] R 4 is -(CH2)2OH or in represents the connection point; and R' is C 1-12 alkyl.

[0238] In some aspects, the present disclosure relates to a compound of formula (Bc):

[0239] or its N-oxide, or its salt or isomer, wherein R aγ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl;

[0240] R bγ is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl; and

[0241] R 4 is -(CH2)2OH or in represents the connection point; and R' is C 1-12 alkyl.

[0242] In some aspects, the present disclosure relates to a compound of formula (Ia):

[0243] where R 2 and R 3 Each independently selected from C 1-14 Alkyl and C 2-14 alkenyl; and R aγ and R bγ Each is independently C 2-6 alkyl.

[0244] 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) include one or more of the following characteristics, where applicable.

[0245] In some embodiments, R 4 yes and n2 is 2. In some embodiments, R 4 yes and n2 is 3. In some embodiments, R 4 yes And n2 is 4.

[0246] In some embodiments, R 10 is -NH2. In some embodiments, R 10 It is -NH(C 1-6 In some embodiments, R 10 Yes-N(C 1-6 In some embodiments, R 10 is -NH(CH3). In some embodiments, R 10 It is -N(CH3)2.

[0247] In some embodiments, R 4 is -(CH2)2OH, -(CH2)3OH or -(CH2)4OH and M and M' are each -C(O)O-. In some embodiments, R 4 is -(CH2)2OH and M and M' are each -C(O)O-. In some embodiments, R 4 is -(CH2)3OH and M and M' are each -C(O)O-. In some embodiments, R 4 is -(CH2)4OH and M and M' are each -C(O)O-.

[0248] In some embodiments, R 4 yes

[0249] In some embodiments, R 4 yes and M and M' are each -C(O)O-. In some embodiments, R 4 yes and M and M' are each -OC(O)-. In some embodiments, R 4yes and M is -OC(O)- and M' is -C(O)O-. In some embodiments, R 4 yes is -C(O)O- and M' is -OC(O)-.

[0250] 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.

[0251] 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.

[0252] In some embodiments, R 5 、R 6 and R 7 Each is H, and m is 5, 6, 7, 8, or 9. In some embodiments, R 5 、R 6 and R 7 Each is H, and m is 5. In some embodiments, R 5 、R 6 and R 7 Each is H, and m is 7.

[0253] In some embodiments, R 2 and R 3 Each is independently C 1-14 Alkyl or C 2-14 In some embodiments, R 2 and R 3 Each is independently C 3-14 Alkyl or C 3-14 In some embodiments, R 2 and R 3 Independently C 5-14 Alkyl or C 5-14 Alkenyl.

[0254] In some embodiments, R 2 and R 3 Each is independently C 1-14 In some embodiments, R 2 and R 3 Each is independently C 3-14 In some embodiments, R 2 and R3 Each is independently C 7-9 alkyl.

[0255] In some embodiments, R 2 and R 3 In some embodiments, R 2 and R 3 In some embodiments, R 2 and R 3 Each is a C9 alkyl group.

[0256] In some embodiments, R' a It's R' 支链 , R aα 、R aγ and R aδ Each is H, and R aβ is a C2-C6 alkyl group. In some embodiments, R' a It's R' 支链 , R aα 、R aβ and R aδ Each is H, and R aγ is a C2-C6 alkyl group. In some embodiments, R' a It's R' 支链 , R aα 、R aβ and R aγ Each is H, and R aδ It is a C2-C6 alkyl group.

[0257] In some embodiments, R bα 、R bγ and R bδ Each is H, and R bβ is C2-C6 alkyl. In some embodiments, R bα 、R bβ and R bδ Each is H, and R bγ is C2-C6 alkyl. In some embodiments, R bα 、R bβ and R bγ Each is H, and R bδ It is a C2-C6 alkyl group.

[0258] In some embodiments, R aα 、R aγ and R aδ H, R respectively aβ is a C2-C6 alkyl group, R bα 、R bγ and R bδEach is H, and R bβ is C2-C6 alkyl. In some embodiments, R aα 、R aγ and R aδ H, R respectively aβ is a C2-C6 alkyl group, R bα 、R bβ and R bδ Each is H, and R bγ is C2-C6 alkyl. In some embodiments, R aα 、R aγ and R aδ H, R respectively aβ is a C2-C6 alkyl group, R bα 、R bβ and R bγ Each is H, and R bδ It is a C2-C6 alkyl group.

[0259] In some embodiments, R aα 、R aβ and R aδ H, R respectively aγ is a C2-C6 alkyl group, R bα 、R bγ and R bδ Each is H, and R bβ is C2-C6 alkyl. In some embodiments, R aα 、R aβ and R aδ H, R respectively aγ is a C2-C6 alkyl group, R bα 、R bβ and R bδ Each is H, and R bγ is C2-C6 alkyl. In some embodiments, R aα 、R aβ and R aδ H, R respectively aγ is a C2-C6 alkyl group, R bα 、R bβ and R bγ Each is H, and R bδ It is a C2-C6 alkyl group.

[0260] In some embodiments, R aα 、R aβ and R aγ H, R respectively aδ is a C2-C6 alkyl group, R bα 、R bγ and R bδ Each is H, and R bβis C2-C6 alkyl. In some embodiments, R aα 、R aβ and R aγ H, R respectively aδ is a C2-C6 alkyl group, R bα 、R bβ and R bδ Each is H, and R bγ is C2-C6 alkyl. In some embodiments, R aα 、R aβ and R aγ H, R respectively aδ is a C2-C6 alkyl group, R bα 、R bβ and R bγ Each is H, and R bδ It is a C2-C6 alkyl group.

[0261] In some embodiments, R' is C 1-12 Alkyl or C 2-12 In some embodiments, R' is a C2 alkyl group. In some embodiments, R' is a C3 or C4 alkyl group. In some embodiments, R' is a C3 alkyl group. In some embodiments, R' is a C4 alkyl group. In some embodiments, R' is a C5 alkyl group.

[0262] In some embodiments, R' is C4 alkyl or C4 alkenyl. In some embodiments, R' is C5 alkyl or C5 alkenyl. In some embodiments, R' is C6 alkyl or C6 alkenyl. In some embodiments, R' is C7 alkyl or C7 alkenyl. In some embodiments, R' is C8 alkyl or C8 alkenyl. In some embodiments, R' is C9 alkyl or C9 alkenyl. In some embodiments, R' is C 10 Alkyl or C 10 In some embodiments, R' is C 11 Alkyl or C 11 Alkenyl.

[0263] In some embodiments, R' a It's R' 支链 , R aα 、R aγ and R aδ H, R respectively aβ is C2-C6 alkyl and R' is C3-C5 alkyl. In some embodiments, R' a It's R' 支链 , R aα 、R aγ and R aδ H, R respectively aβis C2-C6 alkyl and R' is C3 alkyl. In some embodiments, R' a It's R' 支链 , R aα 、R aγ and R aδ H, R respectively aβ is a C2-C6 alkyl group and R' is a C4 alkyl group.

[0264] In some embodiments, R' a It's R' 支链 , R aα 、R aβ and R aδ H, R respectively aγ is C2-C6 alkyl, and R' is C3-C5 alkyl. In some embodiments, R'a is R' 支链 , R aα 、R aβ and R aδ H, R respectively aγ is C2-C6 alkyl, and R' is C3 alkyl. In some embodiments, R' a It's R' 支链 , R aα 、R aβ and R aδ H, R respectively bγ is a C2-C6 alkyl group, and R' is a C4 alkyl group.

[0265] In some embodiments, R' a It's R' 支链 , R aα 、R aβ and R aγ H, R respectively aδ is C2-C6 alkyl and R' is C3-C5 alkyl. In some embodiments, R' a It's R' 支链 , R aα 、R aβ and R aγ H, R respectively aδ is C2-C6 alkyl and R' is C3 alkyl. In some embodiments, R' a It's R' 支链 , R aα 、R aβ and R aγ H, R respectively aδ is a C2-C6 alkyl group and R' is a C4 alkyl group.

[0266] In some embodiments, R*" a It is a C2 alkyl group or a C3 alkyl group.

[0267] In some embodiments, s is 2. In some embodiments, s is 3.

[0268] In some embodiments, s is 2 and R*" a It is a C2 alkyl group or a C3 alkyl group.

[0269] In some embodiments, Y a R*”a is In some embodiments, Y a R*” a yes

[0270] In some embodiments, s is 2, Y a R*” a yes And R*” a is C2 alkyl or C3 alkyl. In some embodiments, s is 2, Y a R*” a yes And R*” a It is a C2 alkyl group or a C3 alkyl group.

[0271] In some embodiments, the compounds of any of the formulae described herein are suitable for use in the preparation of nanoparticle compositions for intramuscular administration.

[0272] 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 its N-oxides, salts or isomers.

[0273] Table 1. Amino lipids.

[0274]

[0275]

[0276]

[0277]

[0278] The central amine moiety of the 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) can be protonated at physiological pH. Thus, the lipid can have a positive charge or a partial positive charge at physiological pH. Such lipids can be referred to as cationic or ionizable (amino) lipids. The lipids can also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.

[0279] definition

[0280] As used herein, the term "alkyl" or "alkyl group" means a straight or branched chain saturated hydrocarbon comprising one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms), which is optionally substituted. 1-14 "Alkyl" means an optionally substituted straight or branched chain saturated hydrocarbon comprising 1 to 14 carbon atoms. Unless otherwise specified, the alkyl groups described herein refer to both unsubstituted and substituted alkyl groups.

[0281] As used herein, the term "alkenyl" or "alkenyl group" means a straight or branched chain hydrocarbon comprising two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one double bond, which is optionally substituted. 2-14 "Alkenyl" means an optionally substituted straight or branched chain hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may contain one, two, three, four or more carbon-carbon double bonds. For example, C 18 Alkenyl groups may include one or more double bonds. 18 The alkenyl group may be linoleyl. Unless otherwise specified, the alkenyl groups described herein refer to both unsubstituted and substituted alkenyl groups.

[0282] As used herein, the term "alkynyl" or "alkynyl group" means a straight or branched chain hydrocarbon comprising two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted.2-14 "Alkynyl" means an optionally substituted straight or branched chain hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon triple bond. Alkyl groups may contain one, two, three, four or more carbon-carbon triple bonds. For example, C 18 Alkynyl groups may include one or more carbon-carbon triple bonds.Unless otherwise specified, alkynyl groups described herein refer to both unsubstituted and substituted alkynyl groups.

[0283] As used herein, the term "carbocycle" or "carbocyclic group" means an optionally substituted monocyclic or polycyclic ring system comprising one or more carbon atom rings. The ring may be three-membered, four-membered, five-membered, six-membered, seven-membered, eight-membered, nine-membered, ten-membered, eleven-membered, twelve-membered, thirteen-membered, fourteen-membered, fifteen-membered, sixteen-membered, seventeen-membered, eighteen-membered, nineteen-membered, or twenty-membered. 3-6 "Carbocycle" means a carbocycle including a monocyclic ring having 3-6 carbon atoms. The carbocycle may include 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" means a non-aromatic carbocycle and may or may not include any double or triple bonds. Unless otherwise specified, the carbocycles described herein refer to unsubstituted and substituted carbocycle groups, i.e., optionally substituted carbocycles. In some embodiments, the carbocycle is C 3-8 In some embodiments, the carbocyclic ring is C 3-6 In some embodiments, the carbocyclic ring is C 6-10 Aryl.

[0284] "Aryl" includes groups with aromatic properties, including "conjugated" or polycyclic ring systems having at least one aromatic ring and not containing any heteroatoms in the ring structure. Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthyl, etc. In some embodiments, "aryl" is a C 6-10 Carbocyclic rings (e.g., "aryl" is C 6-10 aryl).

[0285] As used herein, the term "heterocycle" or "heterocyclic group" means an optionally substituted monocyclic or polycyclic ring system comprising one or more rings, wherein at least one ring comprises at least one heteroatom. The heteroatom may be, for example, a nitrogen, oxygen or sulfur atom. The ring may be a three-membered, four-membered, five-membered, six-membered, seven-membered, eight-membered, nine-membered, ten-membered, eleven-membered, twelve-membered, thirteen-membered or fourteen-membered ring. The heterocycle may include one or more double bonds or triple bonds and may be non-aromatic or aromatic (e.g., heterocycloalkyl or heteroaryl groups). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuranyl, thienyl, pyridyl, piperidyl, quinolinyl and isoquinolinyl groups. As used herein, the term "heterocycloalkyl" means a non-aromatic heterocycle and may or may not include any double or triple bonds. Unless otherwise specified, the heterocycles described herein refer to 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.

[0286] A "heteroaryl" group is an aryl group as defined above, except that it has one to four heteroatoms in the ring structure, and may also be referred to as an "aryl heterocycle" or "heteroaromatic compound". 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 aromatic heterocycle consisting of carbon atoms and one or more heteroatoms independently selected from the group consisting of nitrogen, oxygen, sulfur, and boron, such as 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or such as 1, 2, 3, 4, 5, or 6 heteroatoms. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR, where R is H or other substituents as defined). The nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., N→O and S(O)). p , wherein p=1 or 2). It should be noted that the total number of S and O atoms in the aromatic heterocycle is not more than 1.

[0287] Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.

[0288] In addition, the terms "aryl" and "heteroaryl" include polycyclic aromatic and heteroaryl groups, for example, tricyclic, bicyclic, for example, naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzimidazole, benzothiophene, quinoline, isoquinoline, naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.

[0289] As used herein, a "biodegradable group" is a group that promotes faster metabolism of lipids in mammalian entities. Biodegradable groups can be selected from the group consisting of, but not limited to, -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, aryl groups, and heteroaryl groups. As used herein, an "aryl group" is an optionally substituted carbocyclic group comprising 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 comprising one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furanyl, thienyl, 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 each formula herein, M and M' can be independently selected from the list of biodegradable groups above. Unless otherwise specified, aryl or heteroaryl groups described herein refer to unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.

[0290] Unless otherwise specified, alkyl, alkenyl, and cyclic (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted. Optional substituents may be selected from, 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, or represented by C=O), acyl halides (e.g., -C(O)X, wherein X is a halide selected from bromide, fluoride, chloride, and iodide), carbonates (e.g., -OC(O)OR), alkoxy groups (e.g., -OR), acetals (e.g., -C(OR)2R"", wherein 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)4 3- ), thiols (e.g., -SH), sulfoxides (e.g., -S(O)R), sulfinic acids (e.g., -S(O)OH), sulfonic acids (e.g., -S(O)2OH), thialdehydes (e.g., -C(S)H), sulfates (e.g., S(O)4 2-), sulfonyl (e.g., —S(O)—), amide (e.g., —C(O)NR or —N(R)C(O)R), azido (e.g., —N), nitro (e.g., —NO), cyano (e.g., —CN), isocyano (e.g., —NC), acyloxy (e.g., —OC(O)R), amino (e.g., —NR, —NRH, or —NH), carbamoyl (e.g., —OC(O)NR, —OC(O)NRH, or —OC(O)NH), sulfonamide (e.g., —S(O)NR, —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 groups, alkenyl groups, and cyclic (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 groups themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein. For example, C 1-6 The alkyl group can be further substituted with one, two, three, four, five, or six substituents as described herein.

[0291] Nitrogen-containing compounds of the present disclosure 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 compounds of the present disclosure. Thus, all nitrogen-containing compounds shown and claimed are considered to include the compounds shown and their N-oxide derivatives (which may be designated as N→O or N→O) when valence and structure permit. + -O - ). In addition, in other cases, the nitrogen in the compounds of the present disclosure can be converted into N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds can be prepared by oxidizing the parent amine using an oxidizing agent such as m-CPBA. When valence and structure permit, all nitrogen-containing compounds shown and claimed are also considered to cover the compounds shown and their N-hydroxy (i.e., N-OH) and N-alkoxy (i.e., N-OR, wherein R is a substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3-14 membered carbocyclic ring or 3-14 membered heterocyclic ring) derivatives.

[0292] About, approximately: As used herein, the terms "about" and "approximately" as applied to one or more target values refer to values that are similar to the stated reference value. In certain embodiments, unless otherwise specified or otherwise apparent from the context (except where such a number would exceed 100% of the possible value), the terms "about" or "approximately" refer to a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value in either direction (greater than or less than). For example, when used in the context of the amount of a given compound in a lipid component of a nanoparticle composition, "about" can mean + / - 10% of the stated value. For example, a nanoparticle composition comprising a lipid component having about 40% of a given compound can include 30-50% of the compound.

[0293] As used herein, the term "compound" is intended to include all isomers and isotopes of the depicted structure. "Isotopes" refer to atoms having the same atomic number but different mass numbers due to different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. In addition, the compounds, salts, or complexes of the present disclosure can be prepared by conventional methods in combination with solvents or water molecules to form solvates and hydrates.

[0294] As used herein, term " contact " means setting up physical connection between two or more entities.For example, making mammalian cell contact with nanoparticle composition means making described mammalian cell share physical connection with nanoparticle.In vivo and the method that cell is contacted with external entity in vitro are well-known in biological field.For example, making nanoparticle composition and being placed in mammalian mammalian cell contact can carry out and can relate to the lipid nanometer particle (for example empty LNP or load LNP) of variable amount by the route of administration (for example, intravenous, intramuscular, intradermal and subcutaneous) of variation.In addition, more than a kind of mammalian cell can be contacted by nanoparticle composition.

[0295] As used herein, the term "delivery" means providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic agent to a subject can involve administering a nanoparticle composition comprising the therapeutic and / or prophylactic agent to the subject (e.g., via an intravenous, intramuscular, intradermal, or subcutaneous route). Administering a nanoparticle composition to a mammal or mammalian cell can involve contacting one or more cells with the nanoparticle composition.

[0296] As used herein, the term "enhanced delivery" means that the nanoparticles deliver more (e.g., at least 1.5 times more, at least 2 times more, at least 3 times more, at least 4 times more, at least 5 times more, at least 6 times more, at least 7 times more, at least 8 times more, at least 9 times more, at least 10 times more) therapeutic and / or prophylactic agents to the target tissue of interest (e.g., mammalian liver) than the control nanoparticles deliver the therapeutic and / or prophylactic agent to the target tissue of interest (e.g., MC3, KC2, or DLinDMA). The level of nanoparticle delivery to a specific tissue can be measured by comparing the amount of protein produced in the tissue with the weight of the tissue, the amount of therapeutic and / or prophylactic agent in the tissue with the weight of the tissue, the amount of protein produced in the tissue with the amount of total protein in the tissue, or the amount of therapeutic and / or prophylactic agent in the tissue with the amount of total therapeutic and / or prophylactic agent in the tissue. It should be understood that the enhanced delivery of nanoparticles to the target tissue need not be determined in the subject being treated, and it can be determined in alternatives such as animal models (e.g., rat models). In certain embodiments, nanoparticle compositions comprising a compound according to Formula (I-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc) have substantially the same level of delivery enhancement regardless of the route of administration. For example, certain compounds disclosed herein exhibit similar delivery enhancement when used for intravenous or intramuscular delivery of therapeutic and / or prophylactic agents. In other embodiments, certain compounds disclosed herein exhibit greater levels of delivery enhancement than intravenous delivery when used for intramuscular delivery of therapeutic and / or prophylactic agents.

[0297] As used herein, the terms "specific delivery," "specifically deliver," or "specifically delivering" mean that more (e.g., at least 1.5-fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of the therapeutic and / or prophylactic agent is delivered by the nanoparticles to a target tissue of interest (e.g., the liver of a mammal) as compared to an off-target tissue (e.g., the spleen of a mammal). The level of nanoparticle delivery to a specific tissue can be measured by comparing the amount of protein produced in the tissue to the weight of the tissue, the amount of therapeutic and / or prophylactic agent in the tissue to the weight of the tissue, the amount of protein produced in the tissue to the amount of total protein in the tissue, or 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, with respect to renal vascular targeting, if, following systemic administration of the therapeutic and / or prophylactic agent, 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, or 20-fold more of the therapeutic and / or prophylactic agent per 1 g of tissue is delivered to the kidney as compared to the liver or spleen, then the therapeutic and / or prophylactic agent is specifically provided to the kidney of a mammal as compared to the liver and spleen. It will be understood that the ability of a nanoparticle to be specifically delivered to a target tissue need not be determined in the subject being treated, but can be determined in an alternative, for example, an animal model (e.g., a rat model).

[0298] As used herein, "encapsulation efficiency" refers to the amount of therapeutic and / or prophylactic agent that becomes part of a nanoparticle composition relative to the initial total amount of therapeutic and / or prophylactic agent used to prepare the nanoparticle composition. For example, if 97 mg of a total of 100 mg of therapeutic and / or prophylactic agent initially provided to the nanoparticle composition is encapsulated in the composition, the encapsulation efficiency can be given as 97%. As used herein, "encapsulation" can refer to completely, substantially, or partially enclosing, confining, surrounding, or packaging.

[0299] As used herein, "encapsulation," "encapsulated," "loaded," and "associated" may refer to complete, substantial, or partial encapsulation, confinement, surrounding, or packaging. As used herein, "encapsulation" or "association" may refer to the process of confining individual nucleic acid molecules within a nanoparticle and / or establishing a physiochemical connection between an individual nucleic acid molecule and a nanoparticle. As used herein, "empty nanoparticles" or "empty lipid nanoparticles" may refer to nanoparticles that are substantially free of a therapeutic or prophylactic agent. As used herein, "empty nanoparticles" or "empty lipid nanoparticles" may refer to nanoparticles that are substantially free of nucleic acids. As used herein, "empty nanoparticles" or "empty lipid nanoparticles" may refer to nanoparticles that are substantially free of nucleotides or polypeptides. As used herein, "empty nanoparticles" or "empty lipid nanoparticles" may refer to nanoparticles that are essentially composed only of lipid components. As used herein, "loaded nanoparticles" or "loaded lipid nanoparticles" (also referred to as "complete nanoparticles" or "complete lipid nanoparticles") may refer to nanoparticles that comprise 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 "complete nanoparticles" or "complete lipid nanoparticles") may refer to nanoparticles comprising the components of an empty nanoparticle and a nucleotide or polypeptide. As used herein, "loaded nanoparticles" or "loaded lipid nanoparticles" (also referred to as "complete nanoparticles" or "complete lipid nanoparticles") may refer to nanoparticles comprising the components of an empty nanoparticle and a nucleic acid.

[0300] As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into a polypeptide or protein and / or post-translational modification of the polypeptide or protein.

[0301] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as a test tube or reaction vessel, a cell culture, a petri dish, etc., rather than within an organism (such as an animal, plant, or microorganism).

[0302] As used herein, the term "in vivo" refers to an event that occurs within an organism (eg, an animal, plant, or microorganism, or a cell or tissue thereof).

[0303] As used herein, the term "ex vivo" refers to an event that occurs outside an organism (e.g., an animal, plant, or microorganism, or a cell or tissue thereof). An ex vivo event can occur in an environment that is minimally altered from the natural (e.g., in vivo) environment.

[0304] As used herein, the term "isomer" means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer or diastereomer of a compound. A compound may include one or more chiral centers and / or double bonds and may therefore 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 enantiomers and stereoisomer mixtures (e.g., racemates). Enantiomers and stereoisomer mixtures of compounds and the manner in which they are resolved into their component enantiomers or stereoisomers are well known.

[0305] "Tautomers" are any of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another. This conversion results in a formal migration of hydrogen atoms, accompanied by a switch in adjacent conjugated double bonds. Tautomers exist as a mixture of sets of tautomers in solution. In solutions where tautomerism is possible, a chemical equilibrium of tautomers will be reached. The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH. The concept of tautomers that can interconvert via tautomerism is known as tautomerism.

[0306] Of the several possible types of tautomerism, two are commonly observed. In keto-enol tautomerism, a simultaneous shift of electrons and hydrogen atoms occurs. Ring-chain tautomerism occurs when an aldehyde group (-CHO) in a sugar chain molecule reacts with a hydroxyl group (-OH) in the same molecule, causing it to assume a cyclic (ring-shaped) form, as exhibited by glucose.

[0307] Common tautomer pairs are: keto-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomerism in heterocycles (e.g., in nucleobases such as guanine, thymine, and cytosine), imine-enamine, and enamine-enamine. Examples of tautomerism in disubstituted guanidines are shown below.

[0308]

[0309] It is understood that the compounds of the present disclosure may be depicted as different tautomers. It is also understood that when a compound has tautomeric forms, all tautomeric forms are intended to be included within the scope of the present disclosure, and the naming of the compound does not exclude any tautomeric form.

[0310] As used herein, a "lipid component" is a component of a nanoparticle composition that includes one or more lipids. For example, a lipid component can include one or more cationic / ionizable, PEGylated, structural or other lipids, such as phospholipids.

[0311] As used herein, a "linker" is a portion connecting two parts, such as the connection between two nucleosides of a cap material. The linker may include one or more groups, including but not limited to phosphate groups (e.g., phosphates, borophosphates, phosphorothioates, selenophosphates, and phosphonates), alkyl groups, amidates, or glycerol. For example, the two nucleosides of a cap analog can be connected at their 5' positions by a triphosphate group or by a chain comprising two phosphate moieties and one borophosphate moiety.

[0312] As used herein, "administration method" can include intravenous, intramuscular, intradermal, subcutaneous or other methods of delivering a composition to a subject. An administration method can be selected to target delivery (e.g., specifically deliver) to a particular area or system of the body.

[0313] As used herein, "modified" means non-natural. For example, RNA can be modified RNA. That is, the RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers. A "modified" substance may also be referred to herein as an "altered" substance. A substance can be modified or altered chemically, structurally, or functionally. For example, a modified nucleobase substance can include one or more non-naturally occurring substitutions.

[0314] As used herein, "N:P ratio" is the molar ratio of ionizable (in the physiological pH range) nitrogen atoms in the lipids to phosphate groups in the RNA, eg, in a nanoparticle composition including a lipid component and RNA.

[0315] As used herein, a "nanoparticle composition" is a composition comprising one or more lipids. The size of the nanoparticle composition is typically in the micrometer or smaller order of magnitude and may include a lipid bilayer. The nanoparticle composition encompasses lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and liposome complexes. For example, the nanoparticle composition may be a liposome containing a lipid bilayer with a diameter of 500 nm or less.

[0316] As used herein, "naturally occurring" means occurring in nature without human assistance.

[0317] As used herein, "patient" refers to a subject who may seek or need treatment, requires treatment, is receiving treatment, will receive treatment, or is under the care of a professional trained for a particular disease or condition.

[0318] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that comprises a polyethylene glycol component.

[0319] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0320] As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending, complexing, or dissolving the active compound) and having substantially non-toxic and non-inflammatory properties in patients. Excipients may include, for example, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), softeners, emulsifiers, fillers (diluents), film formers or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinyl pyrrolidone, citric acid, cross-linked povidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized 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 others disclosed herein.

[0321] In this specification, the structural formula of the compound may represent a specific isomer for convenience in some cases, but the present disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc., and it should be understood that not all isomers can have the same level of activity. In addition, regarding the compound represented by the formula, crystal polymorphism may exist. It should be noted that any crystalline form, crystalline form mixture, or its anhydride or hydrate is included in the scope of the present disclosure.

[0322] The terms "crystalline polymorph," "polymorph," or "crystal form" refer to a crystal structure 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 crystal 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 cause one crystal form to dominate. Crystal polymorphs of the compound can be prepared by crystallization under different conditions.

[0323] The composition may also include one or more salts of the compound. The salt may be a pharmaceutically acceptable salt. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl 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 metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like; as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the present disclosure include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the present disclosure can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or in an organic solvent or in a mixture of the two; typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0324] As used herein, "phospholipid" is a lipid comprising a phosphate moiety and one or more carbon chains (e.g., unsaturated fatty acid chains). A phospholipid may comprise one or more multiple (e.g., double or triple) bonds (e.g., one or more degrees of unsaturation). Specific phospholipids may promote fusion with a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cell or intracellular membrane). Fusion of a phospholipid with a membrane may allow one or more elements of a lipid-containing composition to pass through the membrane, thereby allowing, for example, the one or more elements to be delivered to a cell.

[0325] As used herein, the "polydispersity index" or "PDI" is a ratio that describes the homogeneity of the particle size distribution of a system. Small values, such as less than 0.3, indicate a narrow particle size distribution.

[0326] As used herein, the term "polypeptide" or "target polypeptide" refers to a polymer of amino acid residues typically joined by peptide bonds, which can be produced naturally (e.g., isolated or purified) or synthetically. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may contain modified amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and sarcosine), as well as other modified polypeptides known in the art. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include encoded polynucleotide products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a monomer or a multimolecular complex, such as a dimer, trimer, or tetramer. It may also comprise a single-chain or multi-chain polypeptide. The most common disulfide bonds are found in multi-chain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acid. In some embodiments, a "peptide" may be less than or equal to 50 amino acids long, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0327] As used herein, "RNA" refers to ribonucleic acid, which may be naturally occurring or non-naturally occurring. For example, RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA may include a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence encoding a polypeptide of interest.

[0328] As used herein, "DNA" refers to deoxyribonucleic acid, which can occur naturally or non-naturally. For example, DNA can 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 instead produced using molecular biology techniques.

[0329] As used herein, a "single unit dose" is a dose of any therapeutic agent administered in one dose / at the same time / by a single route / at a single point of contact (ie, a single administration event).

[0330] As used herein, a "split dose" is a division of a single unit dose or total daily dose into two or more doses.

[0331] As used herein, the "total daily dose" is the amount given or prescribed for a 24-hour period. It can be administered as a single unit dose.

[0332] As used herein, "size" or "average size" in the context of lipid nanoparticles (eg, empty LNP or loaded LNP) refers to the average diameter of the nanoparticle composition.

[0333] As used herein, the term "subject" or "patient" refers to any organism to which a composition according to the present disclosure can be administered, for example, for experimental, diagnostic, prophylactic and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0334] As used herein, "target cell" refers to any one or more target cells. The cells can be found in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism can be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

[0335] As used herein, "target tissue" refers to any one or more target tissue types, wherein the delivery of the therapeutic and / or preventive agent will result in a desired biological and / or pharmacological effect. Examples of target tissues include specific tissues, organs, and systems or groups thereof. In specific applications, the target tissue may be the kidney, lung, spleen, vascular endothelium in a blood vessel (e.g., within a coronary artery or a femoral artery), or tumor tissue (e.g., via intratumoral injection). "Off-target tissue" refers to any one or more tissue types, wherein the expression of the encoded protein does not result in a desired biological and / or pharmacological effect. In specific applications, off-target tissues may include the liver and spleen.

[0336] The term "therapeutic agent" or "prophylactic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits 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.

[0337] As used herein, the term "therapeutically effective amount" means an amount of an agent (e.g., a nucleic acid, a drug, a composition, a therapeutic agent, a diagnostic agent, a prophylactic agent, etc.) to be delivered which, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of, the infection, disease, disorder, and / or condition.

[0338] As used herein, "transfection" refers to the introduction of a substance (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0339] As used herein, the term "treat" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a particular infection, disease, condition, and / or disorder. For example, "treating" cancer may refer to inhibiting the survival, growth, and / or spread of a tumor. For the purpose of reducing the risk of developing pathology associated with a disease, condition, and / or disorder, treatment may be administered to subjects who do not exhibit symptoms of the disease, condition, and / or disorder, and / or to subjects who exhibit only early signs of the disease, condition, and / or disorder.

[0340] As used herein, "zeta potential" is the zeta potential of, for example, lipids in a particle composition.

[0341] Nanoparticle compositions

[0342] The present disclosure also provides 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) as described herein.

[0343] In some embodiments, the maximum 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), for example, when measured 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 LNPs or loaded LNPs), liposomes, lipid vesicles, and liposome complexes. 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 an aqueous compartment. The lipid bilayers can be functionalized and / or cross-linked to each other. The lipid bilayers can include one or more ligands, proteins, or channels.

[0344] The nanoparticle compositions comprise a lipid component comprising at least one compound according to Formula (I-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 include one or more compounds of Table 1. The nanoparticle compositions can also include various other components. For example, 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), the lipid component of the nanoparticle composition can include one or more additional lipids.

[0345] Cationic / ionizable lipids

[0346] In addition to the 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), the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may also include one or more cationic and / or ionizable lipids (e.g., lipids that can have a positive charge or a partial positive charge at physiological pH). The cationic and / or ionizable lipids may be selected from the non-limiting group consisting of: 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethylamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethylamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-triacontane (KL25), 1, 2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino)butyric acid heptatriacontane-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLi n-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-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)-octadec-9,12-dien-1-yloxy]propan-1-amine In some embodiments, the cationic lipids include (1,2-[(9Z,12Z)-octadec-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)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)). In addition to these lipids, the cationic lipid may also be a lipid including a cyclic amine group.

[0347] Structured lipids

[0348] Lipid nanoparticles (such as empty LNP or load LNP) can include one or more structural lipids.Structural lipids can be selected from the group consisting of but not limited to cholesterol, fecal sterol, phytosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatin, ursolic acid, alpha-tocopherol and their mixture.In some embodiments, structural lipids are cholesterol.In some embodiments, structural lipids include cholesterol and corticosteroids (such as prednisolone (prednisolone), dexamethasone (dexamethasone), prednisone (prednisone) and hydrocortisone (hydrocortisone)) or their combination.In some embodiments, structural lipids are:

[0349] phospholipids

[0350] Lipid nanoparticles (e.g., empty LNP or loaded LNP) can include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids can be assembled into one or more lipid bilayers. Generally, phospholipids can include a phospholipid portion and one or more fatty acid portions. For example, the phospholipid can be a lipid according to formula (IV):

[0351]

[0352] where R p represents the phospholipid portion and R A and R B Represent the fatty acid moiety with or without degree of unsaturation, and described fatty acid moiety can be identical or different.Phospholipid moiety can be selected from the non-restrictive group of free phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin composition.Fatty acid moiety can be selected from the non-restrictive group of free lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid composition.Also contemplate non-natural substances, comprise the natural substances with modification and replacement including side chain, oxidation, cyclization and alkynes.For example, phospholipid can be functionalized by one or more alkynes (for example, the alkenyl group that wherein one or more double bonds are replaced by triple bond) or cross-linked with described one or more alkynes. Under appropriate reaction conditions, the alkyne group can undergo a copper-catalyzed cycloaddition when exposed to azide. Such reactions can be used to functionalize the lipid bilayer of lipid nanoparticles (e.g., empty or loaded LNPs) to facilitate membrane permeation or cell recognition, or can be used to conjugate lipid nanoparticles (e.g., empty or loaded LNPs) to useful components, such as targeting or imaging moieties (e.g., dyes).

[0353] Phospholipids useful in the compositions and methods can be selected from the non-limiting group consisting of 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 (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 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-bis(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-racem-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyl oil In some embodiments, lipid nanoparticles (for example empty LNP or load LNP) include DSPC. In certain embodiments, lipid nanoparticles (for example empty LNP or load LNP) include DOPE. In some embodiments, lipid nanoparticles (for example empty LNP or load LNP) include DSPC. In certain embodiments, lipid nanoparticles (for example empty LNP or load LNP) include DOPE. In some embodiments, lipid nanoparticles (for example empty LNP or load LNP) include DSPC and DOPE.

[0354] PEG lipids

[0355] Lipid nanoparticle (for example empty LNP or load LNP) can comprise one or more PEG lipids or the lipid modified through PEG.This type of material can alternatively be called PEGization lipid.PEG lipid is the lipid modified through polyethylene glycol.PEG lipid can be selected from the non-restrictive group of the phosphatidylethanolamine modified through PEG, the phosphatidic acid modified through PEG, the ceramide modified through PEG (PEG-CER), the dialkylamine modified through PEG, the diacylglycerol (PEG-DEG) modified through PEG, the dialkyl glycerol modified through PEG and their mixture composition.For example, PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipid.

[0356] 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.

[0357] In certain embodiments, the PEG lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-distearylglycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA). For example, in some embodiments, the PEG lipid is PEG-DMG.

[0358] In certain embodiments, the PEG lipid is a compound of formula (PL-I):

[0359]

[0360] or a salt thereof, wherein:

[0361] R 3PL1 Yes-OR OPL1 ;

[0362] R OPL1 is hydrogen, optionally substituted alkyl, or an oxygen protecting group;

[0363] r PL1is an integer between 1 and 100 (inclusive);

[0364] L 1 is an optionally substituted C 1-10 Alkylene, wherein the optionally substituted C 1-10 At least one methylene group of the alkylene group is independently replaced by an optionally substituted carbocyclylene group, an optionally substituted heterocyclylene group, an optionally substituted arylene group, an optionally substituted heteroarylene group, 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 )replace;

[0365] D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions;

[0366] m PL1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0367] A has the following formula:

[0368] L 2 Each instance of is independently a bond or an optionally substituted C 1-6 Alkylene, wherein the optionally substituted C 1-6 A methylene unit of the alkylene group is optionally replaced by 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 )replace;

[0369] R 2SL Each instance of is independently optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 Alkenyl or optionally substituted C 1-30 Alkynyl; optionally wherein R 2SL The one or more methylene units are independently replaced by 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 N PL1 )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 NP L1 )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 instead;

[0370] R NPL1 Each instance of is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

[0371] Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

[0372] p SL It is 1 or 2.

[0373] In certain embodiments, the PEG lipid is a compound of the formula (PL-I-OH): or a salt thereof.

[0374] In certain embodiments, the PEG lipid is a compound of the formula (PL-II-OH):

[0375] or a salt or isomer thereof, wherein:

[0376] R 3PEG Yes-OR O ;

[0377] R O It is hydrogen, C 1-6 Alkyl or oxygen protecting groups;

[0378] r PEG is an integer between 1 and 100;

[0379] R 5PEG It is C 10-40 Alkyl, C 10-40 Alkenyl or C 10-40 Alkynyl; and optionally R 5PEG The one or more methylene groups are independently C 3-10 Carbocyclylene, 4- to 10-membered heterocyclylene, C 6-10 Arylene, 4- to 10-membered heteroarylene, -N(R NPEG )-, -O-, -S-, -C(O)-, -C(O)N(R NPEG )-、-NR NPEG C(O)-、-NR NPEG C(O)N(R NPEG )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R NPEG )-、-NR NPEG C(O)O-, -C(O)S-, -SC(O)-, -C(=NR NPEG )-、-C(=NR NPEG )N(R NPEG )-、-NR NPEG C(=NR NPEG )-、-NR NPEG C(=NR NPEG )N(R NPEG )-、-C(S)-、-C(S)N(R NPEG )-、-NR NPEG C(S)-、-NR NPEG C(S)N(R NPEG )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R NPEG)S(O)-、-S(O)N(R NPEG )-、-N(R NPEG )S(O)N(R NPEG )-、-OS(O)N(R NPEG )-、-N(R NPEG )S(O)O-、-S(O)2-、-N(R NPEG )S(O)2-、-S(O)2N(R NPEG )-、-N(R NPEG )S(O)2N(R NPEG )-、-OS(O)2N(R NPEG )-or-N(R NPEG )S(O)2O- instead; and

[0380] R NPEG Each instance of is independently hydrogen, C 1-6 Alkyl or nitrogen protecting group.

[0381] 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.

[0382] In certain embodiments, in the PEG lipid of formula (PL-II-OH), R 5 It is C 17 alkyl.

[0383] In certain embodiments, the PEG lipid is a compound of formula (PL-II): where r PEG Is an integer between 1 and 100.

[0384] In certain embodiments, the PEG lipid is a compound of formula (PEG-1):

[0385] In certain embodiments, the PEG lipid is a compound of formula (PL-III):

[0386] or a salt or isomer thereof, wherein s PL1 Is an integer between 1 and 100.

[0387] In certain embodiments, the PEG lipid is a compound of the formula:

[0388] In certain embodiments, the formula (PL-I), (PL-I-OH), (PL-II), (PL-II-OH), (PL-III), PEG2k Incorporation of a lipid of one of DMG or PEG-1 into a nanoparticle formulation can improve the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulation. For example, incorporation of a lipid of one of the formulas (PL-II-OH), (PL-IIa-OH), (PL-II), or PEG-1 into a nanoparticle formulation can reduce the accelerated blood clearance (ABC) effect.

[0389] adjuvant

[0390] In some embodiments, lipid nanoparticles (e.g., empty LNPs or loaded LNPs) comprising one or more lipids described herein may further include one or more adjuvants, such as glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxynucleotides (e.g., class A or B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0391] therapeutic agents

[0392] Lipid nanoparticles (e.g., empty LNP or loaded LNP) may include one or more therapeutic and / or prophylactic agents. The present disclosure provides methods for delivering a therapeutic and / or prophylactic agent to a mammalian cell or organ, producing a target polypeptide in a mammalian cell, and treating a disease or condition in a mammal in need thereof, the methods comprising administering to a mammal a lipid nanoparticle (e.g., empty LNP or loaded LNP) comprising a therapeutic and / or prophylactic agent and / or contacting a mammalian cell with a lipid nanoparticle (e.g., empty LNP or loaded LNP) comprising a therapeutic and / or prophylactic agent.

[0393] Therapeutic and / or prophylactic agents include biologically active substances and are alternatively referred to as "active agents." A therapeutic and / or prophylactic agent can be a substance that, once delivered to a cell or organ, causes a desired change in the cell, organ, or other body tissue or system. Such substances can be used to treat one or more diseases, conditions, or disorders. In some embodiments, the therapeutic and / or prophylactic agent is a small molecule drug that can be used to treat a specific disease, condition, or disorder.

[0394] In some embodiments, the therapeutic and / or prophylactic agent is a vaccine, a compound that elicits an immune response (e.g., a polynucleotide or nucleic acid molecule or protein or polypeptide or peptide encoding a protein or polypeptide or peptide), and / or another therapeutic and / or prophylactic agent. Vaccines include compounds and formulations that can provide immunity to one or more conditions associated with an infectious disease and may include mRNA encoding infectious disease-derived antigens and / or epitopes. Vaccines also include compounds and formulations that direct an immune response against cancer cells and may include mRNA encoding tumor cell-derived antigens, epitopes, and / or neo-epitopes. In some embodiments, vaccines and / or compounds that can elicit an immune response are administered intramuscularly via the compositions of the present disclosure.

[0395] In other embodiments, the therapeutic and / or prophylactic agent is a protein, e.g., a protein that is desired to augment or replace a naturally occurring protein of interest. Such proteins or polypeptides may be naturally occurring or may be modified using methods known in the art, e.g., to extend half-life. Exemplary proteins are intracellular, transmembrane, or secreted.

[0396] Polynucleotides and nucleic acids

[0397] In some embodiments, the therapeutic agent is an agent that enhances (i.e., increases, stimulates, upregulates) protein expression. Non-limiting examples of therapeutic agent types 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 can upregulate the expression of naturally occurring or non-naturally occurring proteins (e.g., chimeric proteins that have been modified to improve half-life, or proteins that include desired amino acid changes). Exemplary proteins include intracellular, transmembrane, or secretory proteins, peptides, or polypeptides.

[0398] In some embodiments, the therapeutic agent is a DNA therapeutic agent. The DNA molecule can be double-stranded DNA, single-stranded DNA (ssDNA), or a molecule that is partially double-stranded DNA (i.e., 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.

[0399] DNA therapeutics can be DNA molecules that are capable of transferring genes into cells, e.g., encoding transcripts and expressing the transcripts. In other embodiments, the DNA molecules are synthetic molecules, e.g., synthetic DNA molecules produced in vitro. In some embodiments, the DNA molecules are recombinant molecules. Non-limiting exemplary DNA therapeutics include plasmid expression vectors and viral expression vectors.

[0400] DNA therapeutic agents as described herein (e.g., DNA vectors) may include a variety of different features. DNA therapeutic agents as described herein (e.g., DNA vectors) may include non-coding DNA sequences. For example, a DNA sequence may include at least one regulatory element for a gene, such as a promoter, an enhancer, a termination element, a polyadenylation signal element, a 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, a DNA sequence as described herein may have a non-coding DNA sequence that is operably connected to a transcriptionally active gene. In other embodiments, a DNA sequence as described herein may have a non-coding DNA sequence that is not connected to a gene, i.e., the non-coding DNA does not regulate the gene on the DNA sequence.

[0401] In some embodiments, in the loaded LNP of the present disclosure, the one or more therapeutic and / or prophylactic agents are nucleic acids. In 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).

[0402] For example, in some embodiments, when the therapeutic and / or preventive 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 a mixture thereof.

[0403] In some embodiments, in the loaded LNPs of the present 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.

[0404] For example, in some embodiments, when the therapeutic and / or preventive 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.

[0405] 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.

[0406] 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.

[0407] In some embodiments, the one or more therapeutic and / or prophylactic agents are mRNA. In some embodiments, the one or more therapeutic and / or prophylactic agents are modified mRNA (mmRNA).

[0408] In some embodiments, the one or more therapeutic and / or preventive agents are mRNAs that incorporate microRNA binding sites (miR binding sites). Additionally, in some embodiments, the mRNA includes one or more of a stem-loop, a chain-terminating nucleoside, a polyA sequence, a polyadenylation signal, and / or a 5' cap structure.

[0409] The mRNA may be a naturally occurring or non-naturally occurring mRNA. The mRNA may include one or more modified nucleobases, nucleosides, or nucleotides as described below, in which case it may be referred to as "modified mRNA" or "mmRNA." As used herein, a "nucleoside" is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). As used herein, a "nucleotide" is defined as a nucleoside that includes a phosphate group.

[0410] 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). 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., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000) base pairs. Any number (e.g., all, some, or none) of core bases, nucleosides, or nucleotides can be analogs of standard substances, substituted, modified, or otherwise non-naturally occurring. In certain embodiments, all of a particular core base type can be modified. In some embodiments, all uracils or uridines are modified. An mRNA can be referred to as "fully modified" when all nucleobases, nucleosides, or nucleotides are modified (e.g., all uracils or uridines), e.g., to uracil or uridine.

[0411] In some embodiments, an mRNA as described herein may include a 5' cap structure, a chain-terminating nucleotide, optionally a Kozak sequence (also known as a Kozak consensus sequence), a stem-loop, a polyA sequence, and / or a polyadenylation signal.

[0412] The 5' cap structure or cap material is a compound comprising two nucleoside moieties joined by a linker and can be selected from naturally occurring caps, non-naturally occurring caps or cap analogs or anti-reverse cap analogs (ARCA). The cap material may include one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap may include a guanine nucleotide and a guanine (G) nucleotide methylated at the 7 position, joined by a triphosphate bond at the 5' position of the nucleotide, for example m7G(5')ppp(5')G, typically written as m7GpppG. The cap material may also be an anti-reverse cap analog. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m73'dGpppG, m27,03'GpppG, m27,03'GppppG, m27,02'GppppG, m7Gpppm7G, m73'dGpppG, m27,03'GpppG, m27,03'GppppG, and m27,02'GppppG.

[0413] mRNA may alternatively or additionally include chain-terminating nucleosides. For example, chain-terminating nucleosides may include those nucleosides that are deoxygenated at the 2' and / or 3' positions of their sugar groups. Such substances may include 3' deoxyadenosine (cordycepin), 3' deoxyuridine, 3' deoxycytosine, 3' deoxyguanosine, 3' deoxythymidine, and 2', 3' dideoxynucleosides (e.g., 2', 3' dideoxyadenosine, 2', 3' dideoxyuridine, 2', 3' dideoxycytosine, 2', 3' dideoxyguanosine, and 2', 3' dideoxythymidine). In some embodiments, chain-terminating nucleotides may be incorporated into mRNA, for example, at the 3'-end to achieve stabilization of mRNA.

[0414] mRNA may alternatively or additionally include a stem loop, such as a histone stem loop. The stem loop may include 2, 3, 4, 5, 6, 7, 8 or more nucleotide base pairs. For example, the stem loop may include 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 the untranslated region (5' untranslated region or 3' untranslated region), the coding region or the polyA sequence or the tail end. 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.

[0415] The mRNA may alternatively or additionally include a polyA sequence and / or a polyadenylation signal. The polyA sequence may be entirely or primarily comprised of adenine nucleotides or their analogs or derivatives. The polyA sequence may also include stabilizing nucleotides or analogs. For example, the polyA sequence may include deoxythymidine as a stabilizing nucleotide or analog, such as reverse (or reverse bond) deoxythymidine (dT). Details on the use of reverse dT and other stabilizing polyA sequence modifications can be found in, for example, WO2017 / 049275A2, the contents of which are incorporated herein by reference. The polyA sequence may be a tail end positioned adjacent to the 3' untranslated region of the mRNA. In some embodiments, the polyA sequence may affect nuclear export, translation, and / or stability of the mRNA.

[0416] MRNA may alternatively or additionally include microRNA binding sites. MicroRNA binding sites (or miR binding sites) can be used to regulate the mRNA expression in a variety of tissues or cell types. In an exemplary embodiment, miR binding sites are engineered into the 3'UTR sequence of mRNA to regulate (e.g., enhance) the degradation of mRNA in cells or tissues expressing homologous miR. Such regulation can be used to regulate or control the "off-target" expression of mRNA, i.e., the expression in non-desired cells or tissues in vivo. Details about using mir binding sites can be found in, for example, WO 2017 / 062513 A2, the contents of which are incorporated herein by reference.

[0417] In some embodiments, the mRNA is a bicistronic mRNA comprising a first coding region and a second coding region, wherein the coding region has an intervening sequence comprising an internal ribosome entry site (IRES) sequence that allows internal translation initiation between the first coding region and the second coding region, or an intervening sequence encoding a self-cleaving peptide (e.g., a 2A peptide). IRES sequences and 2A peptides are typically used to enhance expression of multiple proteins from the same vector. Various IRES sequences are known and available in the art and can be used, including, for example, the encephalomyocarditis virus IRES.

[0418] In some embodiments, the mRNA of the present disclosure comprises one or more modified nucleobases, nucleosides, or nucleotides (referred to as "modified mRNA" or "mmRNA"). In some embodiments, the modified mRNA may have useful properties, including enhanced stability, intracellular retention, enhanced translation, and / or lack of substantial induction of an innate immune response in cells into which the mRNA is introduced, as compared to a reference unmodified mRNA. Thus, the use of modified mRNA may enhance protein production efficiency, intracellular retention of nucleic acids, and have reduced immunogenicity.

[0419] In some embodiments, the mRNA includes one or more (e.g., 1, 2, 3, or 4) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the mRNA includes 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 can have reduced degradation in the cell into which it is introduced relative to the corresponding unmodified mRNA.

[0420] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a 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-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-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-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, 1-propynyl-pseudouridine, 5-taurylmethyl-uridine (τm5U), 1-taurylmethyl-pseudouridine, 5-taurylmethyl-2-thio-uridine (τm5s2 U), 1-taurylmethyl-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-methoxy-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 (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um) and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-arabino-uridine, 2'-F-uridine, 2'-OH-arabino-uridine, 5-(2-methoxycarbonylvinyl)uridine and 5-[3-(1-E-propenylamino)]uridine.

[0421] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine 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-hydroxymethyl-cytidine (hmC), 5-hydroxymethyl-cytidine (m ... 5C), 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-zebulin, 5-aza-2-thio-zebulin, 2-thio-zebulin, 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-cytidine (m 5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-arabino-cytidine, 2'-F-cytidine and 2'-OH-arabino-cytidine.

[0422] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides with a modified adenine include α-thio-adenosine, 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 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-glycylcarbamoyl-adenosine (g6A), N6-threonylamino Formyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio 1-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabino-adenosine, 2'-F-adenosine, 2'-OH-arabino-adenosine and N6-(19-amino-pentaoxahedronadecanyl)-adenosine.

[0423] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include α-thio-guanosine, inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methyl wyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyosine (yW), peroxy wyosine (o2yW), hydroxy wyosine (OhyW), undermodified hydroxy wyosine (OhyW*), 7-deaza-guanosine, quercetin (Q), epoxyquercetin (oQ), galactosyl-quercetin ( galQ), mannosyl-braided guanosine (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-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine ( m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 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-arabino-guanosine and 2'-F-guanosine.

[0424] In some embodiments, an mRNA of the present disclosure includes a combination of one or more of the foregoing modified nucleobases (e.g., a combination of 2, 3, or 4 of the foregoing modified nucleobases).

[0425] In some embodiments, the modified nucleobase is pseudouridine (ψ), N1-methyl pseudouridine (m1ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine or 2'-O-methyluridine. In some embodiments, the mRNA of the present disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases). In some embodiments, the modified nucleobase is N1-methylpseudouridine (m1ψ) and the mRNA of the present disclosure is completely modified with N1-methylpseudouridine (m1ψ). In some embodiments, N1-methylpseudouridine (m1ψ) represents 75-100% uracil in the mRNA. In some embodiments, N1-methylpseudouridine (m1ψ) represents 100% uracil in the mRNA.

[0426] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides with 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), 2-thio-5-methyl-cytidine. In some embodiments, mRNA of the present disclosure includes a combination of one or more aforementioned modified nucleobases (e.g., a combination of 2,3, or 4 aforementioned modified nucleobases).

[0427] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides with modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A). In some embodiments, the mRNA of the present disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3, or 4 of the aforementioned modified nucleobases).

[0428] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides with a modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methyl wyosine (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, 7-methyl-8-oxo-guanosine. In some embodiments, the mRNA of the present disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3, or 4 of the aforementioned modified nucleobases).

[0429] 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 mRNA of the present disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3, or 4 of the aforementioned modified nucleobases).

[0430] 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).

[0431] In certain embodiments, the mRNA of the present disclosure is uniformly modified for a particular modification (i.e., completely modified, modified throughout the entire sequence). For example, the mRNA may be uniformly modified with N1-methylpseudouridine (m1ψ) or 5-methyl-cytidine (m5C), meaning that all uridines or all cytosine nucleosides in the mRNA sequence are replaced with N1-methylpseudouridine (m1ψ) or 5-methyl-cytidine (m5C). Similarly, the mRNA of the present disclosure can be uniformly modified for any type of nucleoside residue present in the sequence by replacing it with a modified residue (e.g., those set forth above).

[0432] In some embodiments, the mRNA of the present disclosure may be modified in the coding region (e.g., the open reading frame encoding the polypeptide). In other embodiments, the mRNA may be modified in a region other than the coding region. For example, in some embodiments, a 5'-UTR and / or a 3'-UTR is 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.

[0433] The mmRNA of the present disclosure may include combinations of modifications to sugars, nucleobases, and / or internucleoside linkages. These combinations may include any one or more modifications described herein.

[0434] Where a single modification is listed, the nucleoside or nucleotide listed indicates that 100% of the A, U, G, or C nucleotide or nucleoside has been modified. Where percentages are listed, these represent the percentage of the specific A, U, G, or C nucleobase triphosphate out of the total amount of A, U, G, or C triphosphate 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 triphosphate is 5-aminoallyl-CTP and 75% of the cytosine is CTP; while 25% of the uracil is 5-methoxy UTP and 75% of the uracil is UTP. Where a modified UTP is not listed, then naturally occurring ATP, UTP, GTP, and / or CTP is used in 100% of the sites of those nucleotides found in the polynucleotide. In this example, all GTP and ATP remain unmodified.

[0435] The mRNA or region thereof disclosed herein may be codon optimized. Codon optimization methods are known in the art and can be used for a variety of purposes: matching codon frequencies in the host organism to ensure proper folding, biasing GC content to increase mRNA stability or reduce secondary structure, minimizing tandem repeat codons or base runs that can impair gene construction or expression, customizing transcription and translation 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 reorganizing protein domains, inserting or deleting restriction sites, modifying ribosome binding sites and mRNA degradation sites, regulating translation rates to allow multiple 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; non-limiting examples include services and / or proprietary methods from GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA). In some embodiments, the mRNA sequence is optimized using an optimization algorithm, for example, to optimize expression in mammalian cells or enhance mRNA stability.

[0436] In certain embodiments, the 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 described herein.

[0437] The mRNA disclosed herein can be produced by methods available in the art, including, but not limited to, in vitro transcription (IVT) and synthetic methods. Enzymatic (IVT), solid phase, liquid phase, combinatorial synthesis methods, cell-based synthesis, and conjugation methods can be used. In some embodiments, mRNA is produced using IVT enzymatic synthesis methods. Thus, the present disclosure also includes polynucleotides, such as DNA, constructs, and vectors, that can be used to transcribe the mRNA described herein in vitro.

[0438] Non-naturally modified nucleobases can be introduced into polynucleotides (e.g., mRNA) during or after synthesis. In certain embodiments, the modification can be at an internucleoside bond, a purine or pyrimidine base, or a sugar. In specific embodiments, the modification can be introduced at the terminus of a polynucleotide chain or at any other position in the polynucleotide chain; using chemical synthesis or using a polymerase.

[0439] Enzymatic or chemical conjugation methods can be used to conjugate polynucleotides or regions thereof to various functional moieties (e.g., targeting or delivery agents, fluorescent markers, liquids, nanoparticles, etc.). Therapeutic agents for reducing protein expression

[0440] In some embodiments, the therapeutic agent is a therapeutic agent that reduces (i.e., reduces, inhibits, downregulates) protein expression. Non-limiting examples of the types of therapeutic agents that can be used to reduce protein expression include mRNA, microRNA (miRNA), antagomir, small (short) interfering RNA (siRNA) (including short polymers (shortmer) and dicer-substrate RNA) incorporated into microRNA binding sites (miR binding sites), RNA interference (RNAi) molecules, antisense RNA, ribozymes, small hairpin RNA (shRNA), locked nucleic acid (LNA) and CRISPR / Cas9 technology.

[0441] Peptide / polypeptide therapeutics

[0442] In some embodiments, the therapeutic agent is a peptide therapeutic agent. In some embodiments, the therapeutic agent is a polypeptide therapeutic agent.

[0443] In some embodiments, the peptide or polypeptide is of natural origin, such as isolated from a natural source. In other embodiments, the peptide or polypeptide is a synthetic molecule, such as a synthetic peptide or polypeptide produced in vitro. In some embodiments, the peptide or polypeptide is a recombinant molecule. In some embodiments, the peptide or polypeptide is a chimeric molecule. In some embodiments, the peptide or polypeptide is a fusion molecule. In some embodiments, the peptide or polypeptide therapeutic of the composition is a naturally occurring peptide or polypeptide. In some embodiments, the peptide or polypeptide therapeutic of the composition is a modified form of a naturally occurring peptide or polypeptide (e.g., containing less than 3, less than 5, less than 10, less than 15, less than 20 or less than 25 amino acid substitutions, deletions or additions compared to its wild-type, naturally occurring peptide or polypeptide counterpart).

[0444] In some embodiments, in the loaded LNPs of the present disclosure, the one or more therapeutic and / or prophylactic agents are polynucleotides or polypeptides.

[0445] Other components

[0446] Lipid nanoparticles (e.g., empty LNP or loaded LNP) may include one or more components except those described in the aforementioned sections. For example, lipid nanoparticles (e.g., empty LNP or loaded LNP) may include one or more small hydrophobic molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0447] Lipid nanoparticles (e.g., empty LNP or loaded LNP) may also include one or more permeability enhancer 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).

[0448] Polymers can be included in the nanoparticle composition and / or used to encapsulate or partially encapsulate the nanoparticle composition. The polymer can be biodegradable and / or biocompatible. The polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrile, and polyacrylates.For example, the polymer may include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co- -D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl 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 (e.g., polyethylene and polypropylene), polyalkylene glycols (e.g., poly(ethylene glycol) (PEG)), polyoxyalkylenes (PEO), polyalkylene terephthalates (e.g., poly(ethylene terephthalate)) , polyvinyl alcohol (PVA), polyvinyl ether, polyvinyl ester (e.g., poly(vinyl acetate)), polyvinyl halide (e.g., poly(vinyl chloride) (PVC)), polyvinyl pyrrolidone (PVP), polysiloxane, polystyrene (PS), polyurethane, derivatized cellulose (e.g., alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose), acrylic polymers (e.g., poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly((meth)acrylic acid) and mixtures thereof), polydioxanone and copolymers thereof, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, polyoxyamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol.

[0449] Surface-altering agents can 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., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin beta 4, dornase alfa, neltenexine, and erdosteine), and DNA enzymes (e.g., rhDNase). The surface-altering agent can be disposed within the nanoparticle and / or on the surface of the lipid nanoparticle (eg, empty LNP or loaded LNP) (eg, by coating, adsorption, covalent attachment, or other methods).

[0450] In another embodiment, lipid nanoparticle (for example empty LNP or load LNP) can also comprise one or more functionalized lipids.For example, lipid can be through alkyne group functionalization, and described alkyne group can experience cycloaddition reaction when being exposed to azide under suitable reaction conditions.Particularly, lipid bilayer can be through one or more group functionalizations that can be used for promoting membrane penetration, cell recognition or imaging by this mode.The surface of lipid nanoparticle (for example empty LNP or load LNP) can also be puted together with one or more applicable antibodies.Functional group and the conjugate that can be used for targeted cell delivery, imaging and membrane penetration are well known in the art.

[0451] In some embodiments, the lipid nanoparticle (for example empty LNP or load LNP) can be used for pharmaceutical compositions. For example, the lipid nanoparticle (for example empty LNP or load LNP) can comprise one or more pharmaceutically acceptable excipients or attached components, such as but not limited to one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrating agents, fillers, glidants, liquid vehicles, adhesives, surfactants, isotonic agents, thickening or emulsifying agents, buffer agents, lubricants, oils, antiseptics and other materials. For example, the excipient of wax, butter, coloring agent, coating agent, flavorings and fragrances can also be included.

[0452] 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 potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate. A non-limiting list consists of sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0453] Surfactants and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [ 20], polyoxyethylene sorbitan[ 60], polyoxyethylene sorbitan monooleate[ 80], Sorbitan monopalmitate[ 40], Sorbitan monostearate[ 60], Sorbitan tristearate[ 65], glycerol monooleate, sorbitan monooleate[ 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [ 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. ), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [ 30]), poly (vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, F 68, 188. Cetrimide bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium and / or a combination thereof.

[0454] Binders can 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., gum arabic, sodium alginate, Irish moss extract, panwar gum, ghatti gum, mucilage of isapol husk, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate and larch arabinogalactan); alginate; polyethylene oxide; polyethylene glycol; inorganic calcium salt; silicic acid; polymethacrylate; wax; water; alcohol; and combinations thereof, or any other suitable binder.

[0455] The example of antiseptic may include but is not limited to antioxidant, chelating agent, antimicrobial preservative, antifungal preservative, alcohol preservative, acidic preservative and / or other preservatives. The example of antioxidant includes but is not limited to alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite and / or sodium sulfite. The example of chelating agent includes ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid and / or trisodium edetate. The example of antimicrobial preservative includes but is not limited to benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerol, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol and / or thimerosal. The example of antifungal preservative includes but is not limited to butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate and / or sorbic acid. The example of alcohol preservative includes but is not limited to ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopheryl acetate, deferoxamine 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 Methyl paraben, 115. II. NEOLONE TM KATHON TM and / or

[0456] Examples of buffers include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, calcium glucoheptonate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, dibasic calcium phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, a potassium mixture, dibasic potassium phosphate, monobasic potassium phosphate, a potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, monobasic sodium phosphate, a sodium phosphate mixture, tromethamine, an amino-sulfonate buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and / or combinations thereof. The lubricant may be selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0457] Examples of oils include, but are not limited to, almond, apricot, avocado, carnauba, bergamot, black currant seed, borage, juniper, chamomile, canola, coriander, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, linseed, vanillyl alcohol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, macadamia nut, lavender flower, lavender, lemon, litsea cubeba, macadamia nut, mallow, mango stone, meadowfoam seed, mink, nutmeg, olive , orange, orange snapper, palm, palm kernel, peach kernel, peanut, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, camellia, peppermint, sea buckthorn, sesame, shea butter, silicone, soy, sunflower, tea tree, thistle, tsuba, vetiver, walnut and wheat germ oils and butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, simethicone, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil and / or combinations thereof.

[0458] preparation

[0459] The lipid nanoparticle (for example empty LNP or load LNP) can comprise lipid component and one or more additional components, for example therapeutic agent and / or preventative.The lipid nanoparticle (for example empty LNP or load LNP) can be designed for one or more specific applications or targets.The key element of lipid nanoparticle (for example empty LNP or load LNP) can be based on specific applications or target, and / or select based on effect, toxicity, expense, ease of use, availability or other characteristics of one or more key elements.Similarly, the specific formulation of nanoparticle compositions can be selected for specific applications or target according to effect and the toxicity of for example specific key combination.

[0460] The lipid component of the nanoparticle composition can include, for example, lipids according to Formula (I-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 (e.g., unsaturated lipids such as DOPE or DSPC), PEG lipids, and structural lipids. The elements of the lipid component can be provided in specific fractions.

[0461] In some embodiments, the lipid component of the nanoparticle composition includes 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, PEG lipids, and structural lipids. In certain embodiments, the lipid component of the nanoparticle composition includes about 30 mol% to about 60 mol% of a compound of Formula (I-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% phospholipids, about 18.5 mol% to about 48.5 mol% structural lipids, and about 0 mol% to about 10 mol% PEG lipids, with the proviso 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 (I-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-al), (A-a2), (A-a3), (Ab), (A-bl), (A-b2), (A-b3), (Ac), or (Bc), about 5 mol% to about 25 mol% phospholipids, about 30 mol% to about 40 mol% structural lipids, and about 0 mol% to about 10 mol% PEG lipids. In a specific embodiment, the lipid component comprises about 50 mol% of the compound, about 10 mol% phospholipids, about 38.5 mol% structural lipids, and about 1.5 mol% PEG lipids. In another specific embodiment, the lipid component includes about 40 mol% of the compound, about 20 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In some embodiments, the phospholipids may be DOPE or DSPC. In other embodiments, the PEG lipids may be PEG-1 or PEG 2k -DMG and / or the structural lipid may be cholesterol.

[0462] In some embodiments, 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.

[0463] 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 structured lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents.

[0464] In some embodiments, the empty LNP or loaded LNP comprises a compound of Formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc) in an amount of about 40% to about 60%.

[0465] In some embodiments, the empty LNP or loaded LNP comprises phospholipids in an amount of about 0% to about 20%.For example, in some embodiments, the empty LNP or loaded LNP comprises DSPC in an amount of about 0% to about 20%.

[0466] In some embodiments, the empty LNP or loaded LNP comprises structural lipids in an amount of about 30% to about 50%.For example, in some embodiments, the empty LNP or loaded LNP comprises cholesterol in an amount of about 30% to about 50%.

[0467] In some embodiments, the empty LNP or loaded LNP comprises PEG lipids in an amount of about 0% to about 5%. For example, in some embodiments, the empty LNP or loaded LNP comprises PEG-1 or PEG 2k -DMG.

[0468] In some embodiments, the empty LNP or loaded LNP comprises 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% phospholipids, about 30 mol% to about 50 mol% structural lipids, and about 0 mol% to about 5 mol% PEG lipids.

[0469] In some embodiments, the empty LNP or loaded LNP comprises 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. 2k In some embodiments, the empty LNP or loaded LNP comprises 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.

[0470] In some embodiments, the empty or loaded LNP comprises about 40 mol% to about 60 mol% of a compound of Formula (I-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, the empty or loaded LNP comprises 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-1.

[0471] In some embodiments, the empty LNP or loaded LNP comprises a compound of formula (I-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, the empty LNP or loaded LNP comprises 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.

[0472] In some embodiments, the empty LNP or loaded LNP comprises a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), a phospholipid, a structural lipid, and a PEG lipid, wherein the structural lipid is cholesterol and the PEG lipid is PEG 2k In some embodiments, the empty LNP or loaded LNP comprises 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 2k -DMG.

[0473] In some embodiments, the empty or loaded LNP comprises a compound of Formula (I-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, the empty or loaded LNP comprises 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.

[0474] In some embodiments, the empty LNP or loaded LNP comprises a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), a phospholipid, a structural lipid, and a PEG lipid, wherein the phospholipid is DSPC and the PEG lipid is PEG 2k In some embodiments, the empty LNP or loaded LNP comprises 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 2k -DMG.

[0475] In some embodiments, the empty or loaded LNP comprises a compound of Formula (I-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, the empty or loaded LNP comprises 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.

[0476] In some embodiments, the empty LNP or loaded LNP comprises a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), a phospholipid, a structural lipid, and a PEG lipid, wherein the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG. 2k In some embodiments, the empty LNP or loaded LNP comprises 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 2k -DMG.

[0477] In some embodiments, the empty LNP or loaded LNP comprises 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, the empty LNP or loaded LNP comprises 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 2k -DMG.

[0478] In some embodiments, the empty LNP or loaded LNP comprises a compound of formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), a phospholipid, a structural lipid, and a PEG lipid, wherein the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG-1.

[0479] In some embodiments, empty LNP or load LNP comprises compound of formula (Ac), phospholipid, structural lipid and PEG lipid, wherein said phospholipid is DSPC, said structural lipid is cholesterol, and said PEG lipid is PEG-1.In some embodiments, empty LNP or load LNP comprises compound of table 1, phospholipid, structural lipid and PEG lipid, wherein said phospholipid is DSPC, said structural lipid is cholesterol, and said PEG lipid is PEG-1.

[0480] Lipid nanoparticles (e.g., empty LNP or load LNP) can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed to deliver therapeutic and / or preventative agents, such as RNA, to specific cells, tissues, organs or systems or their groups in mammalian health. The physiochemical properties of lipid nanoparticles (e.g., empty LNP or load LNP) can change to increase the selectivity for specific body targets. For example, granularity can be regulated based on the fenestration size of different organs. The therapeutic and / or preventative agents included in the nanoparticle compositions can also be selected based on one or more required delivery targets. For example, therapeutic and / or preventative agents can be selected for specific indications, illness, disease or disease and / or for delivery (e.g., localization or specific delivery) to specific cells, tissues, organs or systems or their groups. In certain embodiments, nanoparticle compositions can include an mRNA encoding a target polypeptide that can be translated in the cell to produce the target polypeptide. This composition can be designed to be delivered specifically to a specific organ. In some embodiments, composition can be designed to be delivered specifically to the mammalian liver.

[0481] The amount of the therapeutic agent and / or prophylactic in the nanoparticle composition can depend on the size, composition, required target and / or application or other characteristics of the nanoparticle composition, and depends on the characteristic of the therapeutic agent and / or prophylactic.For example, the amount of the RNA that can be used for the nanoparticle composition can depend on the size, sequence and other characteristics of the RNA.The relative amount of the therapeutic agent and / or prophylactic and other key elements (for example lipid) in the nanoparticle composition also can change.In some embodiments, the wt / wt ratio of the liquid component in the nanoparticle composition and the therapeutic agent and / or prophylactic can be about 5:1 to about 60:1, for example 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 wt / wt ratio of the liquid component to the therapeutic and / or prophylactic agent can be about 10: 1 to about 40: 1. In certain embodiments, the wt / wt ratio is about 20:1.

[0482] The amount of therapeutic and / or prophylactic agent in a nanoparticle composition can be measured, for example, using absorption spectroscopy (eg, UV-visible spectroscopy).

[0483] In some embodiments, the nanoparticle composition includes one or more RNAs, and the one or more RNAs, lipids, and amounts thereof can be selected to provide a specific N:P ratio. The N:P ratio of a composition refers to the molar ratio of the number of nitrogen atoms in the one or more lipids to the number of phosphate groups in the RNA. Generally speaking, a lower N:P ratio is preferred. The one or more RNAs, lipids, and amounts thereof can be selected to provide an N:P ratio of about 2:1 to about 30:1, such as 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.

[0484] Physical properties

[0485] The feature of lipid nano particle (for example empty LNP or load LNP) can depend on its component.For example, comprise that cholesterol can have the feature different from the lipid nano particle (for example empty LNP or load LNP) that comprises different structure lipids as the lipid nano particle (for example empty LNP or load LNP) of structural lipid.Similarly, the feature of lipid nano particle (for example empty LNP or load LNP) can depend on the absolute or relative amount of its component.For example, comprise that the lipid nano particle (for example empty LNP or load LNP) of the phospholipid of higher molar fraction can have the feature different from the lipid nano particle (for example empty LNP or load LNP) that comprises the phospholipid of lower molar fraction.Feature also can change according to preparation method and condition of nanoparticle compositions.

[0486] Lipid nanoparticles (e.g., empty LNP or loaded LNP) can be characterized by several methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to check the form and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometric determination (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to measure particle size. The instrument of, for example, Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure the various characteristics of the nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0487] The average size of lipid nanoparticles (e.g., empty LNP or loaded LNP) can be between tens of nm and hundreds of nm, for example, as measured by dynamic light scattering (DLS). For example, the average size can be from about 40 nm to about 150 nm, for example, 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 the lipid nanoparticles (e.g., empty LNPs or loaded 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 lipid nanoparticles (e.g., empty LNP or loaded LNP) can be about 70nm to about 130nm or about 70nm to about 100nm. In a specific embodiment, the average size can be about 80nm. In other embodiments, the average size can be about 100nm. In other embodiments, the average size can be about 120nm.

[0488] In some embodiments, the lipid nanoparticle (for example empty LNP or load LNP) can be relatively homogeneous.Polydispersity index can be used for indicating the homogeneity of nanoparticle compositions, for example the size distribution of lipid nanoparticle (for example empty LNP or load LNP).Little (for example less than 0.3) polydispersity index indicates narrow size distribution usually.Lipid nanoparticle (for example empty LNP or load LNP) can have approximately 0 to approximately 0.25, for example 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 polydispersity index. In some embodiments, the lipid nanoparticles (eg, empty LNPs or loaded LNPs) can have a polydispersity index of about 0.10 to about 0.20.

[0489] The zeta potential of lipid nanoparticles (e.g., empty LNP or loaded LNP) can be used to indicate the zeta potential of a composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Lipid nanoparticles (e.g., empty LNP or loaded LNP) with relatively low charge (positive or negative) are typically desired because higher charge species can interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0490] The encapsulation efficiency of therapeutic and / or prophylactic agent describes the amount of therapeutic and / or prophylactic agent that is encapsulated or otherwise associated with lipid nanoparticles (e.g., empty LNP or loaded LNP) after preparation relative to the initial amount provided. High encapsulation efficiency is desirable (e.g., close to 100%). 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 LNP or loaded LNP) before and after the crushing of lipid nanoparticles (e.g., empty LNP or loaded LNP) with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in solution. With respect to lipid nanoparticles described herein (e.g., empty LNP or loaded LNP), 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%.

[0491] Pharmaceutical composition

[0492] Lipid nanoparticles (e.g., empty LNP or loaded LNP) can be formulated as pharmaceutical compositions in whole or in part. Pharmaceutical compositions can include one or more lipid nanoparticles (e.g., empty LNP or loaded LNP). In one embodiment, a pharmaceutical composition comprises a colony of lipid nanoparticles (e.g., empty LNP or loaded LNP). For example, a pharmaceutical composition can include one or more lipid nanoparticles (e.g., empty LNP or loaded LNP) comprising one or more different therapeutic agents and / or prophylactics. The pharmaceutical composition can also include one or more pharmaceutically acceptable excipients or adjunct ingredients, such as those described herein. General guidelines for the preparation and manufacture of pharmaceutical compositions and medicaments can be found, for example, in Remington's The Science and Practice of Pharmacy, 21st edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and adjunct ingredients can be used for any pharmaceutical composition, unless any conventional excipient or adjunct ingredient is incompatible with one or more components of the nanoparticle composition. An excipient or adjunct ingredient may be incompatible with a component of the lipid nanoparticle (e.g., empty LNP or loaded LNP) if its combination with the component may result in any undesirable biological effect or otherwise cause a deleterious effect.

[0493] In some embodiments, one or more excipients or accessory ingredients may constitute more than 50% of the total mass or volume of the pharmaceutical composition including the nanoparticle composition. For example, the one or more excipients or accessory ingredients may constitute 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical composition. In some embodiments, the 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 Pharmacopoeia.

[0494] The relative amounts of one or more lipid nanoparticles (e.g., empty LNP or loaded LNP), one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary according to the identity, size, and / or condition of the subject being treated and further according to the route of administration of the composition. For example, a pharmaceutical composition can include between 0.1% and 100% (wt / wt) of one or more lipid nanoparticles (e.g., empty LNP or loaded LNP).

[0495] In certain embodiments, the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and / or shipment (e.g., stored at 4°C or lower, e.g., at a temperature between about -150°C and about 0°C or between about -80°C and 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). For example ... , (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), and (Bc) are solutions that are refrigerated for storage and / or shipment at, for example, about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C. In certain embodiments, the present disclosure also relates to a method of preparing a pharmaceutical composition comprising lipid nanoparticles (e.g., empty LNPs or loaded LNPs) and / or a compound of any one of formula (I-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) are stored at 4°C or lower, e.g., between about -150°C and about 0°C or between about -80°C and 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 to increase the lipid nanoparticle (e.g., empty LN) In some embodiments, the present invention provides a method for improving the stability of lipid nanoparticles (such as empty LNP or loaded LNP) and / or pharmaceutical compositions. For example, lipid nanoparticles disclosed herein (such as empty LNP or loaded LNP) and / or pharmaceutical compositions are, for example, stabilized at a temperature of 4 ° C or lower (for example, between about 4 ° C and -20 ° C) for at least 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 formulation is stabilized for at least 4 weeks at about 4 ° C.In certain embodiments, pharmaceutical compositions of the present disclosure comprise lipid nanoparticles disclosed herein (e.g., empty LNP or loaded LNP) 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, pharmaceutical compositions of the present disclosure have a pH value of about 7 to 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 between 7.5 and 8, or between 7 and 7.8). For example, pharmaceutical compositions of the present disclosure comprise lipid nanoparticles disclosed herein (e.g., empty LNP or loaded LNP), Tris, saline, and sucrose, and have a pH of about 7.5-8, which is suitable for storage and / or shipment at, for example, about -20°C. For example, the pharmaceutical compositions of the present disclosure comprise lipid nanoparticles disclosed herein (e.g., empty LNP or loaded LNP) and PBS and have a pH of about 7-7.8, which is suitable for storage and / or shipment at, for example, about 4° C. or below. “Stability,” “stabilization,” and “stable” in the context of the present disclosure refer to the lipid nanoparticles (e.g., empty LNP or loaded LNP) and / or pharmaceutical compositions disclosed herein resisting chemical or physical changes (e.g., degradation, particle size changes, aggregation, encapsulation changes, etc.) under given manufacturing, preparation, transportation, storage, and / or use conditions, for example, when stresses such as shear forces, freeze / thaw stress, etc. are applied.

[0496] In some embodiments, the pharmaceutical compositions of the present disclosure comprise empty or loaded LNPs, a cryoprotectant, a buffer, or a combination thereof.

[0497] In some embodiments, the cryoprotectant comprises one or more cryoprotectants, and each of the one or more cryoprotectants is independently a polyol (e.g., a diol or triol, such as propylene glycol (i.e., 1,2-propylene glycol), 1,3-propylene glycol, 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-pyridyl)-1-propanesulfonate), an osmotic agent (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 2k-DMG, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K 15), pentaerythritol propoxylate or polypropylene glycol P400), an organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), a sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol, inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate or D-(+)-glucose monohydrate) or a salt (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate or any hydrate thereof) or any combination thereof. In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the cryoprotectant and / or excipient is sucrose. In some embodiments, the cryoprotectant comprises sodium acetate. In some embodiments, the cryoprotectant and / or excipient is sodium acetate. In some embodiments, the cryoprotectant comprises sucrose and sodium acetate.

[0498] In some embodiments, the buffer is selected from the group consisting of acetate buffer, citrate buffer, phosphate buffer, tris buffer, and combinations thereof.

[0499] The pharmaceutical composition of lipid nanoparticle (for example empty LNP or load LNP) and / or comprising one or more lipid nanoparticles (for example empty LNP or load LNP) can be applied to any patient or experimenter, including those patients or experimenter who can benefit from the therapeutic effect provided by delivering therapeutic agent and / or preventive to one or more specific cells, tissues, organs or systems or their groups. Although the description of the pharmaceutical composition for lipid nanoparticle (for example empty LNP or load LNP) and comprising lipid nanoparticle (for example empty LNP or load LNP) provided herein relates in principle to compositions that are suitable for being applied to the mankind, it will be understood by those skilled in the art that such compositions are suitable for being applied to any other mammals generally. It should be fully understood that the compositions that are suitable for being applied to the mankind can be modified so that the compositions are suitable for being applied to a variety of animals, and ordinary skilled veterinary pharmacologists only need routine (if present) experiment to design and / or perform such modifications. The experimenter of the expected application of the compositions includes but is not limited to the mankind, other primates and other mammals, including commercially relevant mammals, for example cattle, pigs, horses, sheep, cats, dogs, mice and / or rats. Theme lipid nanoparticles can also be used for in vitro and ex vivo purposes.

[0500] Pharmaceutical compositions comprising one or more lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be prepared by any method known or hereafter developed in the field of pharmacology. In general, such preparation methods include associating the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if desired or necessary, dividing, shaping, and / or packaging the product into desired single or multiple dose units.

[0501] Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is an individual amount of a pharmaceutical composition (e.g., a nanoparticle composition) containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject, and / or a suitable fraction of such a dose, such as one-half or one-third of such a dose.

[0502] Pharmaceutical compositions can be prepared in various forms suitable for a variety of administration routes and methods. For example, pharmaceutical compositions can be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable 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.

[0503] 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, the liquid dosage form may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. In addition to the inert diluent, the oral composition may also include additional therapeutic and / or prophylactic agents, additional agents (such as wetting agents, emulsifying and suspending agents, sweeteners, flavorings and / or aromatics). In certain embodiments for parenteral administration, the composition is combined with, for example, Solubilizer mixtures of alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers and / or combinations thereof.

[0504] Injectable preparations (e.g., sterile injectable aqueous or oily suspensions) can be prepared using suitable dispersants, wetting agents, and / or suspending agents according to known techniques. Sterile injectable preparations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenteral acceptable diluents and / or solvents, such 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 mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used for the preparation of injectables.

[0505] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0506] In order to prolong the effect of the active ingredient, it is usually necessary to slow down the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous materials with poor water solubility. The absorption rate of the drug depends on its dissolution rate, which in turn can depend on the crystal size and crystalline form. Alternatively, delayed absorption of parenteral drug forms is achieved by dissolving or suspending the drug in an oil vehicle. Injectable reservoir forms are prepared by forming a microencapsulation matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the properties of the specific polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Reservoir injectable formulations are prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0507] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the composition with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

[0508] Solid dosage forms for oral administration include capsules, tablets, pills, films, powders and granules. In such solid dosage forms, active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and / or filler or extender (for example, starch, lactose, sucrose, glucose, mannitol and silicic acid), binder (for example, carboxymethyl cellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic), wetting agent (for example glycerol), disintegrant (for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, specific silicate and sodium carbonate), solution delay agent (for example paraffin), absorption accelerator (for example quaternary ammonium compound), wetting agent (for example cetyl alcohol and glycerol monostearate), absorbent (for example kaolin and bentonite, silicate) and lubricant (for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate) and their mixture. In the case of capsules, tablets and pills, dosage form can include buffer.

[0509] Solid compositions of similar types can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose (lactose / milk sugar) and high molecular weight polyethylene glycol. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as other coatings well-known in the field of enteric coatings and pharmaceutical formulations. It can optionally include an opacifier and can have the following composition, which allows it to optionally only or preferentially release the active ingredient in a specific part of the intestinal tract in a delayed manner. The example of spendable embedded composition includes polymeric substances and wax. Solid compositions of similar types can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose and high molecular weight polyethylene glycol.

[0510] The surface and / or transdermal dosage form for compositions can comprise ointment, paste, cream, lotion, gel, powder, solution, spray, inhalant and / or patch.In general, active component is mixed with desirable pharmaceutically acceptable excipient and / or any required preservative and / or buffer under aseptic condition.In addition, the disclosure expects the use of transdermal patch, and the transdermal patch generally has the additional advantage that the control of compound is provided to health and sends.Such dosage form can be prepared, for example, by making the compound dissolve and / or be dispersed in appropriate medium.Or or in addition, speed can be controlled by providing rate control membrane and / or by dispersing the compound in polymer matrix and / or gel.

[0511] Suitable devices for delivering intradermal pharmaceutical compositions as described herein include short needle devices. Intradermal compositions can be applied by limiting the effective penetration length of the needle into the skin. It is suitable to deliver a liquid composition to the dermis via a liquid jet injector and / or via a needle that pierces the stratum corneum and produces a jet that reaches the dermis. It is suitable to use compressed gas to accelerate the ballistic powder / particle delivery device that the vaccine in powder form reaches the dermis through the outer layer of the skin. Alternatively or in addition, conventional syringes can be used in the classic Mantoux method (mantoux method) of intradermal administration.

[0512] Formulations suitable for topical administration include, but are not limited to, liquid and / or semi-liquid formulations, such as 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, for example, contain from about 1% to about 10% (wt / wt) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may also contain one or more additional ingredients as described herein.

[0513] Pharmaceutical compositions can be prepared, packaged and / or sold in the form of formulations suitable for pulmonary administration via the oral cavity. Such formulations may include dry particles comprising an active ingredient. Such compositions are conveniently in dry powder form to be administered using a device comprising a dry powder reservoir (to which a propellant stream may be directed to disperse the powder) and / or using a self-propelling solvent / powder dispensing container (e.g., a device comprising an active ingredient dissolved and / or suspended in a low-boiling-point propellant in a sealed container). Dry powder compositions may include a solid fine powder diluent, such as sugar, and are conveniently provided in unit dosage form.

[0514] Low-boiling-point propellants typically include liquid propellants having a boiling point below 65°F at atmospheric pressure. Typically, the propellant may comprise 50% to 99.9% (wt / wt) of the composition, and the active ingredient may comprise 0.1% to 20% (wt / wt) of the composition. The propellant may also contain additional ingredients, such as liquid nonionic and / or solid anionic surfactants and / or solid diluents (which may have a particle size of the same order as the particles comprising the active ingredient).

[0515] The pharmaceutical composition that is formulated for pulmonary delivery can provide the active ingredient in the form of small droplets of solution and / or suspension. Such preparations can be prepared, packaged and / or sold as aqueous and / or dilute alcohol solutions and / or suspensions that are optionally sterile and comprise active ingredient, and can be conveniently used any spray and / or atomizing device to apply. Such preparations can also include one or more additional ingredients, including but not limited to flavorings (such as saccharin sodium), volatile oils, buffers, surfactants and / or preservatives (such as methyl hydroxybenzoate). The small droplets provided by this route of administration can have an average diameter in the range of about 100 nm to about 200 nm.

[0516] The formulations described herein as being useful for pulmonary delivery can be used to deliver pharmaceutical compositions intranasally. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle size of about 0.2 μm to 500 μm. This formulation is administered by sniffing, i.e., by rapid inhalation through the nasal passages from a powder container held in close proximity to the nose.

[0517] Preparations suitable for nasal administration may, for example, contain as little as about 0.1% (wt / wt) and as much as 100% (wt / wt) of active ingredient, and may contain one or more additional ingredients as described herein. Pharmaceutical compositions can be prepared, packaged, and / or sold in the form of preparations suitable for buccal administration. Such preparations may, for example, be in the form of tablets and / or lozenges prepared using conventional methods and may contain, for example, 0.1% to 20% (wt / wt) active ingredient, the remainder comprising orally soluble and / or degradable compositions and optionally comprising one or more additional ingredients as described herein. Alternatively, preparations suitable for buccal administration may include powders and / or aerosolized and / or atomized solutions and / or suspensions comprising the active ingredient. When dispersed, such powdered, aerosolized, and / or aerosolized preparations may have an average particle and / or droplet size in the range of about 0.1 nm to about 200 nm, and may also include one or more of any additional ingredients as described herein.

[0518] The pharmaceutical composition can be prepared, packaged and / or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops comprising, for example, a 0.1 / 1.0% (wt / wt) solution and / or suspension of the active ingredient in an aqueous or oily liquid excipient. Such drops may also contain a buffer, salts and / or one or more other additional ingredients described herein. Other available ophthalmic formulations include those comprising an active ingredient in microcrystalline form and / or in a liposomal formulation. Ear drops and / or eye drops are contemplated within the scope of the present disclosure.

[0519] mRNA therapy

[0520] As a drug modality, mRNA has the potential for delivering secretory proteins and intracellular proteins and transmembrane proteins. As a drug modality, mRNA has the potential for delivering transmembrane and intracellular proteins (that is, standard biologics cannot cross the cell membrane and cannot approach the target when delivered in protein form). A major challenge in realizing the therapy based on mRNA is the identification of the best delivery vehicle. Due to its large size, chemical instability and potential immunogenicity, mRNA needs to provide protection from endonucleases and exonucleases, and masking cargo to avoid the delivery vehicle of immune markers. In this regard, lipid nanoparticles (LNPs) have been identified as the main option.

[0521] The key performance criteria about lipid nanoparticle delivery systems is to maximize cellular uptake and enable mRNA to be effectively released from endosome. In one embodiment, the theme LNP comprising novel lipids disclosed herein demonstrates an improvement in at least one of cellular uptake and endosome release. Simultaneously, LNP must provide a stable drug product and can safely be administered with treatment-related levels. LNP is a multi-component system typically composed of amino lipids, phospholipids, cholesterol and PEG-lipids. The effective delivery of nucleic acid goods and the aspect of particle stability require every component. It is believed that the key component driving cellular uptake, endosome breakaway and tolerance is amino lipids. Cholesterol and PEG-lipids promote the stability of the drug product in vivo and during shelf life, and phospholipids provide the extra fusogenicity of LNP, therefore helping to drive endosome breakaway and make available nucleic acid in the cytosol of the cell.

[0522] Over the past two decades, several amino lipid series have been developed for oligonucleotide delivery, including amino lipid MC3 (DLin-MC3-DMA). LNP based on MC3 has been shown to effectively deliver mRNA. Such LNP is rapidly conditioned by apolipoprotein E (ApoE) when delivered intravenously, which enables cellular uptake by low-density lipoprotein receptor (LDLr). However, it is still worried that the long tissue half-life of MC3 may promote the adverse side effects that hinder it from being used for long-term therapy. In addition, a large amount of literature evidence shows that the long-term administration of lipid nanoparticles can produce several toxic side effects, including complement activation-related pseudoallergy (CARPA) and liver damage. Therefore, in order to release the potentiality of the therapy based on mRNA and other nucleic acids, nucleotides or peptides for the mankind, it is necessary to have a class of LNPs with increased delivery efficiency together with the metabolism and toxicity profile that will enable long-term administration in the mankind.

[0523] The ability to treat a variety of diseases requires the adaptability of long-term, safe administration at varying dose levels. Through systematic optimization of amino lipid structures, compounds of the present disclosure have been identified as compounds that balance chemical stability, improved delivery efficiency due to improved endosome detachment, rapid in vivo metabolism, and a pure toxicity profile. The combination of these features provides drug candidates that can be administered long-term without activating the immune system. Initial rodent screening resulted in the identification of major lipids with good delivery efficiency and pharmacokinetics. The lead LNP was further profiled for delivery efficiency in non-human primates after single and repeated administration. Finally, the optimized LNP was evaluated in a one-month repeated dose toxicity study in rats and non-human primates. Without wishing to be bound by theory, the novel ionizable lipids of the present disclosure have improved cellular delivery, improved protein expression, and improved biodegradability characteristics, which can result in more than 2-fold, 5-fold, 10-fold, 15-fold, or 20-fold increases in mRNA expression in cells compared to LNPs lacking the disclosed lipids. In another embodiment, LNPs comprising lipids of the present disclosure can result in specific (e.g., preferential) delivery to one or more specific cell types as compared to other cell types, thereby resulting in more than a 2-fold, 5-fold, 10-fold, 15-fold, or 20-fold increase in mRNA expression in specific cells or tissues as compared to LNPs lacking lipids of the present disclosure. These technological improvements allow for the safe and effective use of mRNA-based therapies in acute and chronic diseases.

[0524] method

[0525] In some aspects, the present disclosure provides a method for delivering a therapeutic agent and / or a preventive to a cell (e.g., a mammalian cell). This method includes contacting the cell with a load LNP or a pharmaceutical composition of the present disclosure, whereby the therapeutic agent and / or the preventive are delivered 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 the subject a compound comprising formula (I-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 preventive lipid nanoparticles, whereby the therapeutic agent and / or the preventive are delivered to the cell.

[0526] In some embodiments, the present disclosure provides a method of delivering a therapeutic and / or prophylactic agent to cells in a subject, wherein the method comprises administering to the subject a compound comprising Formula (I-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 -DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method for delivering a therapeutic and / or prophylactic agent to cells in a subject, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (Ac), DSPC, cholesterol and PEG 2k - the step of preparing lipid nanoparticles of DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides and nucleic acids (eg RNA).

[0527] In some embodiments, the present disclosure provides a method of delivering a therapeutic and / or prophylactic agent to cells in a subject, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (I-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-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method of delivering a therapeutic and / or prophylactic agent to cells in a subject, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (Ac), DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0528] In some aspects, the present disclosure provides a method for delivering (e.g., specifically delivering) a therapeutic and / or preventative to a mammalian organ or tissue (e.g., liver, kidney, spleen, or lung). This method includes contacting the cell with a loaded LNP or pharmaceutical composition of the present disclosure, thereby delivering the therapeutic and / or preventative to the target organ or tissue. In some embodiments, the method includes administering to the subject a lipid nanoparticle comprising a compound of formula (I-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 preventative agents, thereby delivering the therapeutic and / or preventative to the target organ or tissue.

[0529] In some embodiments, the present disclosure provides a method for specifically delivering a therapeutic and / or prophylactic agent to an organ of a subject, wherein the method comprises administering to the subject a compound comprising 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 -DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method for delivering a therapeutic and / or prophylactic agent specifically to an organ of a subject, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (Ac), DSPC, cholesterol and PEG 2k - the step of preparing lipid nanoparticles of DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides and nucleic acids (eg RNA).

[0530] In some embodiments, the present disclosure provides a method for specifically delivering a therapeutic and / or prophylactic agent to an organ of a subject, wherein the method comprises administering to the subject lipid nanoparticles comprising a compound of Formula (I-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-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method for specifically delivering a therapeutic and / or prophylactic agent to an organ of a subject, wherein the method comprises administering to the subject lipid nanoparticles comprising a compound of formula (Ac), DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0531] In some aspects, the present disclosure provides a method for enhancing delivery of a therapeutic and / or preventive agent (e.g., mRNA) to a target tissue (e.g., liver, spleen, or lung). This method includes contacting the cell with a loaded LNP or pharmaceutical composition of the present disclosure, thereby delivering the therapeutic and / or preventive agent to the target tissue (e.g., liver, kidney, spleen, or lung). In some embodiments, the method includes the step of administering to the subject lipid nanoparticles comprising a compound of Formula (I-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 agent to the target tissue (e.g., liver, kidney, spleen, or lung).

[0532] In some embodiments, the present disclosure provides a method for enhancing delivery of a therapeutic and / or prophylactic agent to a target tissue, wherein the method comprises administering to the subject a compound comprising 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 -DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method for enhancing delivery of a therapeutic and / or prophylactic agent to a target tissue, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (Ac), DSPC, cholesterol and PEG 2k - the step of preparing lipid nanoparticles of DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides and nucleic acids (eg RNA).

[0533] In some embodiments, the present disclosure provides a method for enhanced delivery of a therapeutic and / or prophylactic agent to a target tissue, wherein the method comprises administering to the subject lipid nanoparticles comprising a compound of formula (I-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-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method for enhanced delivery of a therapeutic and / or prophylactic agent to a target tissue, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (Ac), DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0534] In some aspects, the disclosure provides a method for producing a target polypeptide in a cell (e.g., a mammalian cell). This method includes contacting the cell with a load LNP or a pharmaceutical composition of the present disclosure, wherein the load LNP or pharmaceutical composition include mRNA, and the mRNA can be translated in the cell to produce the polypeptide. In some embodiments, the cell is in an experimenter and the contact includes administering the cell to the experimenter. In some embodiments, the method includes administering to the experimenter a lipid nanoparticle comprising a compound, phospholipid, structural lipid, PEG lipid, and mRNA of formula (I-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 the mRNA can be translated in the cell to produce the polypeptide.

[0535] In some embodiments, the present disclosure provides a method of producing a polypeptide of interest in a cell, wherein the method comprises administering to the subject a compound comprising 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 For example, in some embodiments, the present disclosure provides a method for producing a target polypeptide in a cell, wherein the method comprises administering to the subject a compound of Table 1, DSPC, cholesterol, and PEG. 2k For example, in some embodiments, the present disclosure provides a method for producing a target polypeptide in a cell, wherein the method comprises administering to the subject a compound of formula (Ac), DSPC, cholesterol, and PEG. 2k -DMG and mRNA lipid nanoparticle steps.

[0536] In some embodiments, the present disclosure provides a method for producing a polypeptide of interest in a cell, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (I-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-1, and mRNA. For example, in some embodiments, the present disclosure provides a method for producing a polypeptide of interest in a cell, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (Ac), DSPC, cholesterol, and PEG-1, and mRNA. For example, in some embodiments, the present disclosure provides a method for producing a polypeptide of interest in a cell, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of Table 1, DSPC, cholesterol, and PEG-1, and mRNA.

[0537] In some aspects, the present disclosure provides a method for treating a disease or condition in a mammal (e.g., a human) in need thereof. The method includes administering to the mammal a therapeutically effective amount of a loaded LNP or pharmaceutical composition of the present disclosure. In some embodiments, the method includes administering to the subject a lipid nanoparticle comprising a compound of formula (I-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 preventive agents, thereby delivering the therapeutic and / or preventive agent to the cell. In some embodiments, the disease or condition is characterized by dysfunction or abnormal protein or polypeptide activity. For example, the disease or disorder is selected from the group consisting of rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0538] In some embodiments, the present disclosure provides a method of treating a disease or condition in a subject, wherein the method comprises administering to the subject a compound comprising Formula (I-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 -DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method for treating a disease or condition in a subject, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (Ac), DSPC, cholesterol and PEG 2k -DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method of treating a disease or condition in a subject, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of Table 1, DSPC, cholesterol and PEG 2k - the step of preparing lipid nanoparticles of DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides and nucleic acids (eg RNA).

[0539] In some embodiments, the present disclosure provides a method of treating a disease or condition in a subject, wherein the method comprises administering to the subject lipid nanoparticles comprising a compound of formula (I-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-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method of treating a disease or condition in a subject, wherein the method comprises administering to the subject lipid nanoparticles comprising a compound of formula (Ac), DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method of treating a disease or condition in a subject, wherein the method comprises administering to the subject lipid nanoparticles comprising a compound of Table 1, DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0540] In another aspect, the present disclosure provides a method of reducing immunogenicity, the method comprising introducing a loaded LNP or pharmaceutical composition of the present disclosure into a cell, wherein the loaded LNP or pharmaceutical composition reduces the induction of a cellular immune response in the cell to the loaded LNP or pharmaceutical composition as compared to the induction of a cellular immune response induced in the cell 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 comprises the step of administering to the subject a lipid nanoparticle comprising a compound of Formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), a phospholipid, a 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 lipid nanoparticle comprising a compound of Formula (1-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-a1), (A-a2), (A-a3), (Ab), (A-b1), (A-b2), (A-b3), (Ac), or (Bc), wherein the induction of a cellular immune response in the cell by the lipid nanoparticle comprising 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 compared to the induction of a cellular immune response in the cell by the lipid nanoparticle. The lipid nanoparticles of the compound of Formula (I-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) reduce the induction of a cellular immune response in the cell to the lipid nanoparticles comprising the compound of Formula (I-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 cellular immune response is an innate immune response, an adaptive immune response, or both.

[0541] In some embodiments, the present disclosure provides a method of reducing immunogenicity in a subject, wherein the method comprises administering to the subject a compound comprising 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 -DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity in a subject, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (Ac), DSPC, cholesterol and PEG 2k -DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method of reducing immunogenicity in a subject, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of Table 1, DSPC, cholesterol and PEG 2k - the step of preparing lipid nanoparticles of DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides and nucleic acids (eg RNA).

[0542] In some embodiments, the present disclosure provides a method of reducing immunogenicity in a subject, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (I-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-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method of reducing immunogenicity in a subject, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (Ac), DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the present disclosure provides a method of reducing immunogenicity in a subject, wherein the method comprises administering to the subject a lipid nanoparticle comprising a compound of Table 1, DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

[0543] The present disclosure also includes methods for 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 for preparing compounds comprising 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) are methods of producing lipid nanoparticles (e.g., empty LNPs or loaded LNPs) comprising a lipid component of a compound of the invention.

[0544] Methods for producing polypeptides in cells

[0545] The present disclosure provides a method for producing a target polypeptide in a mammalian cell. The method for producing a polypeptide relates to contacting a cell with a lipid nanoparticle (e.g., empty LNP or loaded LNP) comprising an mRNA encoding the target polypeptide. When the cell contacts the nanoparticle composition, the mRNA can be dissolved in the cell and translated to produce the target polypeptide.

[0546] In general, the step of contacting mammalian cells with the lipid nano particle (for example empty LNP or load LNP) that comprises the mRNA of coding target polypeptide can be in vivo, in vitro, in culture or externally carried out.Can depend on the type of the cell or tissue contacted, mode of administration, lipid nano particle (for example empty LNP or load LNP) and physiochemical characteristics (for example, size, electric charge and chemical composition) and other factors of mRNA wherein with the amount of the lipid nano particle (for example empty LNP or load LNP) of cell contact and / or the amount of mRNA wherein.In general, the effective dose of lipid nano particle (for example empty LNP or load LNP) will allow the effective polypeptide in the cell to produce.Can comprise level and the immunoreaction indicator of polypeptide translation (by polypeptide expression indication), mRNA degraded about the tolerance of efficiency.

[0547] The step of making the lipid nanoparticle (for example empty LNP or load LNP) comprising mRNA contact with cells can relate to or cause transfection.The phospholipid that comprises in the lipid component of lipid nanoparticle (for example empty LNP or load LNP) can for example promote transfection and / or increase transfection efficiency by interacting and / or merging with cell or intracellular membrane.Transfection can allow intracellular mRNA translation.

[0548] In some embodiments, lipid nanoparticle as herein described (for example empty LNP or load LNP) can be used in treatment.For example, the mRNA encoding therapeutic polypeptide (for example, in a translatable region) included in the lipid nanoparticle (for example empty LNP or load LNP) and produces the therapeutic polypeptide when contacting and / or entering (for example, transfection) into the cell.In other embodiments, the mRNA encoding therapeutic polypeptide included in the lipid nanoparticle (for example empty LNP or load LNP) can improve or increase the immunogenic polypeptide of the experimenter.For example, mRNA encoding granulocyte-colony stimulating factor or trastuzumab (trastuzumab) can be used.

[0549] In certain embodiments, the mRNA included in the lipid nanoparticle (e.g., empty LNP or loaded LNP) can encode a recombinant polypeptide that can supplement one or more polypeptides that are substantially absent in the cell in contact with the nanoparticle composition. The one or more substantially absent polypeptides can be lacking due to mutations in the coding gene or its regulatory pathway. Alternatively, the recombinant polypeptide produced by mRNA translation can antagonize the activity of an endogenous protein present in the cell, on the surface of the cell, or secreted from the cell. Antagonistic recombinant polypeptides may need to combat the harmful effects caused by the activity of the endogenous protein, such as altered activity or the positioning caused by mutation. In another alternative, the recombinant polypeptide produced by mRNA translation can indirectly or directly antagonize the activity of a biological part present in the cell, on the surface of the cell, or secreted from the cell. The biological part antagonized can include, but is not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoproteins), nucleic acids, carbohydrates, and small molecule toxins. The recombinant polypeptide produced by mRNA translation can be engineered to be located within the cell, such as within a specific compartment (e.g., nucleus), or can be engineered to be secreted from the cell or translocated to the plasma membrane of the cell.

[0550] In some embodiments, cell is contacted with the lipid nanoparticle (for example empty LNP or load LNP) comprising mRNA and can reduce the innate immune response of cell to exogenous nucleic acid.Cell can contact with the first lipid nanoparticle (for example empty LNP or load LNP) comprising the first exogenous mRNA comprising a first amount and can measure the level of the innate immune response of described cell to the first exogenous mRNA.Subsequently, described cell can contact with the second composition comprising the first exogenous mRNA of a second amount, and the second amount is the first exogenous mRNA of a smaller amount compared with the first amount.Or, the second composition can comprise the second exogenous mRNA that is different from the first exogenous mRNA of a first amount.The step of contacting described cell with the first composition and the second composition can be repeated one or many times.In addition, the polypeptide in the cell can optionally be measured to produce (for example translate) efficiency, and described cell can repeatedly contact with the first composition and / or the second composition again, until realizing target protein production efficiency.

[0551] Methods for delivering therapeutic agents to cells and organs

[0552] The disclosure provides the method for delivering therapeutic agent and / or prophylactic to mammalian cell or organ.Delivering therapeutic agent and / or prophylactic to cell relates to using the lipid nanoparticle (for example empty LNP or load LNP) that comprises described therapeutic agent and / or prophylactic to experimenter, wherein the using of said composition relates to making described cell contact with described composition.For example, protein, cytotoxic agent, radioactive ion, chemotherapeutic agent or nucleic acid (for example RNA, for example mRNA) can be delivered to cell or organ.When therapeutic agent and / or prophylactic is mRNA, when cell contacts with described nanoparticle composition, translatable mRNA can be translated in cell to produce target polypeptide.But, basically non-translatable mRNA also can be delivered to cell.Basically non-translatable mRNA can be used as vaccine and / or can isolate the translation component of cell to reduce the expression of other substances in cell.

[0553] In some embodiments, lipid nanoparticle (for example empty LNP or load LNP) can target the cell (for example, the cell of specific organ or its system) of specific type or classification.For example, the lipid nanoparticle (for example empty LNP or load LNP) comprising target therapeutic agent and / or preventive can be specifically delivered to mammal liver, kidney, spleen or lung.Specific delivery to the cell of specific classification, organ or its system or group hint relative to other destinations, and the lipid nanoparticle (for example load LNP) comprising therapeutic agent and / or preventive of higher ratio is delivered to target destination (for example tissue).In some embodiments, the specific delivery of the load LNP comprising mRNA can result in as compared with the cell of another destination (for example spleen), the mRNA expression in the cell of target destination (for example target tissue, for example liver) exceeds 2 times, 5 times, 10 times, 15 times or 20 times increase.In some embodiments, target tissue is selected from the group consisting of liver, kidney, lung, spleen and tumor tissue (for example, via intratumoral injection).

[0554] In some embodiments, specific delivery of mRNA contained in a loaded LNP of the present disclosure (i.e., lipid nanoparticles formulated with a compound of the present disclosure) can result in a greater than 2-fold, 5-fold, 10-fold, 15-fold, or 20-fold increase in mRNA expression as compared to delivery of mRNA contained in an LNP formulated with another lipid (i.e., without any of the lipids of Formula (I-1), (2-1), (Ia), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (Aa), (A-al), (A-a2), (A-a3), (Ab), (A-bl), (A-b2), (A-b3), (Ac), or (Bc)).

[0555] As another example of targeting or specific delivery, the mRNA of protein binding partner (for example, antibody or its functional fragment, skeletal protein or peptide) or acceptor on the coding cell surface can be included in the nanoparticle composition.MRNA can be used for guiding the synthesis and extracellular location of lipid, carbohydrate or other biological parts in addition or alternatively.Or, other therapeutic agents and / or preventive agents or key elements (for example lipid or part) of lipid nanoparticle (for example empty LNP or load LNP) can be selected based on its affinity to specific receptor (for example low-density lipoprotein receptor), so that lipid nanoparticle (for example empty LNP or load LNP) can more easily interact with the target cell colony comprising the acceptor. For example, ligands can 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 forms thereof; multivalent binding agents including monospecific or bispecific antibodies, such as disulfide-stabilized Fv fragments, scFv tandems, diabodies, triabodies, or tetrabodies; as well as aptamers, receptors, and fusion proteins.

[0556] In some embodiments, the ligand may be a surface-bound antibody, which can allow for adjustment of the cell targeting specificity. This is particularly useful because highly specific antibodies can be generated against the target epitope relative to the desired targeting site. In some embodiments, multiple antibodies are expressed on the surface of the cell, and each antibody may have a different specificity for the desired target. Such an approach can increase the avidity and specificity of the targeting interaction.

[0557] The ligand can be selected, for example, by one skilled in the biological arts based on the desired location or function in the cell.

[0558] Target cells can 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, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticular cells, leukocytes, granulocytes, and tumor cells.

[0559] In some embodiments, lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be targeted to hepatocytes. Apolipoproteins such as apolipoprotein E (apoE) have been shown to associate with lipid nanoparticles (e.g., empty LNPs or loaded LNPs) containing neutral or near-neutral lipids in the body and are known to associate with receptors found on the surface of hepatocytes (e.g., low-density lipoprotein receptors (LDLRs)). Therefore, lipid nanoparticles (e.g., empty LNPs or loaded LNPs) comprising lipid components having a neutral or near-neutral charge administered to a subject can obtain apoE in the subject's body and can subsequently deliver therapeutic and / or prophylactic agents (e.g., RNA) to hepatocytes comprising LDLRs in a targeted manner.

[0560] Methods of treating diseases and conditions

[0561] Lipid nanoparticle (for example empty LNP or load LNP) can be used for treating disease, disease or illness.Particularly, this type of composition can be used for treating disease, disease or illness that is characterized by lack or abnormal protein or polypeptide activity.For example, the lipid nanoparticle (for example empty LNP or load LNP) that comprises the mRNA of coding lack or abnormal polypeptide can be used or be delivered to cell.The subsequent translation of mRNA can produce described polypeptide, reduces or eliminates thus because described polypeptide does not exist or the problem that causes of abnormal activity caused by described polypeptide.Because translation can occur rapidly, described method and composition can be used for treating acute disease, disease or illness, for example sepsis, apoplexy and myocardial infarction.The therapeutic agent and / or the preventive agent that comprise in lipid nanoparticle (for example empty LNP or load LNP) also can change the transcription rate of given substance, affect gene expression thus.

[0562] Diseases, conditions and / or disorders characterized by dysfunctional or abnormal protein or polypeptide activity to which compositions can be administered include, but are not limited to, rare diseases, infectious diseases (both as vaccines and therapeutic agents), cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases. Various diseases, conditions and / or disorders may be characterized by the absence (or substantial attenuation, such that the proper protein function does not occur) of protein activity. Such proteins may not be present, or they may be substantially non-functional. The present disclosure provides a method of treating such diseases, conditions and / or disorders in a subject by administering lipid nanoparticles (e.g., empty LNPs or loaded LNPs) comprising RNA and a lipid component, wherein the lipid component 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 (optionally unsaturated), a PEG lipid, and a structured lipid, wherein the RNA may be an mRNA encoding a polypeptide that antagonizes or otherwise overcomes the activity of an abnormal protein present in the cells of the subject.

[0563] The method provided by the present disclosure relates to the use of lipid nanoparticles (such as empty LNP or loaded LNP) and pharmaceutical compositions comprising the lipid nanoparticles including one or more therapeutic and / or prophylactic agents. The terms therapeutic agent and prophylactic agent can be used interchangeably herein with respect to the features and embodiments of the present disclosure. Its therapeutic composition or imaging, diagnosis or prophylactic composition can be used to be administered to the subject using any reasonable amount and any route of administration that is effective for preventing, treating, diagnosing a disease, condition and / or illness or imaging a disease, condition and / or illness and / or any other purpose. The specific amount administered to a given subject can vary according to the following: the species, age and general condition of the subject; the purpose of administration; a specific composition; a mode of administration; etc. For ease of administration and the uniformity of dosage, compositions according to the present disclosure can be prepared in dosage unit form. However, it should be understood that the total daily dosage of the disclosed compositions will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective, prophylactically effective, or otherwise appropriate dosage level (e.g., for imaging) for any particular patient will depend on a variety of factors, including the severity and identification of the condition being treated, if any; the therapeutic and / or prophylactic agent(s) employed; the specific composition employed; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and rate of excretion of the specific pharmaceutical composition employed; the duration of the treatment; drugs used in combination or concomitantly with the specific pharmaceutical composition employed; and like factors well known in the medical arts.

[0564] Load LNP can be administered by any route. In some embodiments, the composition comprising one or more load LNP as described herein (including prevention, diagnosis or imaging compositions) is 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, by inhalation; As oral spray and / or powder, nasal spray and / or aerosol, and / or via portal vein catheter. In some embodiments, the composition can be intravenous, intramuscular, intradermal, intraarterial, intratumoral, subcutaneous or by any other parenteral administration route or by inhalation. However, considering the possible progress of drug delivery science, the present disclosure encompasses delivering or administering compositions as described herein by any appropriate route. In general, the most appropriate route of administration will depend on a variety of factors, including the properties (for example, its stability in a variety of body environments such as bloodstream and gastrointestinal tract) of the load LNP comprising one or more therapeutic agents and / or prophylactics, patient condition (for example, whether the patient can tolerate a specific route of administration) etc.

[0565] In certain embodiments, the compositions according to the present disclosure may be sufficient to deliver, in a given dose, about 0.0001 mg / kg to about 10 mg / kg, about 0.001 mg / kg to about 10 mg / kg, about 0.005 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 0.00 0.01 mg / kg to about 5 mg / kg, about 0.001 mg / kg to about 5 mg / kg, about 0.005 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 5 mg / kg, about 0.0001 mg / kg to about 2.5 mg / kg, about 0.001 mg / kg to about 2.5 mg / kg, about 0.005 mg / kg to about 2.5 mg / kg kg, about 0.01 mg / kg to about 2.5 mg / kg, about 0.05 mg / kg to about 2.5 mg / kg, about 0.1 mg / kg to about 2.5 mg / kg, about 1 mg / kg to about 2.5 mg / kg, about 2 mg / kg to about 2.5 mg / kg, about 0.0001 mg / kg to about 1 mg / kg, about 0.001 mg / kg to about 1 mg / kg, about 0.005 mg / kg to about 1 mg / kg, about 0.01 mg / kg to about 1 mg / kg, about 0.05 mg / kg to about 1 mg / kg, about 0.1 mg / kg In some embodiments, the dosage of the therapeutic agent and / or preventive agent (e.g., mRNA) of about 0.05mg / kg to about 0.25mg / kg, about 0.01mg / kg to about 0.25mg / kg, about 0.05mg / kg to about 0.25mg / kg, or about 0.1mg / kg to about 0.25mg / kg is used, wherein 1mg / kg (mpk) dosage provides 1mg therapeutic agent and / or preventive agent / 1kg subject body weight. In some embodiments, the dosage of the therapeutic agent and / or preventive agent of the load LNP of about 0.001mg / kg to about 10mg / kg can be used. In other embodiments, the dosage of the therapeutic agent and / or preventive agent of about 0.005mg / kg to about 2.5mg / kg can be used. In certain embodiments, the dosage of about 0.1mg / kg to about 1mg / kg can be used.In other embodiments, the dosage of about 0.05mg / kg to about 0.25mg / kg can be used. Dosage can be applied once or repeatedly with the same or different amounts every day to obtain the desired level of mRNA expression and / or treatment, diagnosis, prevention or imaging. The desired dosage can 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 can be delivered using multiple administrations (for example, secondary, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times or more administrations). In some embodiments, a single dose can be applied, for example, before or after an operating procedure or in the case of an acute disease, illness or disorder.

[0566] In some embodiments, the lipid nanoparticles (for example empty LNP or load LNP) comprising one or more therapeutic and / or prophylactic agents can be combined with one or more other therapeutic agents, prophylactics, diagnostic agents or imaging agents." with ... combination (in combination with) " is not intended to imply that the agent must be used simultaneously and / or be formulated for sending together, although these delivery methods are within the scope of the present disclosure. For example, one or more lipid nanoparticles (for example empty LNP or load LNP) comprising one or more different therapeutic and / or prophylactic agents can be used in combination. Compositions can be used in parallel with one or more other required therapeutic agents or medical procedures, before or after one or more other required therapeutic agents or medical procedures. In general, every kind of agent will be used with the dosage and / or time course determined for the agent. In some embodiments, the present disclosure contains that its composition or imaging, diagnosis or prophylactic composition and the agent combination that improves its bioavailability, reduces and / or regulates its metabolism, suppresses its excretion and / or modifies its distribution in health are sent.

[0567] It will be further understood that the therapeutic, prophylactic, diagnostic, or imaging agents used in combination may be administered together in a single composition or separately in different compositions. In general, it is contemplated that the agents used in combination are used at levels no greater than their levels when used individually. In some embodiments, the levels used in combination may be lower than the levels used individually.

[0568] The specific combination of therapies (therapeutics or procedures) to be used in a combination regimen will take into account the compatibility of the desired therapeutics and / or procedures and the desired therapeutic effect to be achieved. It will also be understood that the therapies employed may achieve the desired effect for the same condition (e.g., a composition useful for treating cancer may be administered concurrently with a chemotherapeutic agent), or they may achieve different effects (e.g., control of any adverse effects such as infusion-related reactions).

[0569] Lipid nanoparticles (e.g., empty LNP or load LNP) can be used in combination with an agent to increase the effectiveness and / or therapeutic window of the composition. This agent can be, for example, an anti-inflammatory compound, a steroid (e.g., a corticosteroid), a statin, an estradiol, a BTK inhibitor, an S1P1 agonist, a glucocorticoid receptor modulator (GRM), or an antihistamine. In some embodiments, lipid nanoparticles (e.g., empty LNP or load LNP) can be used in combination with dexamethasone, methotrexate, acetaminophen, an H1 receptor blocker, or an H2 receptor blocker. In some embodiments, a method for treating a subject in need or delivering a therapeutic agent and / or a prophylactic agent to a subject (e.g., a mammal) can be directed to pre-treating the subject with one or more agents before administering the nanoparticle composition. For example, the subject can be pretreated with dexamethasone, methotrexate, acetaminophen, H1 receptor blockers or H2 receptor blockers with an applicable 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 applicable amount). Pretreatment can occur 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) before the administration of lipid nanoparticles (e.g., empty LNP or loaded LNP) and can occur once, twice or more at an increased dosage.

[0570] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein.The scope of the present disclosure is not intended to be limited to the specific embodiments set forth above, but is set forth in the claims appended hereto.

[0571] Unless otherwise indicated or otherwise apparent from the text, in the claims, articles such as "a," "an," and "the" may mean one or more than one. Unless otherwise indicated or otherwise apparent from the text, claims or descriptions that include "or" between one or more members of a group are deemed satisfied when one, more than one, or all of the group members are present in, used in, or otherwise related to a given product or process. The present disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or process. The present disclosure includes embodiments in which more than one or all of the group members are present in, used in, or otherwise related to a given product or process. Unless otherwise specified, as used herein, the expressions "one or more of A, B or C", "one or more A, B or C", "one or more of A, B and C", "one or more A, B and C", "selected from A, B and C", "selected from the group consisting of A, B and C", etc. are used interchangeably and all refer to selected 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.

[0572] It should also be noted that the term "comprising" is intended to be open and allows but does not require the inclusion of additional elements or steps. When the term "comprising" is used in this article, the terms "consisting essentially of and "consisting of" are also encompassed and disclosed. Throughout the description, where a composition is described as having, including or comprising a particular component, it is contemplated that the composition is also essentially composed of the stated components, or is composed of the stated components. Similarly, where a method or process is described as having, including or comprising a particular process step, the process is also essentially composed of the stated process steps, or is composed of the stated process steps. In addition, it should be understood that the order of the steps or the order in which certain actions are performed is not important, as long as the disclosure remains operable. In addition, two or more steps or actions can be performed simultaneously.

[0573] Where ranges are given, the endpoints are included. Furthermore, it should be understood that unless otherwise indicated or otherwise apparent from the text and understanding of one of ordinary skill in the art, values expressed in ranges may assume any specific value or sub-range within the stated range in different embodiments of the present disclosure, up to the tenth of the lower limit of the range unless otherwise clearly indicated herein.

[0574] The synthetic process disclosed herein can tolerate a variety of functional groups, and thus a variety of substituted starting materials can be used. The process generally provides the desired final compound at or near the end of the overall process, although in some cases it may be necessary to further convert the compound into a pharmaceutically acceptable salt thereof.

[0575] The disclosed compounds 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 which will be apparent to the skilled artisan in view of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and for functional group transformations and manipulations can be obtained from the relevant scientific literature or standard textbooks in the field. Although not limited to any one or several sources, classic texts (e.g., Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th ed., John Wiley & Sons: New York, 2001; Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3rd ed., 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), incorporated herein by reference) are useful and recognized reference textbooks on organic synthesis known to those skilled in the art. The following descriptions of synthetic methods are designed to illustrate, but not to limit, general procedures for the preparation of compounds of the present disclosure.

[0576] Compounds of the present disclosure having any of the formulae described herein can be prepared according to the procedures illustrated in Schemes 1, 2, and 3 below from commercially available starting materials or starting materials that can be prepared using literature procedures. The variables in the schemes (e.g., R 1 、R 2 and R 3

[00155] Those of ordinary skill in the art will note that during the reaction sequences and synthetic schemes described herein, the order of certain steps may be varied, such as the introduction and removal of protecting groups.

[0577] Those skilled in the art will recognize that certain groups may need to be protected from reaction conditions by using protecting groups. Protecting groups can also be used to distinguish similar functional groups in a molecule. A list of protecting groups and how to introduce and remove these groups can be found in Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999.

[0578] Preferred protecting groups include, but are not limited to:

[0579] Regarding the hydroxyl moiety: TBS, benzyl, THP, Ac.

[0580] About carboxylic acids: benzyl ester, methyl ester, ethyl ester, allyl ester.

[0581] About amines: Fmoc, Cbz, BOC, DMB, Ac, Bn, Tr, Ts, trifluoroacetyl, phthalimide, benzylamine.

[0582] For the diol: Ac(×2)TBS(×2), or when taken together the acetonide.

[0583] About thiols: Ac.

[0584] About benzimidazole: SEM, benzyl, PMB, DMB.

[0585] Regarding aldehydes: dialkyl acetals, such as dimethoxyacetal or diethylacetyl.

[0586] In the reaction schemes described herein, a variety of stereoisomers may be produced. When a specific stereoisomer is not indicated, it is understood that all possible stereoisomers that may be produced by the reaction are intended. One of ordinary skill in the art will recognize that the reaction can be optimized to preferentially generate one isomer, or a new scheme can be designed to produce a single isomer. If a mixture is produced, the isomers can be separated using techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC.

[0587] Solution 1

[0588]

[0589] As illustrated in Scheme 1 above, 8-bromooctanoic acid is reacted with alcohol a1 (e.g., heptadecan-9-ol) to provide ester b1 (e.g., heptadecan-9-yl 8-bromooctanoate). Step 1 can occur in an organic solvent (e.g., dichloromethane) in the presence of, for example, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine, and DMAP. Step 1 can occur at room temperature for 18 hours. Ester b1 is then reacted with 2-aminoethan-1-ol to provide amine c1 (e.g., heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate). Step 2 can occur in ethanol at a temperature of, for example, about 60°C. Amine c1 is then reacted with a bromoalkyl group R 1 -Br (eg, 1-bromotetradecane) to provide compound d1 (eg, heptadecan-9-yl 8-((2-hydroxyethyl)(tetradecyl)amino)octanoate). Step 3 can occur in ethanol in the presence of N,N-diisopropylethylamine.

[0590] Option 2

[0591]

[0592]

[0593] As illustrated in Scheme 2 above, acid a2(x 3 is an integer between 1 and 7; for example, 8-bromooctanoic acid) reacts with alcohol b2 (for example, nonan-1-ol) to provide ester c2 (for example, nonyl-8-bromooctanoate). Step 1 can occur in an organic solvent (for example, dichloromethane) in the presence of, for example, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine and DMAP. Alcohol e2 (for example, heptadecan-9-ol) can be obtained via step 2 from the reaction of aldehyde d2 (for example, nonanal) with a Grignard reagent R 3 -MgX (e.g. n-C8H 17MgBr). 8-Bromooctanoic acid is then reacted with alcohol e2 (e.g., heptadecan-9-ol) to provide ester f2 (e.g., heptadecan-9-yl 8-bromooctanoate). Step 3 can occur in an organic solvent (e.g., dichloromethane) in the presence of, for example, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine, and DMAP. Ester f2 is then reacted with 2-aminoethan-1-ol to provide amine g2 (e.g., heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate). Step 4 can occur in ethanol in the presence of i-Pr2EtN. Amine g2 is then reacted with ester c2 (e.g., nonyl-8-bromooctanoate) to provide compound h2 (e.g., heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate). Step 5 can occur 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., K2CO3) or a non-nucleophilic organic base (e.g., i-Pr2EtN)) and a catalyst (e.g., an iodide, such as KI or NaI) at, for example, elevated temperature (e.g., at about 70-90°C, e.g., about 80°C).

[0594] Option 3

[0595]

[0596] As illustrated in Scheme 3 above, the haloalkanol (x 3 is an integer between 1 and 12, such as 6-bromohexan-1-ol) and the starting material a3 (x 2 is an integer between 1 and 6, such as 4-(hexyloxy)-4-oxobutanoic acid) to provide a halogenated diester b3 (such as 6-bromohexyl succinate). Compound a3 can be obtained by the reaction of an alcohol (such as hexan-1-ol) with an acid anhydride (such as succinic anhydride, dihydro-2H-pyran-2,6(3H)-dione, 3-(tert-butoxy)-3-oxopropionic acid, 4-(tert-butoxy)-3-methyl-4-oxobutanoic acid or 4-(tert-butoxy)-2-methyl-4-oxobutanoic acid). Step 1 can occur in an organic solvent (such as dichloromethane) in the presence of, for example, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine and DMAP. Then, the halogenated diester b3 is reacted with an amine c3 (x 4 is an integer between 5 and 13, x 5 is an integer between 1 and 5, for example, 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-yl ester) to provide the product d3. Step 2 can occur in an organic solvent (for example, a mixture of CPME and MeCN) in the presence of a base (for example, an inorganic base (for example, K2CO3) and a catalyst (for example, an iodide such as KI) and an ether solvent (for example, cyclopentyl methyl ether) at an elevated temperature (for example, about 90°C).

[0597] One of ordinary skill in the art will recognize that in the above schemes, the order of certain steps may be interchanged.

[0598] In certain aspects, the present disclosure also includes methods for synthesizing compounds of any 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), and intermediates for synthesizing the compounds.

[0599] In some embodiments, the method of synthesizing the disclosed compound comprises making a compound of formula (X2): With R 1 -Br reaction to provide compounds of the present disclosure, 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 unsubstituted C 1-3 Alkyl or -(CH2) n Q, wherein 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 of -Br occurs 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 occurs 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 occurs at an elevated temperature (e.g., about 50-100°C, 70-90°C, or about 80°C).

[0600] The method may further comprise making a compound of formula (X1): With R 4 NH2 is reacted to provide a compound of formula (X2), wherein each variable is as defined herein.

[0601] In some embodiments, the intermediates include those having any of formula (X1) and (X2): wherein each variable is as defined herein.For example, the intermediates include heptadecan-9-yl 8-bromooctanoate and heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate, and morphological forms (eg, crystalline forms) thereof.

[0602] Furthermore, it should be understood that any particular embodiment of the present disclosure that is prior art may be explicitly excluded from any one or more of the claims. Such embodiments are deemed to be known to one of ordinary skill in the art and therefore may be excluded even if such exclusion is not explicitly stated herein.

[0603] All cited sources (e.g., references, publications, databases, database entries, and techniques cited herein) are incorporated herein by reference, even if not explicitly stated in the citation. In the event of a conflict between a cited source and the statements in this application, the statements in this application shall prevail.

[0604] Example

[0605] Example 1: Synthesis of the compounds in Table 1

[0606] A. General considerations

[0607] Unless otherwise noted, all solvents and reagents used were obtained commercially and used as received. 1 H NMR spectra were recorded in CDCl3 at 300 K using a Bruker Ultrashield 300 MHz instrument. 1 H, chemical shifts are reported as parts per million (ppm) relative to TMS (0.00). Silica gel chromatography was performed on an ISCO CombiFlash Rf+Lumen instrument using ISCO RediSep Rf Gold Flash Cartridges (particle size: 20-40 microns). Reverse phase chromatography was performed on an ISCO CombiFlash Rf+Lumen instrument using a RediSep Rf Gold C18 high performance column. All final compounds were determined to be greater than 85% pure by reverse phase UPLC-MS (retention time, RT, in minutes) analysis using a Waters Acquity UPLC instrument with DAD and ELSD and a ZORBAX Rapid Resolution High Precision (RRHD) SB-C18 LC column (2.1 mm, 50 mm, 1.8 μm) and a 5 minute gradient of 65-100% acetonitrile / water with 0.1% TFA 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) performed in positive mode using a Waters SQD mass spectrometer (Milford, MA, USA) and an evaporative light scattering detector.

[0608] LCMS method:

[0609] Instrument information: HPLC / MS-Agilent 1100

[0610] Column: Agela Technologies Durashell C18 3.5μm, 4.6×50mm Mobile phase A: water / 0.1% trifluoroacetic acid

[0611] Mobile phase B: acetonitrile / 0.1% trifluoroacetic acid

[0612] Flow rate: 1 mL / min

[0613] Gradient: 70% B-100% B in 5 min, hold 100% B for 10 min, 100% B-70% B in 1 min, then stop.

[0614] Column temperature: ambient

[0615] Detector: ELSD

[0616] The procedures described below can be used to synthesize the compounds in Table 1.

[0617] The following abbreviations are used in this article:

[0618] THF: Tetrahydrofuran

[0619] MeCN: acetonitrile

[0620] LAH: lithium aluminum hydride

[0621] DCM: dichloromethane

[0622] DMAP: 4-dimethylaminopyridine

[0623] LDA: lithium diisopropylamide

[0624] rt: room temperature

[0625] DME: 1,2-dimethoxyethane

[0626] n-BuLi: n-butyllithium

[0627] CPME: Cyclopentyl methyl ether

[0628] i-Pr2EtN:N,N-diisopropylethylamine

[0629] Representative syntheses of compounds 7, 12, and 13

[0630]

[0631] Representative Procedure A: 1,4-Addition of Grignard Reagent RMgX to Methyl (E)-Non-2-enoate 1A1. Compound 2a: Methyl 3-Butylnonanoate

[0632]

[0633] Into a 100mL round-bottom flask dried, copper bromide (I) (421.3mg, 2.93mmol) and lithium chloride (249mg, 5.87mmol) were added, followed by anhydrous THF (15mL) and the mixture was stirred for 10min, during which time the solid dissolved. The flask was placed in an ice bath and (E)-non-2-enoic acid methyl ester 1 (5g, 29.37mmol) was added, followed by TMSCl (4mL, 32.31mmol). The reaction was stirred for 15min. A THF solution of butylmagnesium bromide (17.6mL, 35.2mmol, 2.0M in THF) was slowly added and the reaction was stirred for 2h. The reaction was quenched with saturated NH4Cl (10mL) and extracted with diethyl ether (100mL) and dried over anhydrous sodium sulfate. After removal of the solvent, the crude material was purified by flash chromatography (SiO 2 : ethyl acetate / hexane 0-100%) and the product 2a (3 g, 45%) was obtained as a colorless oil. 1 H NMR (300MHz, CDCl3): δppm 3.64 (s, 3H); 2.21 (d, 2H, J = 6.9Hz); 1.85-1.81 (m, 1H); 1.23-1.20 (m, 16H); 0.86-0.84 (m, 6H).

[0634] A2. Compound 2b: 3-isopropylnonanoic acid methyl ester

[0635]

[0636] Same as procedure A1, but using isopropylmagnesium bromide.Yield = 2.4 g (38%). 1 HNMR (300MHz, CDCl3): δppm 3.65 (s, 3H); 2.25 (dd, 1H, J = 15.1, 6.1Hz); 2.14 (dd, 1H, J = 15.1, 7.1Hz); 1.23-1.20 (m, 12H); 0.87-0.84 (m, 9H).

[0637] A3. Compound 2c: 3-propylnonanoic acid methyl ester

[0638]

[0639] Same as procedure A1, but using n-propylmagnesium bromide.Yield = 2.2 g (35%). 1HNMR (300MHz, CDCl3): δppm 3.64 (s, 3H); 2.23 (d, 2H, J = 6.8Hz); 1.85-1.84 (m, 1H); 1.23-1.20 (m, 14H); 0.87-0.84 (m, 6H).

[0640] Representative procedure B: LAH reduction

[0641] B1. Compound 3a: 3-Butylnonan-1-ol

[0642]

[0643] A solution of methyl 3-butyl nonanoate 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 N2. The mixture was heated under reflux for 5 h. The reaction was cooled to room temperature. Under a 0°C ice-water bath, 0.7 mL of H2O, 0.7 mL of 15% NaOH, and 2.1 mL of H2O were added sequentially. The white precipitate was filtered and the filtrate was concentrated. The crude product was purified by flash chromatography (SiO2: ethyl acetate / hexane 0-100%) to obtain the product 3a (980 mg, 51%) as a colorless oil. 1 H NMR (300MHz, CDCl3): δppm 3.64 (t, 2H, J = 6.8Hz); 1.52 (q, 2H, J = 7.1Hz); 1.32-1.30 (m, 1H); 1.23-1.20 (m, 17H); 0.88-0.84 (m, 6H).

[0644] B2. Compound 3b: 3-Isopropylnonan-1-ol

[0645]

[0646] Same as procedure B1, but using methyl 3-isopropylnonanoate 2b. Yield = 1.7 g (81%). 1 H NMR (300MHz, CDCl3): δppm 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).

[0647] B3. Compound 3c: 3-propylnonan-1-ol

[0648]

[0649] Same as procedure B1, but using methyl 3-propylnonanoate 2c. Yield = 1.28 g (67%). 1 H NMR (300MHz, CDCl3): δppm 3.64 (t, 2H, J = 6.6Hz); 1.52 (q, 2H, J = 6.3Hz); 1.23-1.20 (m, 16H); 0.88-0.84 (m, 6H).

[0650] Representative Procedure C for the esterification of 8-bromooctanoic acid 4

[0651] C1. Compound 5a: 3-Butylnonyl 8-bromooctanoate

[0652]

[0653] To a solution of 3-butyl nonan-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 that the reaction was complete. The reaction mixture was cooled to 0 ° C. and 1N hydrochloric acid (3 mL) was slowly added, and then the mixture was 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 material was purified by flash chromatography (SiO 2: hexane / diethyl ether 0-100%) and a colorless oily product 5a (680 mg.73%) was obtained. 1 H NMR (300MHz, CDCl3): δppm 4.07(t,2H,J=6.8Hz); 3.39(t,2H,J=6.8Hz); 2.28(t,2H,J=7.6Hz); 1.88 -1.79(m,2H); 1.70-1.42(m,6H); 1.38-1.17(m,21H); 0.88-0.82(m,6H).

[0654] C2. Compound 5b: 3-Isopropylnonyl 8-bromooctanoate

[0655]

[0656] Same as procedure C1, but using 3-isopropylnonan-1-ol 3b. Yield = 297 mg (71%). 1H NMR (300MHz, CDCl3): δppm 4.05(dd,2H,J=14.3,6.6Hz); 3.39(t,2H,J=6.8Hz); 2.28(t,2H,J=7.7Hz); 1 .86-1.81(m,2H); 1.70-1.42(m,6H); 1.38-1.17(m,16H); 0.84-0.82(m,9H).

[0657] C3. Compound 5c: 8-bromooctanoic acid 3-propylnonyl ester

[0658]

[0659] Same as procedure C1, but using 3-propylnonan-1-ol 3c. Yield = 430 mg (68%). 1 H NMR (300MHz, CDCl3): δppm 3.96(d,2H,J=5.8Hz); 3.38(t,2H,J=5.5Hz); 2.27(t,2H,J=7.4Hz); 1.88 -1.79(m,2H); 1.70-1.42(m,6H); 1.38-1.17(m,19H); 0.88-0.82(m,6H).

[0660] Representative Procedure D: N-Alkylation of Heptadec-9-yl 8-((2-hydroxyethyl)amino)octanoate 6

[0661] D1. Compound 7: 8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoic acid 3-butylnonyl ester (86-g-nBu)

[0662]

[0663] In a 500 mL round-bottom flask connected to a condenser, 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-yl ester 6 (601 mg, 1.36 mmol), 8-bromooctanoic acid 3-butyl nonyl ester 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), and the reaction mixture was heated to 85 ° C for 18 h. MS showed complete conversion, and the mixture was cooled to room temperature and diluted with hexane. The mixture was filtered through a celite pad. After washing with hexane, the filtrate was concentrated to give a brown oil, which was purified by flash chromatography (SiO2: hexane / diethyl ether 0-100%) to provide 7 (588 mg.56%) as a colorless oil. HPLC / ELSD: RT=7.07 min. About C48 H 95 NO5, MS (CI): m / z (MH + )766.7. 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).

[0664] D2. Compound 12: 8-((2-hydroxyethyl)(8-((3-isopropylnonyl)oxy)-8-oxooctyl)amino)octanoate heptadecan-9-yl ester (86-g-iPr)

[0665]

[0666] Same as procedure D1, but using 3-isopropylnonyl 8-bromooctanoate 5b. Yield = 258 mg (50%). HPLC / ELSD: RT = 6.98 min. About C 47 H 93 NO5, MS (CI): m / z (MH + )752.6. 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).

[0667] D3. Compound 13: 8-((2-hydroxyethyl)(8-oxo-8-((3-propylnonyl)oxy)octyl)amino)octanoate heptadecan-9-yl ester (86-g-nPr)

[0668]

[0669] Same as procedure D1, but using 3-propylnonyl 8-bromooctanoate 5c. Yield = 510 mg (68%). HPLC / ELSD: RT = 7.01 min. About C47 H 93 NO5, MS (CI): m / z (MH + )752.6. 1 H 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).

[0670] Synthetic scheme for the preparation of compound 8

[0671]

[0672] C4. Compound 5d: 2-propylnonyl 8-bromooctanoate

[0673]

[0674] Same as procedure C1, but using 2-propylnonan-1-ol 15a. Yield = 1.67 g (79%). 1 H NMR (300MHz, CDCl3): δppm 3.96(d,2H,J=5.8Hz); 3.38(t,2H,J=5.5Hz); 2.27(t,2H,J=7.4Hz); 1.88 -1.79(m,2H); 1.70-1.42(m,6H); 1.38-1.17(m,19H); 0.88-0.82(m,6H).

[0675] D4. Compound 8: 8-((2-hydroxyethyl)(8-oxo-8-((2-propylnonyl)oxy)octyl)amino)octanoate heptadecan-9-yl ester (86-b-nPr)

[0676]

[0677] Same as procedure D1, but using 2-propylnonyl 8-bromooctanoate 5d. Yield = 355 mg (68%). HPLC / ELSD: RT = 7.0 min. About C 47 H 93 NO5, MS (CI): m / z (MH + )752.6. 1H 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).

[0678] synthetic intermediates :

[0679] Intermediate AA: 3-propylhex-2-enoic acid ethyl ester

[0680]

[0681] Triethyl phosphonoacetate (11.3mL, 56.9mmol) is added dropwise to a suspension of sodium hydride (2.28g, 56.9mmol) in THF (17mL) over 20 minutes and the mixture is stirred at room temperature until gas evolution stops (approximately 30min). The reaction mixture is cooled to 0°C and divided into multiple portions to add 4-heptanone (6.12mL, 43.8mmol). The reaction is gradually warmed to room temperature and allowed to stir under reflux for 24h. The reaction is cooled to room temperature and then quenched with saturated sodium bicarbonate aqueous solution. The aqueous phase is extracted with diethyl ether, and the organic extract is washed with salt water, dried (MgSO4), and concentrated. The crude material is purified by silica gel chromatography (0-20% EtOAc: hexane) to provide 3-propyl hex-2-enoic acid ethyl ester (8.07g, 43.8mmol, 100%) as a clear oil and as a mixture of regioisomers. 1 H NMR (300 MHz, CDCl3) showed a mixture of regioisomers δ: ppm 5.63 (s, 1H); 5.38-5.25 (m, 0.74H); 4.19-4.07 (m, 3.40H); 3.02 (s, 0.81H); 2.96 (s, 0.59H); 2.57 (ddd, 2H, J = 6.0, 6.0, 3.0 Hz); 2.16-1.98 (m, 4.87H); 1.57-1.35 (m, 6.10H); 1.34-1.21 (m, 7.59H); 1.01-0.82 (m, 12.9H).

[0682] Intermediate AB: 3-propylhexanoate

[0683]

[0684] The ethanol (44mL) containing 3-propyl hexamethylene-2-enoate (8.07g, 43.8mmol) is loaded into the steel Parr reactor equipped with a stirring rod. Palladium hydroxide / carbon (922mg, 6.57mmol) is added and the container is sealed, evacuated, and H is used Gas backfill (3 times), and the pressure is set to 200psi. The reaction is at 500rpm, at 200psi H Under gas, stir for 2h at room temperature. Then the container is evacuated, N Gas backfill, and opened. The crude reaction mixture is filtered through diatomaceous earth pad. The diatomaceous earth pad is washed with EtOH and the crude material is concentrated to obtain 3-propyl hexanoic acid ethyl ester (6.55g, 35.2mmol, 80%) in a transparent oily state. The compound is continued for the next step without further purification. 1 H NMR (300MHz, CDCl3) δ: ppm 4.12 (q, 2H, J = 6.0Hz); 2.22 (d, 2H, J = 9.0Hz); 1.95-1.81 (m, 1H); 1.38-1.18 (m, 11H); 0.89 (br.t, 6H, J = 6.0Hz).

[0685] Intermediate AC: 3-propylhexan-1-ol

[0686]

[0687] Under N2, at 0 ° C, to a mixture of lithium aluminum hydride (1.60 g, 42.2 mmol) in anhydrous ether (42 mL) was added dropwise anhydrous ether (28 mL) containing 3-propyl hexanoate (6.55 g, 35.2 mmol). The mixture was stirred at room temperature for 2.5 h and then cooled to 0 ° C. Water (1 mL / g LiAlH4) was added dropwise to the solution, followed by the slow addition of 15% sodium hydroxide (1 mL / g LiAlH4) and water (3 mL / g LiAlH4). The solution was stirred at room temperature for several minutes and filtered through a diatomaceous earth pad. The diatomaceous earth pad was washed with diethyl ether and the filtrate was concentrated. The crude material was purified by silica gel chromatography (0-40% EtOAc: hexane) to provide 3-propyl hexan-1-ol (4.82 g, 33.4 mmol, 95%) as a clear oil. 1 H NMR (300MHz, CDCl3) δ: ppm 3.67 (t, 2H, J = 6.0Hz); 1.57-1.39 (m, 3H); 1.37-1.18 (m, 9H); 0.88 (t, 6H, J = 6.0Hz).

[0688] Intermediate AD: ethyl 3-butylhept-2-enoate

[0689]

[0690] Triethyl phosphonoacetate (9.07mL, 45.7mmol) is added dropwise to a suspension of sodium hydride (1.83g, 45.7mmol) in THF (14mL) over 20 minutes and the mixture is stirred at room temperature until gas evolution stops (approximately 30min). The reaction mixture is cooled to 0°C and divided into multiple portions to add 5-nonanone (6.05mL, 35.2mmol). The reaction is gradually warmed to room temperature and allowed to stir under reflux for 24h. The reaction is cooled to room temperature and then quenched with saturated sodium bicarbonate aqueous solution. The aqueous phase is extracted with diethyl ether, and the organic extract is washed with salt water, dried (MgSO4), and concentrated. The crude material is purified by silica gel chromatography (0-20% EtOAc: hexane) to provide 3-butyl hept-2-enoic acid ethyl ester (5.27g, 24.8mmol, 71%) in a clear oily state. 1 H NMR (300MHz, CDCl3) δ: ppm 5.62 (s, 1H); 4.14 (q, 2H, J = 6.0Hz); 2.59 (t, 2H, J = 6.0Hz); 2.14 (t, 2H, J = 6.0Hz); 1.50-1.23 (m, 11H); 0.99-0.82 (m, 6H).

[0691] Intermediate AE: 3-butylheptanoate

[0692]

[0693] The ethanol (50mL) containing 3-butyl hept-2-enoic acid ethyl ester (10.5g, 49.5mmol) is loaded into the steel Parr reactor equipped with a stirring rod. Palladium hydroxide / carbon (1.04g, 7.42mmol) is added and the container is sealed, evacuated, and H is used Gas backfill (3 times), and the pressure is set to 200psi. The reaction is at 500rpm, at 200psi H Under gas, stir for 2h at room temperature. The container is then evacuated, N Gas backfill, and opened. The crude reaction mixture is filtered through a diatomaceous earth pad. The diatomaceous earth pad is washed with EtOH and the crude material is concentrated to obtain 3-butyl heptanoic acid ethyl ester (9.69g, 45.2mmol, 91%) in a transparent oily state. The compound is continued for the next step without further purification. 1 H NMR (300MHz, CDCl3) δ: ppm 4.12 (q, 2H, J = 9.0Hz); 2.22 (d, 2H, J = 6.0Hz); 1.90-1.76 (m, 1H); 1.38-1.19 (m, 15H); 0.88 (br.t, 6H, J = 6.0Hz).

[0694] Intermediate AF: 3-Butylheptan-1-ol

[0695]

[0696] To a mixture of lithium aluminum hydride (850 mg, 22.4 mmol) in anhydrous ether (23 mL) was added dropwise anhydrous ether (15 mL) containing ethyl 3-butylheptanoate (4.00 g, 18.7 mmol) under N at 0 ° C. The mixture was stirred at room temperature for 2.5 h and then cooled to 0 ° C. Water (1 mL / g LiAlH4) was added dropwise to the solution, followed by the slow addition of 15% sodium hydroxide (1 mL / g LiAlH4) and water (3 mL / g 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: hexane) to provide 3-butyl heptanoate (3.19 g, 18.5 mmol, 99%) as a clear oil. 1 H NMR (300MHz, CDCl3) δ: ppm3.66 (t, 2H, J = 6.0Hz); 1.53 (q, 2H, J = 6.0Hz); 1.46-1.36 (m, 1H); 1.35-1.21 (m, 12H); 1.18 (br.s, 1H); 0.89 (br.t, 6H, J = 6.0Hz).

[0697] Intermediate AG: Ethyl 3-pentyloct-2-enoate

[0698]

[0699] Triethyl phosphonoacetate (10.6mL, 53.4mmol) is added dropwise to a suspension of sodium hydride (2.13g, 53.4mmol) in THF (16mL) over 20 minutes and the mixture is stirred at room temperature until gas evolution stops (approximately 30min). The reaction mixture is cooled to 0°C and divided into multiple portions to add 6-undecanone (8.42mL, 41.1mmol). The reaction is gradually warmed to room temperature and stirred at reflux for 60h. The reaction is cooled to room temperature and then quenched with saturated sodium bicarbonate aqueous solution. The aqueous phase is extracted with diethyl ether, and the organic extract is washed with salt water, dried (MgSO4), and concentrated. The crude material is purified by silica gel chromatography (0-20% EtOAc: hexane) to provide 3-pentyl octyl-2-enoic acid ethyl ester (8.76g, 36.5mmol, 89%) as a clear oil. 1H NMR(300MHz, CDCl3)δ:ppm 5.61(s,1H); 4.14(q,2H,J=6.0Hz); 2.58(ddd,2H,J=9.0,9.0,6.0Hz); 2.13(ddd, 2H, J=6.0, 6.0, 3.0Hz); 1.52-1.38 (m, 3H); 1.38-1.23 (m, 12H); 0.93-0.86 (m, 6H).

[0700] Intermediate AH: ethyl 3-pentyloctanoate

[0701]

[0702] The ethanol (37mL) containing 3-amyl octane-2-enoic acid ethyl ester (8.76g, 36.5mmol) is loaded into the steel Parr reactor equipped with a stirring rod. Palladium hydroxide / carbon (768mg, 5.47mmol) is added and the container is sealed, evacuated, and H is used Gas backfill (3 times), and the pressure is set to 200psi. The reaction is at 500rpm, at 200psi H Under gas, stir for 2h at room temperature. Then the container is evacuated, N Gas backfill, and opened. The crude reaction mixture is filtered through a diatomaceous earth pad. The diatomaceous earth pad is washed with EtOH and the crude material is concentrated to obtain 3-amyl octane ethyl ester (8.45g, 34.9mmol, 96%) in a transparent oily state. The compound is continued for the next step without further purification. 1 H NMR (300MHz, CDCl3) δ: ppm 4.12 (q, 2H, J = 6.0Hz); 2.22 (d, 2H, J = 6.0Hz); 1.92-1.77 (br.m, 1H); 1.37-1.19 (m, 19H); 0.88 (t, 6H, J = 6.0Hz).

[0703] Intermediate AI: 3-pentyloctan-1-ol

[0704]

[0705] To a mixture of lithium aluminum hydride (1.59 g, 41.8 mmol) in anhydrous ether (42 mL) was added dropwise anhydrous ether (28 mL) containing ethyl 3-pentyl octanoate (8.45 g, 34.9 mmol) under N at 0 ° C. The mixture was stirred at room temperature for 2.5 h and then cooled to 0 ° C. Water (1 mL / g LiAlH4) was added dropwise to the solution, followed by the slow addition of 15% sodium hydroxide (1 mL / g LiAlH4) and water (3 mL / g 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: hexane) to provide 3-pentyl octan-1-ol (6.98 g, 34.9 mmol, 100%) as a clear oil. 1 H NMR (300MHz, CDCl3) δ: ppm3.66 (t, 2H, J = 6.0Hz); 1.53 (q, 2H, J = 6.0Hz); 1.47-1.37 (br.s, 1H); 1.36-1.15 (m, 17H); 0.88 (t, 6H, J = 6.0Hz).

[0706] Intermediate AJ: 3-pentyloctanal

[0707]

[0708] To a stirred suspension of pyridinium chlorochromate (9.02 g, 41.8 mmol) and silica gel (9.02 g, 1 g / g pyridinium chlorochromate) in dichloromethane (90 mL) was added 3-pentyloctan-1-ol (6.98 g, 34.9 mmol) under N2 atmosphere. The suspension was stirred at room temperature for 1 h. The reactant was then filtered through a celite pad, which was washed with dichloromethane, and the filtrate was concentrated. The crude material was purified by silica gel chromatography (0-20% EtOAc: Hexane) to provide 3-pentyloctanal (4.66 g, 23.5 mmol, 67%) as a clear oil. 1 H NMR (300MHz, CDCl3) δ: ppm 9.76 (t, 1H, J = 3.0Hz); 2.33 (dd, 2H, J = 6.0, 3.0Hz); 2.01-1.86 (br.m, 1H); 1.40-1.19 (m, 16H); 0.88 (t, 6H, J = 6.0Hz).

[0709] Intermediate AK: 6-allylundecane

[0710]

[0711] To a suspension of methyltriphenylphosphonium bromide (4.68g, 13.1mmol) in anhydrous ether (190mL) was added potassium tert-butoxide (1.47g, 13.1mmol) at one time under N2. The mixture was stirred at room temperature for 15 minutes, and then anhydrous ether (26mL) containing 3-pentyloctanal (2.00g, 10.1mmol) was added dropwise over 15min. The resulting mixture was stirred at room temperature for 90min. The reaction mixture was diluted with ice water, the layers were separated, and the organic layer was extracted with ether. The combined organic matter was dried (MgSO4), filtered, and concentrated. The crude material was purified by silica gel chromatography (0-10% EtOAc: hexane) to provide 6-allyl undecane (1.65g, 8.38mmol, 83%) as a clear oil. 1 H NMR(300MHz, CDCl3)δ:ppm 5.77 (dddd, 1H, J = 15.0, 12.0, 9.0, 9.0Hz); 5.03-4.94 (m, 2H); 2.02 (dddd, 2H, J = 9.0, 6.0, 6.0Hz); 1.43-1.16 (m, 17H); 0.88 (d, 6H, J = 6.0Hz).

[0712] Intermediate AL: 4-pentylnonan-1-ol

[0713]

[0714] To a stirred solution of sodium borohydride (131 mg, 3.46 mmol) in anhydrous diglyme (3.6 mL) was added a solution of 6-allyl undecane (2.26 g, 11.5 mmol) in anhydrous diglyme (2.3 mL) under an N atmosphere. Subsequently, a solution of boron trifluoride etherate (569 μL, 4.61 mmol) in 1.2 mL of anhydrous diglyme was added at room temperature over 15 min. The resulting mixture was stirred for 1 hour, and then water (1.2 mL) was added dropwise. When gas evolution stopped, 2.3 mL of 3M NaOH was added at room temperature, followed by 2.3 mL of 30% H2O2 at 40°C. After stirring for 1 hour at 40°C, the reactant was poured into 10 mL of water. The reaction vessel was washed with additional water. The combined aqueous solution was extracted with ether (2 times). The combined ether extracts were washed with water (× 5). The ether extracts were dried (MgSO4), filtered, and concentrated.The crude residue was purified by silica gel chromatography (0-40% EtOAc:hexanes) to provide 4-pentylnonan-1-ol (1.88 g, 8.77 mmol, 76%) as a clear oil. 1H NMR (300MHz, CDCl3) δ: ppm 3.62 (t, 2H, J = 6.0Hz); 1.60-1.48 (m, 2H); 1.37-1.19 (m, 20H); 0.88 (t, 6H, J = 6.0Hz).

[0715] Intermediate AM: 8-bromooctanoic acid 3-propylhexyl ester

[0716]

[0717] At 0 ℃, EDCI (9.60g, 50.1mmol) is added to a solution of 3-propyl hexan-1-ol (4.82g, 33.4mmol), 8-bromooctanoic acid (8.94g, 40.1mmol) and DMAP (816mg, 6.68mmol) in dichloromethane (58mL). The reaction mixture is stirred at room temperature overnight. The reaction mixture is then cooled to 0 ℃ and 10% hydrochloric acid solution (180mL) is slowly added over 20 minutes. Each layer is separated, and the organic layer is concentrated in a vacuum to obtain a thick oil. The oil is dissolved in hexane (180mL) and washed with a mixture of acetonitrile (180mL) and 5% sodium bicarbonate (180mL). The hexane layer is separated, dried (MgSO ), and filtered. Solvent is removed under vacuum to obtain 8-bromooctanoic acid 3-propyl hexyl ester (10.9g, 31.2mmol, 93%) in a transparent 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).

[0718] Intermediate AN: 8-bromooctanoic acid 3-butylheptyl ester

[0719]

[0720] At 0 ℃, EDCI (5.33g, 27.8mmol) is added to a solution of 3-butyl heptane-1-ol (3.19g, 18.5mmol), 8-bromooctanoic acid (4.96g, 22.2mmol) and DMAP (453mg, 3.71mmol) in dichloromethane (32mL). The reaction mixture is stirred at room temperature overnight. The reaction mixture is then cooled to 0 ℃ and 10% hydrochloric acid solution (150mL) is slowly added over 20 minutes. Each layer is separated, and the organic layer is concentrated in a vacuum to obtain a thick oil. The oil is dissolved in hexane (150mL) and washed with a mixture of acetonitrile (150mL) and 5% sodium bicarbonate (150mL). The hexane layer is separated, dried (MgSO ), and filtered. Solvent is removed under vacuum to obtain 8-bromooctanoic acid 3-butyl heptyl esters (6.90g, 18.3mmol, 99%) in a transparent 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).

[0721] Intermediate AO: 8-bromooctanoate 3-pentyloctyl ester

[0722]

[0723] At 0 ℃, EDCI (2.87g, 15.0mmol) is added to a solution of 3-pentyl octan-1-ol (2.00g, 9.98mmol), 8-bromooctanoic acid (2.67g, 12.0mmol) and DMAP (244mg, 2.00mmol) in dichloromethane (18mL) and the reaction mixture is stirred at room temperature overnight. The reaction mixture is then cooled to 0 ℃ and 10% hydrochloric acid solution (70mL) is slowly added over 20 minutes. Each layer is separated, and the organic layer is concentrated in a vacuum to obtain a thick oil. The oil is dissolved in hexane (70mL) and washed with a mixture of acetonitrile (70mL) and 5% sodium bicarbonate (70mL). The hexane layer is separated, dried (MgSO ), and filtered. Solvent is removed under vacuum to obtain 8-bromooctanoic acid 3-pentyl octyl esters (3.94g, 9.72mmol, 97%) in a transparent oil. The compound was carried on to the next step without further purification. 1H 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).

[0724] Intermediate AP: 4-pentylnonyl-8-bromooctanoate

[0725]

[0726] At 0 ℃, EDCI (2.52g, 13.2mmol) is added to a solution of 4-pentyl nonyl-1-alcohol (1.88g, 8.77mmol), 8-bromooctanoic acid (2.35g, 10.5mmol) and DMAP (214mg, 1.75mmol) in dichloromethane (15mL) and the reaction mixture is stirred at room temperature overnight. The reaction mixture is then cooled to 0 ℃ and 10% hydrochloric acid solution (60mL) is slowly added over 20 minutes. Each layer is separated, and the organic layer is concentrated in a vacuum to obtain a thick oil. The oil is dissolved in hexane (60mL) and washed with a mixture of acetonitrile (60mL) and 5% sodium bicarbonate (60mL). The hexane layer is separated, dried (MgSO ), and filtered. Solvent is removed under vacuum to obtain 4-pentyl nonyl-8-bromooctanoate (3.68g, 8.77mmol, 100%) in a transparent 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).

[0727] Intermediate AQ: 8-bromooctanoate pentadecane-8-yl ester

[0728]

[0729] 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 pentadecane-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 slowly added 10% hydrochloric acid solution. 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 NaHCO (aqueous solution). The hexane layer was separated, dried over MgSO4, then filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% ethyl acetate / hexanes) to afford a approximately 13.2:1 mixture of 8-bromooctanoate and 8-chlorooctanoate (3.19 g, 83.1%) as a colorless liquid. 1 H NMR(300MHz, 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).

[0730] Intermediate AR: 8-bromooctanoic acid tridecyl-7-yl ester

[0731]

[0732]

[0733] 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 tridecane-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 NaHCO (aqueous solution). The organic layer was separated and washed with brine, dried over MgSO, then filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% ethyl acetate / hexane) to obtain a mixture of about 12.3:1 of 8-bromooctanoic acid tridecane-7-yl ester and 8-chlorooctanoic acid tridecane-7-yl ester (2.10 g, 59.4%). 1H NMR(300MHz, 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).

[0734] Intermediate AS: 8-bromooctanoic acid undecyl-6-yl ester

[0735]

[0736] 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 slowly added 10% hydrochloric acid solution. 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 NaHCO (aqueous solution). The hexane layer was separated, dried over MgSO4, then filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% ethyl acetate / hexanes) to afford a ca. 19:1 mixture of undec-6-yl 8-bromooctanoate and undec-6-yl 8-chlorooctanoate as a colorless liquid (4.33 g, 64.01%). 1 H NMR(300MHz, 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).

[0737] Intermediate AT: 8-bromooctanoic acid non-5-yl ester

[0738]

[0739] 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (4.31g, 22.5mmol), 4-(dimethylamino)pyridine (0.438g, 3.59mmol) and 5-nonanol (2.59g, 17.9mmol) are added to a solution of 8-bromooctanoic acid (4.00g, 17.9mmol) in dichloromethane (60mL). The reaction is stirred at room temperature for 18 hours. The reaction mixture is cooled to 0°C and slowly added with 10% hydrochloric acid solution. The organic layer is separated and evaporated under vacuum. The residue is dissolved in hexane and washed with a 1:1 mixture of acetonitrile and saturated NaHCO (aqueous solution). The hexane layer is separated, dried over MgSO4, then filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% ethyl acetate / hexanes) to afford a approximately 7:1 mixture of nonan-5-yl 8-bromooctanoate and nonan-5-yl 8-chlorooctanoate as a colorless liquid (5.23 g, 83.5%). 1 HNMR(300MHz,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).

[0740] Intermediate AU: 3-propylhexyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate

[0741]

[0742] To the solution of tert-butyl N-(3-aminopropyl)carbamate (23.9g, 137mmol) in EtOH (60mL) was added EtOH (55mL) containing 8-bromooctanoic acid 3-propyl hexyl ester (8.00g, 22.3mmol) over 20min process. The reaction was heated to 60 ℃ and stirred at this temperature for 16h. When cooling, the solvent was evaporated and the residue was diluted with ethyl acetate and washed with saturated NaHCO3 aqueous solution and salt water (5 times) until no white precipitate was observed in the water layer. The organic layer was separated, washed with salt water, dried (MgSO4), filtered, and concentrated. The residue was purified by flash chromatography (0-5-10-25-50-100% (1% NH4OH, 20% MeOH in dichloromethane) / dichloromethane) to give 3-propylhexyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate (5.81 g, 13.1 mmol, 57%) as a clear oil. 1H NMR(300MHz, CDCl3)δ:ppm 5.16(br.s,1H); 4.08(t,2H,J=6.0Hz); 3.19(br.q,2H,J=6.0Hz); 2.65(t,2H,J=6.0Hz); 2.56(t,2H,J=6.0Hz); 2. 27(t,2H,J=6.0Hz); 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=6.0Hz).

[0743] Intermediate AV: 8-((2-hydroxyethyl)amino)octanoic acid 3-propylhexyl ester

[0744]

[0745] 8-bromooctanoic acid 3-propyl hexyl ester (2.82g, 8.06mmol), ethanolamine (14.6mL, 242mmol) and ethanol (6mL) are added into a round-bottom flask equipped with a stirring bar. The resulting mixture is stirred at 40 ° C for 16h. The reaction is diluted with dichloromethane, washed with water (2 times), and each layer is separated. The organic layer (MgSO4) is dried, filtered and concentrated. The crude material is purified by silica gel chromatography (0-5-10-25-50-100% (1% NH4OH, 20% MeOH in dichloromethane) / dichloromethane) to obtain 8- ((2-hydroxyethyl) amino) 3-propyl hexyl caprylate (876mg, 2.66mmol, 33%) in a clear oily state. 1 H NMR(300MHz, CDCl3)δ:ppm 4.08(t,2H,J=6.0Hz); 3.63(t,2H,J=6.0Hz); 2.77(t,2H,J=6.0Hz); 2.61(t,2H,J=6.0Hz); 2.28 (t, 2H, J = 6.0Hz); 1.91 (br.s, 2H); 1.68-1.39 (m, 7H); 1.38-1.18 (m, 14H); 0.88 (t, 6H, J = 6.0Hz).

[0746] Intermediate AW: 3-pentyloctyl 8-((3-((tert-Butoxycarbonyl)amino)propyl)amino)octanoate

[0747]

[0748] To a solution of tert-butyl N-(3-aminopropyl)carbamate (15.5 g, 88.8 mmol) in EtOH (38 mL) was added EtOH (36 mL) containing 3-pentyl octyl 8-bromooctanoate (6.00 g, 14.8 mmol) over a 20 min process. The reaction was heated to 60 ° C and stirred at this temperature for 16 h. Upon cooling, the solvent was evaporated and the residue was diluted with ethyl acetate and washed with saturated NaHCO aqueous solution and salt water (5 times) until no white precipitate was observed in the aqueous layer. The organic layer was separated, washed with salt water, dried (MgSO ), filtered, and concentrated. The residue was purified by flash chromatography (0-5-10-25-50-100% (1% NH4OH, 20% MeOH in dichloromethane) / 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(300MHz, CDCl3)δ:ppm 5.17(br.s,1H); 4.07(t,2H,J=6.0Hz); 3.19(br.q,2H,J=6.0Hz); 2.66(t,2H,J=6.0Hz); 2.56(t,2H,J=6.0Hz); 2. 28(t,2H,J=6.0Hz); 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=6.0Hz).

[0749] Intermediate AX: 4-pentylnonyl 8-((2-hydroxyethyl)amino)octanoate

[0750]

[0751] 8-bromooctanoic acid 4-pentyl nonyl ester (600mg, 1.43mmol), ethanolamine (2.59mL, 42.9mmol) and ethanol (1mL) were added to a round-bottom flask equipped with a stirring bar. The resulting mixture was stirred at 40 ° C for 16h. The reaction was diluted with dichloromethane, washed with water (2 times), and the layers were separated. The organic layer was dried (MgSO4), filtered and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% (1% NH4OH, 20% MeOH in dichloromethane) / dichloromethane) to obtain 8- ((2-hydroxyethyl) amino) octanoic acid 4-pentyl nonyl ester (306mg, 0.77mmol, 54%) as a clear oil. UPLC / ELSD: RT = 1.66min. About C 24 H 49NO3, MS (ES): m / z (MH + )400.31. 1 H NMR(300MHz, CDCl3)δ:ppm 3.97(t,2H,J=6.0Hz); 3.57(br.t,2H,J=6.0Hz); 2.81(br.s,2H),2.67(br.t,2H,J=6.0Hz); 2.53(t ,2H,J=6.0Hz); 2.22(t,2H,J=6.0Hz); 1.61-1.35(m,6H); 1.32-1.10(m,25H); 0.81(t,6H,J=6.0Hz).

[0752] Intermediate AY: 8-((3-hydroxypropyl)amino)octanoate 3-pentyloctyl ester

[0753]

[0754] 8-bromooctanoic acid 3-pentyloctyl ester (1.00g, 2.47mmol), propanolamine (5.66mL, 74.0mmol) and ethanol (2mL) were added to a round-bottom flask equipped with a stirring bar. The resulting mixture was stirred at 40 ° C for 16h. The reaction was diluted with dichloromethane, washed with water (2 times), and the layers were separated. The organic layer was dried (MgSO4), filtered and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% (1% NH4OH, 20% MeOH in dichloromethane) / dichloromethane) to obtain 8- ((3-hydroxypropyl) amino) octanoic acid 3-pentyloctyl ester (374mg, 0.94mmol, 38%) as a clear oil. UPLC / ELSD: RT = 1.64min. About C 24 H 49 NO3, MS (ES): m / z (MH + )400.18.

[0755] Intermediate AZ: 8-((3-hydroxypropyl)amino)octanoic acid heptadecan-9-yl ester

[0756]

[0757] 8-bromooctanoic acid heptadecan-9-yl ester (1.00g, 2.17mmol), propanolamine (4.97mL, 65.0mmol) and ethanol (2mL) were added to a round-bottom flask equipped with a stirring bar. The resulting mixture was stirred at 40 ° C for 16h. The reaction was diluted with dichloromethane, washed with water (2 times), and the layers were separated. The organic layer was dried (MgSO4), filtered and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% (1% NH4OH, 20% MeOH in dichloromethane) / dichloromethane) to obtain 8- ((3-hydroxypropyl) amino) octanoic acid heptadecan-9-yl ester (723mg, 1.59mmol, 73%) as a clear oil. UPLC / ELSD: RT = 2.06min. About C 28 H 57 NO3, MS (ES): m / z (MH + )456.17.

[0758] Intermediate BA: 8-((4-hydroxybutyl)amino)octanoic acid heptadecan-9-yl ester

[0759]

[0760] 8-bromooctanoic acid heptadecan-9-yl ester (1.00g, 2.17mmol), 4-aminobutan-1-ol (5.99mL, 65.0mmol) and ethanol (2mL) were added to a round-bottom flask equipped with a stirring bar. The resulting mixture was stirred at 40 ° C for 16h. The reaction was diluted with dichloromethane, washed with water (2 times), and the layers were separated. The organic layer was dried (MgSO4), filtered and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% (1% NH4OH, 20% MeOH in dichloromethane) / dichloromethane) to obtain 8- ((4-hydroxybutyl) amino) octanoic acid heptadecan-9-yl ester (773mg, 1.65mmol, 76%) as a clear oil. UPLC / ELSD: RT = 2.02min. About C 29 H 59 NO3, MS (ES): m / z (MH + )470.23.

[0761] Intermediate BB: 8-((4-hydroxybutyl)amino)octanoate 3-pentyloctyl ester

[0762]

[0763] 8-bromooctanoic acid 3-pentyloctyl ester (1.00g, 2.47mmol), 4-aminobutan-1-ol (6.82mL, 74.0mmol) and ethanol (2mL) were added to a round-bottom flask equipped with a stirring bar. The resulting mixture was stirred at 40 ° C for 16h. The reaction was diluted with dichloromethane, washed with water (2 times), and the layers were separated. The organic layer was dried (MgSO4), filtered and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% (1% NH4OH, 20% MeOH in dichloromethane) / dichloromethane) to obtain 8- ((4-hydroxybutyl) amino) octanoic acid 3-pentyloctyl ester (501mg, 1.21mmol, 49%) as a clear oil. UPLC / ELSD: RT = 1.67min. About C 25 H 51 NO3, MS (ES): m / z (MH + )414.24.

[0764] Intermediate BC: 3-pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate

[0765]

[0766] 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 acetonitrile (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 under vacuum. 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% (1% NH4OH, 20% MeOH in dichloromethane) / 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. 46 H 90 N2O6,MS(ES):m / z(MH + )767.59.

[0767] Intermediate BD: 3-Butylheptyl 8-((3-((tert-Butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate

[0768]

[0769] 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 acetonitrile (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 under vacuum. 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% (1% NH4OH, 20% MeOH in dichloromethane) / 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. 44 H 86 N2O6,MS(ES):m / z(MH + )739.46.

[0770] Intermediate BE: 8,8'-((3-((tert-Butoxycarbonyl)amino)propyl)azanediyl)dioctanoate bis(3-propylhexyl) ester

[0771]

[0772] 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 acetonitrile (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 under vacuum. 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% (1% NH4OH, 20% MeOH in dichloromethane) / 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. 42 H 82 N2O6,MS(ES):m / z(MH + )711.59.

[0773] Intermediate BF: 3-Butylheptyl 8-((3-((tert-Butoxycarbonyl)amino)propyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate

[0774]

[0775] 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 acetonitrile (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 under vacuum. 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% (1% NH4OH, 20% MeOH in dichloromethane) / 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. 48 H94 N2O6,MS(ES):m / z(MH + )795.59.

[0776] Intermediate BG: 3-pentyloctyl 8-((3-((tert-Butoxycarbonyl)amino)propyl)(8-oxo-8-(undec-6-yloxy)octyl)amino)octanoate

[0777]

[0778] UPLC / ELSD:RT=2.93min. MS(ESI):C 48 H 95 N2O6 + (M+H) m / z calculated value 795.288; found value 795.71. 1H NMR (300 MHz, CDCl3) δ: ppm 5.66 (br.s, 1H); 4.88 (p, 1H); 4.09 (t, 2H); 3.18 (br.d, 2H); 2.50 (br.d, 2H); 2.32 (br.d, 3H); 2.29 (t, 4H); 1.65-1.46 (m, 28H); 1.27 (m, 44H); 0.90 (t, 12H).

[0779] Intermediate BH: 8-((3-((tert-Butoxycarbonyl)amino)propyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoic acid nonan-5-yl ester

[0780]

[0781] 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 acetonitrile (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 under vacuum. 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% (1% NH4OH, 20% MeOH in dichloromethane) / 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(300MHz, CDCl3)δ:ppm 5.65 (br.s, 1H); 4.87 (quintet, 2H, J = 6.0Hz); 4.08 (t, 2H, J = 6.0Hz); 3.18 (br.q, 2H, J = 6.0Hz); 2.44 (br.s , 2H); 2.35 (br.s, 2H); 2.28 (t, 4H, J = 6.0Hz); 1.71-1.17 (m, 53H); 1.43 (s, 9H); 0.88 (t, 12H, J = 6.0Hz).

[0782] Intermediate BI: Pentadecyl-8-yl 8-((3-((tert-Butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate

[0783]

[0784] UPLC / ELSD:RT=2.68min. MS(ESI):C 48 H 95 N2O6 + (M+H) m / z calculated value 795.288; found value 795.71. 1H NMR (300 MHz, CDCl3) δ: ppm 5.66 (br.s, 1H); 4.89 (p, 1H); 4.10 (t, 2H); 3.19 (br.d, 2H); 2.56-2.35 (br.d, 5H); 2.30 (t, 5H); 1.66-1.39 (m, 66H); 0.90 (t, 12H).

[0785] Intermediate BJ: 3-propylhexyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-(tridec-7-yloxy)octyl)amino)octanoate

[0786]

[0787] 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 tridecyl-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 and filtered, and the filtrate was evaporated under vacuum. The residue was purified by silica gel chromatography [0-70% (1% NH4OH, 20% MeOH in dichloromethane) / dichloromethane] to afford 3-propylhexyl 8-({3-[(tert-butoxycarbonyl)amino]propyl}[8-oxo-8-(tridec-7-yloxy)octyl]amino)octanoate (1.32 g, 55.1%) as a yellow oil. UPLC / ELSD: RT = 2.70 min found, 767.34. 1 H NMR(300MHz, CDCl3)δ:ppm5.67(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).

[0788] Intermediate BK: 3-propylhexyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-(undec-6-yloxy)octyl)amino)octanoate

[0789]

[0790] 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 undecyl-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 and filtered, and the filtrate was evaporated under vacuum. The residue was purified by silica gel chromatography [0-70% (1% NH4OH, 20% MeOH in dichloromethane) / dichloromethane] to afford 3-propylhexyl 8-({3-[(tert-butoxycarbonyl)amino]propyl}[8-oxo-8-(undecan-6-yloxy)octyl]amino)octanoate (1.53 g, 61.2%) as a yellow oil. UPLC / ELSD: RT = 2.56 min found, 739.46. 1 H NMR(300MHz, CDCl3)δ:ppm5.66(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).

[0791] Intermediate BL: 8-((3-((tert-Butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoic acid nonan-5-yl ester

[0792]

[0793] 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 non-5-yl 8-bromooctanoate (1.18 g, 3.39 mmol) in CMPE (10 mL). The reaction was stirred at 77 ° C for 18 hours. The reaction was cooled to room temperature and filtered, and the filtrate was evaporated under vacuum. The residue was purified by silica gel chromatography [0-70% (1% NH4OH, 20% MeOH in dichloromethane) / dichloromethane] to afford 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 yellow oil. UPLC / ELSD: RT = 2.45 min found, 711.46. 1 H NMR (300MHz, CDCl3) δ: ppm5.66(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).

[0794] Intermediate BM: 3-pentyloctyl 8-((3-aminopropyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate

[0795]

[0796] 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 dichloromethane (18 mL) was added trifluoroacetic acid (1.39 mL, 18.2 mmol). The reaction was stirred at room temperature for 4 h. 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% (1% NH4OH, 20% MeOH in dichloromethane) / 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. 41 H 82 N2O4,MS(ES):m / z(MH + )667.56.

[0797] Intermediate BN: 3-Butylheptyl 8-((3-aminopropyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate

[0798]

[0799] 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 dichloromethane (27 mL) was added trifluoroacetic acid (2.01 mL, 26.3 mmol). The reaction was stirred at room temperature for 4 h. 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% (1% NH4OH, 20% MeOH in dichloromethane) / 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. 39 H 78 N2O4,MS(ES):m / z(MH + )639.31.

[0800] Intermediate BO: 8,8'-((3-aminopropyl)azanediyl)dioctanoate bis(3-propylhexyl) ester

[0801]

[0802] To a solution of 8,8'-((3-((tert-butoxycarbonyl)amino)propyl)azanediyl)bis(3-propylhexyl)dioctanoate (730 mg, 1.03 mmol) in dichloromethane (21 mL) was added trifluoroacetic acid (1.57 mL, 20.5 mmol). The reaction was stirred at room temperature for 4 h. The reaction was quenched with saturated NaHCO3 aqueous solution and extracted with dichloromethane. The organic layer was separated, washed with brine, dried (MgSO4), filtered and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% (1% NH4OH, 20% MeOH in dichloromethane) / dichloromethane) to give 8,8'-((3-aminopropyl)azanediyl)bis(3-propylhexyl)dioctanoate (499 mg, 0.82 mmol, 80%) as a clear oil. UPLC / ELSD: RT = 1.93 min. 37 H 74 N2O4,MS(ES):m / z(MH + )611.44.

[0803] Intermediate BP: 3-Butylheptyl 8-((3-aminopropyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate

[0804]

[0805] 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 dichloromethane (23 mL) was added trifluoroacetic acid (1.72 mL, 22.5 mmol). The reaction was stirred at room temperature for 4 h. The reaction was quenched with saturated NaHCO3 aqueous solution and extracted with dichloromethane. The organic layer was separated, washed with brine, dried (MgSO4), filtered and concentrated. The crude material was purified by silica gel chromatography (0-5-10-25-50-100% (1% NH4OH, 20% MeOH in dichloromethane) / 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. 43 H 86N2O4,MS(ES):m / z(MH + )695.68.

[0806] Intermediate BQ: 3-pentyloctyl 8-((4-aminobutyl)(8-oxo-8-(undec-6-yloxy)octyl)amino)octanoate

[0807]

[0808] UPLC / ELSD:RT=2.49min. MS(ESI):C 44 H 89 N2O6 + (M+H)m / z

[0809] Calculated value 709.198; experimental value 695.43. 1 H NMR (300MHz, CDCl3) δ: ppm4.88(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).

[0810] Intermediate BR: 8-((3-aminopropyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoic acid nonan-5-yl ester

[0811]

[0812] 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 dichloromethane (28 mL) was added trifluoroacetic acid (2.12 mL, 27.7 mmol). The reaction was stirred at room temperature for 4 h. 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% (1% NH4OH, 20% MeOH in dichloromethane) / 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. 41 H 82 N2O4,MS(ES):m / z(MH +)667.43.

[0813] Intermediate BS: 8-((3-aminopropyl)(8-oxo-8-(undec-6-yloxy)octyl)amino)octanoate 3-pentyloctyl ester

[0814]

[0815] UPLC / ELSD:RT=2.34min. MS(ESI):C 43 H 87 N3O6 + (M+H) m / z calculated value 695.171; found 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).

[0816] Intermediate BT: 8-((3-aminopropyl)(8-oxo-8-(tridec-7-yloxy)octyl)amino)octanoic acid 3-propylhexyl ester

[0817]

[0818] To a solution of 3-propylhexyl 8-({3-[(tert-butoxycarbonyl)amino]propyl}[8-oxo-8-(tridec-7-yloxy)octyl]amino)octanoate (1.32 g, 1.72...

Claims

1. A compound of formula (A), (A) or an N-oxide thereof, or a salt or isomer thereof, where R' a It's R' 支链 or R' 环状 ;in R' 支链 yes: R' 环状 yes: in Indicates a connection point; where R aα is H, and R aβ 、R aγ and R aδ Each independently selected from H, C 2-12 Alkyl and C 2-12 A group consisting of alkenyl groups, wherein R aβ 、R aγ and R aδ At least one of which is selected from C 2-12 Alkyl and C 2-12 a group consisting of alkenyl groups; R 2 and R 3 Each is C 1-14 alkyl; R 4 Selected from -(CH2)2OH, -(CH2)3OH, -(CH2)4OH, -(CH2)5OH and The group consisting of R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each R 5 Independently selected from OH, C 1-3 Alkyl, C 2-3 A group consisting of an alkenyl group and H; Each R 6 Independently selected from OH, C 1-3 Alkyl, C 2-3 A group consisting of an alkenyl group and H; R 7 It is H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 alkenyl; Y a It is C 3-6 carbon ring; R*” a Choose from C 1-15 Alkyl and C 2-15 a group consisting of alkenyl groups; l is selected from the group consisting of 1, 2, 3, 4 and 5; s is 2 or 3; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

2. A compound of formula (B), (B) or an N-oxide thereof, or a salt or isomer thereof, where R' a It's R' 支链 or R' 环状 ;in R' 支链 yes: R' 环状 yes: in Indicates a connection point; where R aα and R aβ Each is H, and R aγ and R aδ Each independently selected from H, C 2-12 Alkyl and C 2-12 A group consisting of alkenyl groups, wherein R aγ and R aδ At least one of which is selected from C 2-12 Alkyl and C 2-12 a group consisting of alkenyl groups; R bα 、R bβ 、R bγ and R bδ Each independently selected from H, C 2-30 Alkyl and C 5-20 A group consisting of alkenyl groups, wherein R bα 、R bβ 、R bγ and R bδ At least one of which is selected from C 2-30 Alkyl and C 5-20 a group consisting of alkenyl groups; R 4 Selected from -(CH2)2OH, -(CH2)3OH, -(CH2)4OH, -(CH2)5OH and The group composed of in Indicates a connection point; R 10 is N(R)2; each R is independently selected from C 1-6 Alkyl, C 2-3 and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each R 5 Independently selected from OH, C 1-3 Alkyl, C 2-3 A group consisting of an alkenyl group and H; Each R 6 Independently selected from OH, C 1-3 Alkyl, C 2-3 A group consisting of an alkenyl group 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 alkenyl; Y a It is C 3-6 carbon ring; R*” a Choose from C 1-15 Alkyl and C 2-15 a group consisting of alkenyl groups; l is selected from the group consisting of 1, 2, 3, 4 and 5; s is 2 or 3; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

3. A compound of formula (1-1), (1-1) or its N-oxide, or its salt or isomer, where R' a It's R' 支链 or R' 环状 ;in R' 支链 yes: And R' 环状 yes: and R' b yes: in Indicates a connection point; where R aγ and R bγ Each is independently C 2-12 Alkyl or C 2-12 alkenyl; R 2 and R 3 Each independently selected from C 1-14 Alkyl and C 2-14 a group consisting of alkenyl groups; R 4 is -(CH2)2OH; Each R' is independently C 1-12 Alkyl or C 2-12 alkenyl; Y a It is C 3-6 carbon ring; R*” a Choose from C 1-15 Alkyl and C 2-15 alkenyl; and s is 2 or 3.

4. A compound according to any one of the preceding claims, having one of the following structures:

5. A compound as claimed in any one of the preceding claims, wherein R aγ It is C 2-6 alkyl.

6. A compound as claimed in any one of the preceding claims, wherein R bγ It is C 2-6 alkyl.

7. A compound as claimed in any one of the preceding claims, wherein R aγ and R bγ Each is independently C 2-6 alkyl.

8. A compound as claimed in any one of the preceding claims, wherein R bγ It is C 4-6 alkyl.

9. A compound as claimed in any one of the preceding claims, wherein R 2 and R 3 Each is a C8 alkyl group.

10. A compound as claimed in any one of the preceding claims, wherein Y a It is cyclohexyl or cyclopentyl.

11. A compound as claimed in any one of the preceding claims, wherein R*" a It is a C2 alkyl group or a C3 alkyl group.

12. A compound as claimed in any one of the preceding claims, wherein each R' is independently C 2-5 alkyl.

13. A compound selected from the group consisting of:

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

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

16. The empty LNP of any one of the preceding claims, wherein the phospholipid is selected from the group consisting of: 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 (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-bis(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 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-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof.

17. The empty LNP of any one of the preceding claims, wherein the structural lipid is selected from the group consisting of cholesterol, coprosterol, phytosterol, ergosterol, campesterol, stigmasterol, brassicasterol, and mixtures thereof.

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

19. An empty LNP as claimed in any one of the preceding claims, wherein the PEG lipid is selected from PEG 2k -DMG and PEG-1: and mixtures thereof.

20. A loaded lipid nanoparticle (loaded LNP) comprising the empty LNP of any one of the preceding claims and one or more therapeutic and / or prophylactic agents.

21. The loaded LNP of any one of the preceding claims, wherein the one or more therapeutic and / or prophylactic agents is a nucleic acid.

22. The loaded LNP of any of the preceding claims, wherein the nucleic acid is RNA, and wherein 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.

23. The loaded LNP of any one of the preceding claims, wherein the RNA is mRNA.

24. A pharmaceutical composition comprising the loaded LNP of any one of the preceding claims and a pharmaceutically acceptable carrier.

25. A method of delivering a therapeutic and / or prophylactic agent to cells in a subject, the method comprising administering to the subject the loaded LNP of any one of the preceding claims.

26. A method of delivering a therapeutic and / or prophylactic agent specifically to an organ of a subject, the method comprising administering to the subject the loaded LNP of any one of the preceding claims.

27. A method of producing a polypeptide of interest in cells within a subject, the method comprising administering to the subject the loaded LNP of any preceding claim.

28. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the loaded LNP of any one of the preceding claims.

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

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

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