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

Abstract

To provide novel compounds for delivering one or more therapeutic and / or prophylactic agents to mammalian cells or organs and / or for producing polypeptides, compositions comprising such compounds, and lipid nanoparticle compositions.SOLUTION: The present invention provides a compound of a formula (A), or an N-oxide thereof, or a salt or an isomer thereof, for example. In the formula, R'a is R'branched or R'cyclic.SELECTED DRAWING: None

Description

Technical Field

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

[0002] The present disclosure provides novel compounds for delivering one or more therapeutic and / or prophylactic agents and / or generating polypeptides to mammalian cells or organs, compositions comprising such compounds, and methods involving lipid nanoparticle compositions. In addition to novel lipids, the disclosed lipid nanoparticle compositions can include one or more cationic and / or ionic amino lipids, phospholipids containing polyunsaturated lipids, PEG lipids, structural lipids, and / or therapeutic and / or prophylactic agents at specific fractions.

Background Art

[0003] The effective targeted delivery of bioactive substances such as small molecule drugs, proteins, and nucleic acids is a representative of ongoing medical challenges. In particular, the delivery of nucleic acids to cells is complicated by the relative instability and low cell permeability of such species. Therefore, there is a need to develop methods and compositions for promoting the delivery of therapeutic and / or prophylactic agents such as nucleic acids to cells.

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

Summary of the Invention

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

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

Chem.

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Chem.

Chem.

Chem.

[0007] In some embodiments, the disclosure relates to a compound of formula (2-1):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0008] In some embodiments, the disclosure relates to a compound of formula (A):

Chemical formula

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

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

[0010] In some embodiments, the disclosure relates to a compound of formula (A-a): [Chemical formula] or its N-oxide, or its salt or isomer. Wherein R aβ , Raγ and R aδ each independently represents H, C 2-12 alkyl, and C 2-12 alkenyl, and at least one of R aβ , R aγ , and R aδ is selected from the group consisting of C 2-12 alkyl and C 2-12 alkenyl, R 4 is -(CH 2 ) 2 OH, -(CH 2 ) 3 OH, -(CH 2 ) 4 OH, -(CH 2 ) 5 OH, and is selected from the group consisting of M and M' each independently represent -C(O)O- and -OC(O)-, R' is C 1-12 alkyl or C 2-12 alkenyl.

[0011] In some embodiments, the disclosure relates to a compound of formula (A-b):

Chemical formula

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

[0013] The present disclosure provides a lipid comprising a central amine moiety and at least one biodegradable group. The lipids described herein can be advantageously used in lipid nanoparticles (e.g., empty LNPs or loaded LNPs) for delivering therapeutic and / or prophylactic agents to mammalian cells or organs. For example, the lipids described herein have little or no immunogenicity. For example, lipid compounds of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c) have low immunogenicity compared to reference lipids (e.g., MC3, KC2, or DLinDMA). For example, formulations comprising the lipids and therapeutic or prophylactic agents disclosed herein have an increased therapeutic index compared to corresponding formulations comprising reference lipids (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.

[0014] In some embodiments, the disclosure relates to a compound of formula (A-1):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0015] In some embodiments, the disclosure relates to a compound of formula (A-2):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

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Chem.

[0016] In some embodiments, the disclosure is of formula (A-3):

Chem.

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

Chemical formula

[0018] In some aspects, the disclosure relates to a compound of formula (B-1):

Chemical formula

[0019] In some embodiments, the disclosure relates to a compound of formula (B-2):

Chemical formula

Chemical formula

Chemical formula

[0020] In some embodiments, the disclosure relates to a compound of formula (B-3):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0021] In some embodiments, the disclosure relates to a compound of formula (A-a1):

Chemical formula

Chemical formula

Chemical formula

[0022] In some embodiments, the disclosure relates to a compound of formula (A-a2):

Chemical formula

Chemical formula

Chemical formula

[0023] In some embodiments, the disclosure relates to a compound of formula (A-a3):

Chemical formula

Chemical formula

Chemical formula

[0024] In some embodiments, the disclosure relates to a compound of formula (A-b1):

Chemical formula

Chemical formula

Chemical formula

[0025] In some embodiments, the disclosure relates to a compound of formula (A-b2):

Chemical formula

Chemical formula

Chemical formula

[0026] In some embodiments, the disclosure relates to a compound of formula (A-b3):

Chemical formula

Chemical formula

Chemical formula

[0027] In some embodiments, the disclosure relates to a compound of formula (A-c):

Chemical formula

Chemical formula

[0028] In some embodiments, the disclosure relates to a compound of formula (B-c): [Chemical formula] or its N-oxide, or its salt or isomer, wherein R aγ is C 2 alkyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, or C 6 alkyl, and R bγ is C 2 alkyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, or C 6 alkyl, and R 4 is -(CH 2 ) 2 OH or [Chemical formula] and [Chemical formula] represents a bonding point, and R’ is C 1-12 alkyl.

[0029] In some embodiments, the disclosure relates to a compound of formula (I-a): [Chemical formula] Regarding the compound, in the formula, R 2 and R 3 are each independently selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl, and R aγ and R bγ are each independently C 2-6 alkyl.

[0030] Any of the compounds of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), and (B-c), when applicable, includes one or more of the following features.

[0031] In some embodiments, R 4 is

Chemical formula

Chemical formula

Chemical formula

[0032] In some embodiments, R 10 is -NH 2 . In some embodiments, R 10 is -NH(C 1-6 alkyl). In some embodiments, R 10 is -N(C 1-6 alkyl) 2It is. In some embodiments, R 10 is -NH(CH 3 ). In some embodiments, R 10 is -N(CH 3 ) 2 .

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

[0034] In some embodiments, R 4 is

Chemical formula

[0035] In some embodiments, R 4 is

Chemical formula

Chemical formula

Chem.

Chem.

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

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

[0038] In some embodiments, R 5 , R 6 、 and R 7 are each H and m is 5, 6, 7, 8, or 9. In some embodiments, R 5 , R 6 、 and R 7 are each H and m is 5. In some embodiments, R 5 , R 6 、 and R 7 are each H and m is 7.

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

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

[0041] In some embodiments, R 2 and R 3 are each C 7 alkyl. In some embodiments, R 2 and R 3 are each C 8 alkyl. In some embodiments, R 2 and R 3 are each C 9 alkyl.

[0042] In some embodiments, R’ a is R’ 分岐状 where R aα , R aγ , and R aδ are each H, and R aβ is C 2 -C 6is alkyl. In some embodiments, R’ a is R’ 分岐状 and R aα 、R aβ 、and R aδ are each H, and R aγ is C 2 -C 6 alkyl. In some embodiments, R’ a is R’ 分岐状 and R aα 、R aβ 、and R aγ are each H, and R aδ is C 2 -C 6 alkyl.

[0043] In some embodiments, R bα 、R bγ 、and R bδ are each H, and R bβ is C 2 -C 6 alkyl. In some embodiments, R bα 、R bβ 、and R bδ are each H, and R bγ is C 2 -C 6 alkyl. In some embodiments, R bα 、R bβ 、and R bγ are each H, and R bδ is C 2 -C 6 alkyl.

[0044] In some embodiments, R aα 、R aγ 、and R aδ are each H, and R aβ is C 2 -C 6 alkyl, and R bα 、R bγ 、and R bδ are each H, and R bβ is C 2 -C 6 alkyl. In some embodiments, Raα , R aγ , and R aδ are each H, and R aβ is C 2 -C 6 alkyl, and R bα , R bβ , and R bδ are each H, and R bγ is C 2 -C 6 alkyl. In some embodiments, R aα , R aγ , and R aδ are each H, and R aβ is C 2 -C 6 alkyl, and R bα , R bβ , and R bγ are each H, and R bδ is C 2 -C 6 alkyl.

[0045] In some embodiments, R aα , R aβ , and R aδ are each H, and R aγ is C 2 -C 6 alkyl, and R bα , R bγ , and R bδ are each H, and R bβ is C 2 -C 6 alkyl. In some embodiments, R aα , R aβ , and R aδ are each H, and R aγ is C 2 -C 6 alkyl, and R bα , R bβ , and R bδ are each H, and R bγ is C 2 -C 6 alkyl. In some embodiments, R aα , R aβ , and R aδis each H, and R aγ is C 2 -C 6 is alkyl, and R bα , R bβ , and R bγ are each H, and R bδ is C 2 -C 6 is alkyl.

[0046] In some embodiments, R aα , R aβ , and R aγ are each H, and R aδ is C 2 -C 6 is alkyl, and R bα , R bγ , and R bδ are each H, and R bβ is C 2 -C 6 is alkyl. In some embodiments, R aα , R aβ , and R aγ are each H, and R aδ is C 2 -C 6 is alkyl, and R bα , R bβ , and R bδ are each H, and R bγ is C 2 -C 6 is alkyl. In some embodiments, R aα , R aβ , and R aγ are each H, and R aδ is C 2 -C 6 is alkyl, and R bα , R bβ , and R bγ are each H, and R bδ is C 2 -C 6 is alkyl.

[0047] In some embodiments, R’ is C 1-12 alkyl or C 2-12is alkenyl. In some embodiments, R’ is C 2 is alkyl. In some embodiments, R’ is C 3 or C 4 is alkyl. In some embodiments, R’ is C 3 is alkyl. In some embodiments, R’ is C 4 is alkyl. In some embodiments, R’ is C 5 is alkyl.

[0048] In some embodiments, R’ is C 4 alkyl or C 4 is alkenyl. In some embodiments, R’ is C 5 alkyl or C 5 is alkenyl. In some embodiments, R’ is C 6 alkyl or C 6 is alkenyl. In some embodiments, R’ is C 7 alkyl or C 7 is alkenyl. In some embodiments, R’ is C 8 alkyl or C 8 is alkenyl. In some embodiments, R’ is C 9 alkyl or C 9 is alkenyl. In some embodiments, R’ is C 10 alkyl or C 10 is alkenyl. In some embodiments, R’ is C 11 alkyl or C 11 is alkenyl.

[0049] In some embodiments, R’ a is, R’ 分岐状 and R aα , R aγ , and R aδ are each H, and R aβ is C 2 -C 6 is alkyl, and R’ is C 3 -C 5 is alkyl. In some embodiments, R’ ais R’ 分岐状 and R aα R aγ and R aδ are each H, R aβ is C 2 -C 6 alkyl, and R’ is C 3 alkyl. In some embodiments, R’ a is R’ 分岐状 and R aα R aγ and R aδ are each H, R aβ is C 2 -C 6 alkyl, and R’ is C 4 alkyl.

[0050] In some embodiments, R’ a is R’ 分岐状 and R aα R aβ and R aδ are each H, R aγ is C 2 -C 6 alkyl, and R’ is C 3 -C 5 alkyl. In some embodiments, R’ a is R’ 分岐状 and R aα R aβ and R aδ are each H, R aγ is C 2 -C 6 alkyl, and R’ is C 3 alkyl. In some embodiments, R’ a is R’ 分岐状 and R aα R aβ and R aδ are each H, R bγ is C 2 -C 6 alkyl, and R’ is C 4 alkyl.

[0051] In some embodiments, R’ ais R’ 分岐状 and R aα 、R aβ 、and R aγ are each H, and R aδ is C 2 -C 6 alkyl, and R’ is C 3 -C 5 alkyl. In some embodiments, R’ a is R’ 分岐状 and R aα 、R aβ 、and R aγ are each H, and R aδ is C 2 -C 6 alkyl, and R’ is C 3 alkyl. In some embodiments, R’ a is R’ 分岐状 and R aα 、R aβ 、and R aγ are each H, and R aδ is C 2 -C 6 alkyl, and R’ is C 4 alkyl.

[0052] In some embodiments, R * ” a is C 2 -alkyl or C 3 -alkyl.

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

[0054] In some embodiments, s is 2 and R * ” a is C 2 alkyl or C 3 -alkyl.

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

Chemical Formula

Chem.

[0056] In some embodiments, s is 2 and Y a R * ” a is

Chem.

Chem.

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

[0058] In some embodiments, the compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c) is selected from the compounds of Table 1, and their N-oxides, salts, or isomers.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

[0059] The central amine moiety of the lipid by formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c) can be protonated at physiological pH. Thus, the lipid can have a positive or partially positive charge at physiological pH. Such lipids may be referred to as cationic or ionic (amino) lipids. The lipid may also be zwitterionic, i.e., a neutral molecule having both a positive and a negative charge.

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

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

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

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

[0064] "Aryl" includes a "complex", or polycyclic system having at least one aromatic ring that does not contain heteroatoms in the ring structure. Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthalenyl, and the like. In some embodiments, "aryl" is an aromatic C6-10 a carbon ring (e.g., "aryl" is C 6-10 is aryl).

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

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

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

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

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

[0070] Alkyl, alkenyl, and cyclil (e.g., carbocyclil and heterocyclil) groups may be optionally substituted unless otherwise specified. Any substituent is not limited, but includes a halogen atom (e.g., chloride, bromide, fluoride, or iodide group), carboxylic acid (e.g., -C(O)OH), alcohol (e.g., hydroxyl, -OH), ester (e.g., -C(O)OR or -OC(O)R), aldehyde (e.g., -C(O)H), carbonyl (e.g., -C(O)R, alternatively represented by C=O), acyl halide (e.g., -C(O)X where X is a halide selected from bromide, fluoride, chloride, and iodide), carbonate (e.g., -OC(O)OR), alkoxy (e.g., -OR), acetal (e.g., -C(OR) 2 R””) where each OR may be the same or different alkoxy groups and R”” is an alkyl or alkenyl group, phosphate (e.g., P(O) 4 3- ), thiol (e.g., -SH), sulfoxide (e.g., -S(O)R), sulfinic acid (e.g., -S(O)OH), sulfonic acid (e.g., -S(O) 2 OH), thial (e.g., -C(S)H), sulfate (e.g., S(O) 4 2- ), sulfonyl (e.g., -S(O) 2 -), amide (e.g., -C(O)NR 2 or -N(R)C(O)R), azide (e.g., -N 3 ), nitro (e.g., -NO 2 ), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(O)R), amino (e.g., -NR 2 , -NRH, or -NH 2 ), carbamoyl (e.g., -OC(O)NR 2 , -OC(O)NRH, or -OC(O)NH 2 ), sulfonamide (e.g., -S(O) 2 NR 2 , -S(O) 2 NRH, -S(O) 2 NH 2 , -N(R)S(O)2 R, -N(H)S(O) 2 R, -N(R)S(O) 2 H, or -N(H)S(O) 2 H), an alkyl group, an alkenyl group, and a cyclil (e.g., carbocyclil or heterocyclil) group. In any of the foregoing, R is an alkyl or alkenyl group as defined herein. In some embodiments, the substituent itself may be further substituted with, for example, 1, 2, 3, 4, 5, or 6 substituents as defined herein. For example, C 1-6 The alkyl group may be further substituted with 1, 2, 3, 4, 5, or 6 substituents as described herein.

[0071] The disclosed compounds containing nitrogen can be converted to N-oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (mCPBA) and / or hydrogen peroxide) to obtain other compounds of the disclosure. Accordingly, all nitrogen-containing compounds shown and claimed are considered to include both the shown compounds and their N-oxide derivatives (designated as N→O or N + -O - if so specified). Further, in other cases, the nitrogen in the disclosed compounds can be converted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine with an oxidizing agent such as m-CPBA. All nitrogen-containing compounds shown and claimed are also considered to cover both the shown compounds and their N-hydroxy (i.e., N-OH) and N-alkoxy (i.e., where R is substituted or unsubstituted C 1 -C 6 alkyl, C 1 -C 6 alkenyl, C 1 -C 6 alkynyl, a 3- to 14-membered carbocyclic ring, or a 3- to 14-membered heterocyclic ring, N-OR) derivatives.

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

[0073] As used herein, the term "compound" is meant to include all isomers and isotopes of the indicated structure. "Isotope" refers to an atom having the same atomic number but a different mass number, arising from a different number of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. Further, the compounds, salts, or complexes of the present disclosure can be combined with a solvent or water molecules by conventional methods to form solvates and hydrates.

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

[0075] As used herein, the term "delivering" means providing to a location of interest. For example, delivering a therapeutic and / or prophylactic agent may involve administering a nanoparticle composition comprising the therapeutic and / or prophylactic agent to a subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administration of a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

[0076] As used herein, the term "enhanced delivery" means that the delivery level of a therapeutic agent and / or prophylactic agent by nanoparticles to a target cell of interest (e.g., mammalian liver) is higher (e.g., at least 1.5-fold or more, at least 2-fold or more, at least 3-fold or more, at least 4-fold or more, at least 5-fold or more, at least 6-fold or more, at least 7-fold or more, at least 8-fold or more, at least 9-fold or more, at least 10-fold or more) compared to the delivery level of the therapeutic agent and / or prophylactic agent by control nanoparticles to a target cell of interest (e.g., MC3, KC2, or DLinDMA). The delivery level of nanoparticles to a specific tissue can be measured by comparing the amount of protein produced in the tissue with the weight of the tissue, comparing the amount of therapeutic agent and / or prophylactic agent in the tissue with the weight of the tissue, comparing the amount of protein produced in the tissue with the total amount of protein in the tissue, or comparing the amount of therapeutic agent and / or prophylactic agent in the tissue with the total amount of therapeutic agent and / or prophylactic agent in the tissue. It will be understood that enhanced delivery of nanoparticles to a target cell need not be determined in the subject being treated, but can be determined in a surrogate such as an animal model (e.g., a rat model). In certain embodiments, nanoparticle compositions comprising a compound according to formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c) have substantially the same level of enhanced delivery regardless of the route of administration. For example, certain compounds disclosed herein exhibit similar enhanced delivery whether used to deliver a therapeutic agent and / or prophylactic agent intravenously or intramuscularly. In other embodiments, certain compounds disclosed herein exhibit a higher level of enhanced delivery when used to deliver a therapeutic agent and / or prophylactic agent intramuscularly rather than intravenously.

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

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

[0079] As used herein, "encapsulated", "encapsulating", "filled", and "associated" can refer to complete, substantial, or partial sealing, confinement, enclosure, or stuffing. As used herein, "encapsulation" or "association" can refer to the process of confining individual nucleic acid molecules within nanoparticles and / or the process of establishing a physicochemical relationship between individual nucleic acid molecules and nanoparticles. As used herein, "empty nanoparticles" can refer to nanoparticles that are substantially free of therapeutic or prophylactic agents. As used herein, "empty nanoparticles" or "empty lipid nanoparticles" can refer to nanoparticles that are substantially free of nucleic acids. As used herein, "empty nanoparticles" or "empty lipid nanoparticles" can refer to nanoparticles that are substantially free of nucleotides or polypeptides. As used herein, "empty nanoparticles" or "empty lipid nanoparticles" can refer to nanoparticles consisting essentially of only a lipid component. As used herein, "filled nanoparticles" or "filled lipid nanoparticles" (also referred to as "complete nanoparticles" or "complete lipid nanoparticles") can refer to nanoparticles that contain the components of empty nanoparticles and a therapeutic or prophylactic agent. As used herein, "filled nanoparticles" or "filled lipid nanoparticles" (also referred to as "complete nanoparticles" or "complete lipid nanoparticles") can refer to nanoparticles that contain the components of empty nanoparticles and a nucleotide or polypeptide. As used herein, "filled nanoparticles" or "filled lipid nanoparticles" (also referred to as "complete nanoparticles" or "complete lipid nanoparticles") can refer to nanoparticles that contain the components of empty nanoparticles and a nucleic acid.

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

[0081] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within a living organism (e.g., an animal, a plant, or a microorganism).

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

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

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

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

[0086] Of the various types of tautomerism that are possible, two are commonly observed. In keto-enol tautomerism, a simultaneous shift of electrons and hydrogen atoms occurs. Ring-chain tautomerism results from the reaction of the aldehyde group (-CHO) of a sugar molecule with one of the hydroxy groups (-OH) of the same molecule, giving rise to the cyclic (ring-shaped) form presented by glucose.

[0087] Common tautomeric pairs are the keto-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomerism, imine-enamine, and enamine-enamine of heterocyclic rings (such as nucleic acid bases like guanine, thymine, and cytosine). An example of tautomerism in a disubstituted guanidine is shown below. [Chemical formula]

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

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

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

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

[0092] As used herein, "modified" means non-natural. For example, RNA can be modified RNA. That is, RNA can contain one or more nucleobases, nucleosides, nucleotides, or linkers that are not naturally occurring. "Modified" species can also be referred to herein as "altered" species. Species can be modified or altered chemically, structurally, or functionally. For example, a modified nucleobase species can contain one or more substitutions that do not occur naturally.

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

[0094] As used herein, a "nanoparticle composition" is a composition containing one or more lipids. Nanoparticle compositions are typically on the order of a few micrometers or less in size and can include a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0095] As used herein, "naturally occurring" means occurring in nature without artificial assistance.

[0096] As used herein, "patient" refers to an individual who may seek, be in need of, require, be receiving, be undergoing, or be treated by a professional trained in a particular disease or condition.

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

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

[0099] As used herein, the term "pharmaceutically acceptable excipient" refers to any component other than the compounds described herein (e.g., a vehicle capable of suspending, complexing, or dissolving the active compound) and has the properties of being substantially non-toxic and non-inflammatory in a patient. Excipients include, for example, antiadherents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film formers or coating agents, flavors, fragrances, glidants, 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, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and other species disclosed herein.

[0100] The structural formulas of the compounds herein may, in some cases, represent certain isomers for convenience, but it is understood that the present disclosure includes all isomers such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc., and not all isomers may have the same level of activity. Additionally, crystal polymorphs may exist for the compounds represented by the formulas. It should be noted that any crystalline form, mixture of crystalline forms, or their anhydrates or hydrates are included within the scope of the present disclosure.

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

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

[0103] As used herein, "phospholipid" is a lipid that contains a phosphate moiety and one or more carbon chains such as unsaturated fatty acid chains. Phospholipids can contain one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). Certain phospholipids can promote fusion to a membrane. For example, cationic phospholipids can interact with one or more negatively charged phospholipids of a membrane (e.g., a cell membrane or an intracellular membrane). By fusing the phospholipid to the membrane, one or more components of the lipid-containing composition can pass through the membrane, for example, enabling delivery of one or more components to a cell.

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

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

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

[0107] As used herein, "DNA" refers to deoxyribonucleic acid, which can be naturally or non-naturally occurring. For example, DNA can be a synthetic molecule, such as a synthetic DNA molecule generated in vitro. In some embodiments, the DNA molecule is a recombinant molecule. As used herein, "recombinant DNA molecule" refers to a DNA molecule that does not exist as a natural product but is generated using molecular biology techniques.

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

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

[0110] As used herein, "total daily dose" is the amount given or prescribed over a 24-hour period. This can be administered as a single unit dose.

[0111] As used herein, "size" or "average size" in the context of lipid nanoparticles (e.g., empty LNP or loaded LNP) refers to the average diameter of the nanoparticle composition.

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

[0113] As used herein, "target cell" refers to any one or more cells of interest. Cells can be found in vitro, in vivo, in situ, or in the tissues or organs of an organism. The organism can be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

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

[0115] The term "therapeutic agent" or "prophylactic agent" refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect. A therapeutic agent is also referred to as an "active agent" or "active ingredient". Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0116] As used herein, the term "therapeutically effective amount" means an amount of a delivered agent (e.g., nucleic acid, drug, composition, therapeutic, diagnostic agent, prophylactic agent, etc.) that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat the infection, disease, disorder, and / or condition, ameliorate, diagnose, prevent, and / or delay the onset thereof.

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

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

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

[0120] Nanoparticle composition The disclosure also features lipid nanoparticles comprising a compound according to formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c) described herein.

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

[0122] The nanoparticle composition comprises a lipid component comprising at least one compound according to formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c). For example, the lipid component of the nanoparticle composition can comprise one or more of the compounds in Table 1. The nanoparticle composition can also include various other components. For example, in addition to the lipid according to formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), the lipid component of the nanoparticle composition can include one or more other lipids.

[0123] Cationic / Ionic Lipid Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can include one or more cationic and / or ionic lipids (e.g., lipids that can have a positive or partial positive charge at physiological pH) in addition to the lipids according to formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c).Cationic and / or ionic lipids can be selected from the non-limiting group consisting of 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), octatriacontan-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-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)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)), and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)). In addition to these, the cationic lipid can also be a lipid containing a cyclic amine group.

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

Chemical formula

[0125] Phospholipid Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may contain one or more phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids may be assembled into one or more lipid bilayers. Generally, a phospholipid may contain a phospholipid moiety and one or more fatty acid moieties. For example, a phospholipid may be of formula (IV):

Chemical formula

[0126] Phospholipids useful in compositions and methods include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-didecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2 cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof. In some embodiments, the lipid nanoparticles (e.g., empty LNP or loaded LNP) comprise DSPC. In certain embodiments, the lipid nanoparticles (e.g., empty LNP or loaded LNP) comprise DOPE. In some embodiments, the lipid nanoparticles (e.g., empty LNP or loaded LNP) comprise both DSPC and DOPE.

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

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

[0129] In certain embodiments, the PEG lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (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.

[0130] In certain embodiments, the PEG lipid has the formula (PL-I):

Chemical formula

Chemical formula

[0131] In certain embodiments, the PEG lipid is of the formula (PL-I-OH): [Chemical formula] a compound of, or a salt thereof.

[0132] In certain embodiments, the PEG lipid is of the formula (PL-II-OH): [Chemical formula] a compound of, or a salt or isomer thereof, wherein R 3PEG is -OR O and R O is hydrogen, C 1-6 alkyl, or an oxygen protecting group, r PEG is an integer from 1 to 100, R 5PEG is C 10-40 alkyl, C 10-40 alkenyl, or C10-40 is alkynyl, and optionally, one or more of the methylene groups of R 5PEG 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) 2 O-, -OS(O) 2 O-, -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)-, -S(O) 2 -, -S(O) 2 N(R NPEG )-, -N(R NPEG )S(O)-, -S(O) 2 N(R NPEG )-, -OS(O) 2 N(R NPEG)-, or -N(R NPEG )S(O) 2 is replaced by O-, and R NPEG in each case is, independently, hydrogen, C 1-6 alkyl, or a nitrogen protecting group.

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

[0134] In certain embodiments, for the PEG lipid of formula (PL-II-OH), R 5 is C 17 alkyl.

[0135] In certain embodiments, the PEG lipid is of formula (PL-II):

Chemical formula

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

Chemical formula

[0137] In certain embodiments, the PEG lipid is of formula (PL-III):

Chemical formula

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

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

[0140] adjuvant In some embodiments, lipid nanoparticles (e.g., empty LNPs or loaded LNPs) comprising one or more of the lipids described herein may further comprise 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.

[0141] therapeutic agent Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may contain one or more therapeutic and / or prophylactic agents. The disclosure features methods of delivering a therapeutic and / or prophylactic agent to mammalian cells or organs, methods of producing a polypeptide of interest in mammalian cells, and methods of treating a disease or disorder in a mammal in need of treatment for the disease or disorder, comprising administering to the mammal a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) containing the therapeutic and / or prophylactic agent, and / or contacting mammalian cells with a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) containing the therapeutic and / or prophylactic agent.

[0142] Therapeutic and / or prophylactic agents include biologically active substances and are alternatively referred to as "active agents". Once delivered to a cell or organ, a therapeutic and / or prophylactic agent can be a substance that brings about a desirable change in the cell, organ, or other body tissue or system. Such agents can be useful for the treatment of one or more diseases, disorders, or conditions. In some embodiments, the therapeutic and / or prophylactic agent is a small molecule drug useful for the treatment of a particular disease, disorder, or condition.

[0143] In some embodiments, the therapeutic and / or prophylactic agent is a vaccine that induces an immune response, a compound (e.g., a polynucleotide or nucleic acid molecule encoding a protein or polypeptide or peptide, or a protein or polypeptide or protein), and / or another therapeutic and / or prophylactic agent. Vaccines include compounds and preparations capable of providing immunity against one or more conditions associated with infectious diseases and may include mRNA encoding an antigen and / or epitope derived from an infectious disease. Vaccines can also include compounds and preparations that induce an immune response against cancer cells and may include mRNA encoding an antigen, epitope, and / or neoepitope derived from a tumor cell. In some embodiments, the vaccine and / or compound capable of inducing an immune response is administered intramuscularly via the disclosed composition.

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

[0145] Polynucleotides and Nucleic Acids In some embodiments, the therapeutic agent is an agent that improves (i.e., increases, stimulates, upregulates) protein expression. Non-limiting examples of types of therapeutic agents that can be used to improve 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 modified to improve half-life, or those containing desired amino acid changes). Exemplary proteins include intracellular, transmembrane, or secreted proteins, peptides, or polypeptides.

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

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

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

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

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

[0151] In some embodiments, in the disclosed filled LNPs, one or more therapeutics and / or prophylactics are selected from the group consisting of plasmid expression vectors, viral expression vectors, and mixtures thereof.

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

[0153] For example, in some embodiments, when the therapeutic agent 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, ribozyme, dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), locked nucleic acid (LNA), and CRISPR / Cas9 technology, and mixtures thereof.

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

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

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

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

[0158] The 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). The mRNA can contain any suitable number of base pairs, including tens (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100), hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900), or thousands (e.g., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000) of base pairs. Any number (e.g., all, some, or none) of the nucleobases, nucleosides, or nucleotides can be orthologs, substitutions, modifications, or other analogs of non-naturally occurring ones. In certain embodiments, all types of a particular nucleobase can be modified. In some embodiments, all uracil or uridine is modified. When all nucleobases, nucleosides, or nucleotides are modified, e.g., when all uracil or uridine is modified, the mRNA can be referred to as, e.g., "fully modified" uracil or uridine.

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

[0160] The 5’ cap structure or cap species 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 species can include one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap can include a guanine nucleotide and a 7-methylated guanine (G) nucleotide joined by a triphosphate bond at the 5’ position, e.g., m7G(5′)ppp(5′)G, generally written as m7GpppG. The cap species can also be an anti-reverse cap analog. A non-limiting listing of possible cap species includes m7GpppG, m7Gpppm7G, m73′dGpppG, m27,O3′GpppG, m27,O3′GppppG, m27,O2′GppppG, m7Gpppm7G, m73′dGpppG, m27,O3′GpppG, m27,O3′GppppG, and m27,O2′GppppG.

[0161] The mRNA can alternatively or additionally include a chain-terminating nucleoside. For example, chain-terminating nucleosides can include nucleosides that are deoxygenated at the 2’ and / or 3’ positions of the sugar moiety. Such species can include 3’-deoxyadenosine (cordycepin), 3’-deoxyuridine, 3’-deoxycytosine, 3’-deoxyguanosine, 3’-deoxythymidine, and 2’,3’-dideoxynucleosides such as 2’,3’-dideoxyadenosine, 2’,3’-dideoxyuridine, 2’,3’-dideoxycytosine, 2’,3’-dideoxyguanosine, and 2’,3’-dideoxythymidine. In some embodiments, for example, incorporation of a chain-terminating nucleotide at the 3’ end of the mRNA can result in stabilization of the mRNA.

[0162] The 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 within, before, or after the untranslated region (5' untranslated region or 3' untranslated region), the coding region, or the polyA sequence, or the tail. In some embodiments, the stem loop may affect one or more functions of the mRNA, such as translation initiation, translation efficiency, and / or transcription termination.

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

[0164] The mRNA may alternatively or additionally contain a microRNA binding site. The microRNA binding site (or miR binding site) can be used to regulate mRNA expression in various tissues or cell types. In an exemplary embodiment, the miR binding site is engineered into the 3’UTR sequence of the mRNA to regulate, e.g., enhance, the degradation of the mRNA in cells or tissues that express the cognate miR. Such regulation is useful for modulating or controlling “off-target” expression of the ir mRNA, i.e., in vivo expression in unwanted cells or tissues. Details regarding the use of mir binding sites can be found, for example, in WO2017 / 062513A2, the content of which is incorporated herein by reference.

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

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

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

[0168] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-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-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having 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-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine) are included.,

[0169] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having 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 (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2’-O-methyl-cytidine (Cm), 5,2’-O-dimethyl-cytidine (m5Cm), N4-acetyl-2’-O-methyl-cytidine (ac4Cm), N4,2’-O-dimethyl-cytidine (m4Cm), 5-formyl-2’-O-methyl-cytidine (f5Cm), N4,N4,2’-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2’-F-ara-cytidine, 2’-F-cytidine, and 2’-OH-ara-cytidine.

[0170] In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having modified adenines include α-thio-adenosine, 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m1A), 2-methyl-adenosine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenosine, 2-methoxy-adenosine, α-thio-adenosine, 2’-O-methyl-adenosine (Am), N6,2’-O-dimethyl-adenosine (m6Am), N6,N6,2’-O-trimethyl-adenosine (m62Am), 1,2’-O-dimethyl-adenosine (m1Am), 2’-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2’-F-ara-adenosine, 2’-F-adenosine, 2’-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0171] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanosine include α-thio-guanosine, inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), webtosine (yW), peroxwebtosine (o2yW), hydroxywebtosine (OhyW), hypomodified hydroxywebtosine (OhyW *) 7-Deazaguanosine, queosine (Q), epoxyqueosine (oQ), galactosyl-queosine (galQ), mannosyl-queosine (manQ), 7-cyano-7-deazaguanosine (preQ0), 7-aminomethyl-7-deazaguanosine (preQ1), archaeosine (G+), 7-deaza-8-azaguanosine, 6-thio-guanosine, 6-thio-7-deazaguanosine, 6-thio-7-deaza-8-azaguanosine, 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-ara-guanosine, and 2'-F-guanosine are included.

[0172] In some embodiments, the disclosed mRNA comprises one or more combinations of the foregoing modified nucleobases (e.g., combinations of 2, 3, or 4 of the foregoing modified nucleobases).

[0173] In some embodiments, the modified nucleobase is pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2'-O-methyluridine. In some embodiments, the disclosed mRNA comprises one or more combinations of the foregoing modified nucleobases (e.g., combinations of 2, 3, or 4 of the foregoing modified nucleobases). In some embodiments, the modified nucleobase is N1-methylpseudouridine (m1ψ), and the disclosed mRNA is fully modified with N1-methylpseudouridine (m1ψ). In some embodiments, N1-methylpseudouridine (m1ψ) corresponds to 75-100% of the uracil in the mRNA. In some embodiments, N1-methylpseudouridine (m1ψ) corresponds to 100% of the uracil in the mRNA.

[0174] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a 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, the disclosed mRNA comprises one or more combinations of the foregoing modified nucleobases (e.g., combinations of 2, 3, or 4 of the foregoing modified nucleobases).

[0175] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenosine (m2A), N6-methyl-adenosine (m6A). In some embodiments, the disclosed mRNA comprises one or more combinations of the foregoing modified nucleobases (e.g., combinations of 2, 3, or 4 of the foregoing modified nucleobases).

[0176] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine. In some embodiments, the disclosed mRNA comprises one or more combinations of the foregoing modified nucleobases (e.g., combinations of 2, 3, or 4 of the foregoing modified nucleobases).

[0177] In some embodiments, the modified nucleobase is 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine, or α-thio-adenosine. In some embodiments, the disclosed mRNA comprises one or more combinations of the foregoing modified nucleobases (e.g., combinations of 2, 3, or 4 of the foregoing modified nucleobases).

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

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

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

[0181] The disclosed mmRNA may include combinations of modifications to the sugar, nucleobase, and / or the internucleoside linkage. These combinations may include any one or more of the modifications described herein.

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

[0183] The mRNA of the present disclosure or a region thereof can be codon-optimized. Methods of codon optimization are known in the art and serve various purposes as follows: to match codon frequencies in the host organism to ensure proper folding; to bias the G / C content to increase mRNA stability or reduce secondary structure; to minimize tandem repeat codons or runs of bases that can impair gene construction or expression; to customize transcriptional and translational control regions; to insert or remove protein transport sequences; to remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein; to add, remove, or shuffle protein domains; to insert or delete restriction sites; to modify ribosome binding sites and mRNA degradation sites; to adjust the translation rate to allow proper folding of various domains of the protein; or to reduce or eliminate problematic secondary structure within the polynucleotide. Codon optimization tools, algorithms, and services are known in the art and include, by way of non-limiting example, services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA), and / or proprietary methods. In some embodiments, the mRNA sequence is optimized using an optimization algorithm to optimize expression, for example, in mammalian cells or to improve mRNA stability.

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

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

[0186] Non-natural modified nucleobases can be introduced into polynucleotides, such as mRNA, during or after synthesis. In certain embodiments, the modification can be present on the internucleoside linkage, purine or pyrimidine base, or sugar. In specific embodiments, the modification can be introduced using chemical synthesis or polymerase enzymes at the ends of the polynucleotide chain or at any other location within the polynucleotide chain.

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

[0188] Therapeutic agents for reducing protein expression In some embodiments, the therapeutic agent is a therapeutic agent that reduces (i.e., decreases, inhibits, downregulates) protein expression. Non-limiting examples of types of therapeutic agents that can be used to reduce protein expression include microRNA binding site(s) (miR binding site), microRNA (miRNA), antagomir, small (short) interfering RNA (siRNA) (including shortmers and dicer substrate RNAs), RNA interference (RNAi) molecules, antisense RNA, ribozyme, small hairpin RNA (shRNA), locked nucleic acid (LNA), and mRNA incorporating CRISPR / Cas9 technology.

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

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

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

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

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

[0194] A polymer may be included and / or used to encapsulate or partially encapsulate the nanoparticle composition. The polymer may be biodegradable and / or biocompatible. The polymer may be selected from, but not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polyethylacrylates, polyacrylonitriles, and polyarylates. For example, polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-glycolic acid) (PLGA), poly(L-lactic acid-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,Examples may include polyalkylenes such as 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, polyethylene, and polypropylene; polyalkylene glycols such as poly(ethylene glycol) (PEG); polyalkylene oxides (PEO); polyalkylene terephthalates such as poly(ethylene terephthalate); polyvinyl alcohol (PVA); polyvinyl ethers; polyvinyl esters such as poly(vinyl acetate); polyvinyl halides such as poly(vinyl chloride) (PVC); polyvinyl pyrrolidone (PVP); polysiloxanes; polystyrene (PS); polyurethanes; derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose; poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), and polymers of acrylic acid, copolymers and mixtures thereof; polydioxanone and copolymers thereof; polyhydroxyalkanoates; polypropylene fumarate; polyoxymethylene; poloxamers; poloxamines; poly(ortho)esters; poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone); trimethylene carbonate; poly(N-acryloylmorpholine) (PAcM); poly(2-methyl-2-oxazoline) (PMOX); poly(2-ethyl-2-oxazoline) (PEOZ); and polyglycol.

[0195] Examples of surface modifiers include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, magnwort, bromelain, papain, clerodendrum, bromhexine, carbocysteine, epratrizone, mesna, ambroxol, sobrerol, domiodol, restelin, stepronin, thioproline, gelrin, thymosin, β4, dornase, alpha, nertenexin, and erdostein), and DNases (e.g., rhDNase). The surface modifier can be disposed (e.g., by coating, adsorption, covalent bonding, or other processes) within the nanoparticles and / or on the surface of lipid nanoparticles (e.g., empty LNPs or loaded LNPs).

[0196] Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can also contain one or more functionalized lipids. For example, the lipid may be functionalized with an alkyne group that can undergo an addition cyclization reaction when exposed to azide under appropriate reaction conditions. In particular, the lipid bilayer may be functionalized in this way with one or more groups useful for promoting membrane penetration, cell recognition, or imaging. The surface of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful in targeted cell delivery, imaging, and membrane penetration are well known in the art.

[0197] In addition to these components, the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can contain any substance useful in a pharmaceutical composition. For example, the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can contain, without limitation, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, fluids, liquid vehicles, binders, surfactants, isotonic agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives, and one or more pharmaceutically acceptable excipients or accessory components such as other species. Excipients such as waxes, butters, colorants, coating agents, flavors, and fragrances may also be included.

[0198] Examples of diluents can 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, dried starch, corn starch, powdered sugar, and / or combinations thereof. Granulating and dispersing agents can be selected from the non-limiting list consisting of potato starch, corn starch, tapioca starch, sodium glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM (registered trademark)), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0199] As the surfactant and / or emulsifier, although not limited thereto, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, condurrox, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM (registered trademark) [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 polymer, and carboxyvinyl polymer), carrageenan, cellulose derivatives (e.g., sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN (registered trademark) 20], polyoxyethylene sorbitan [TWEEN (registered trademark) 60], polyoxyethylene sorbitan monooleate [TWEEN (registered trademark) 80], sorbitan monopalmitate [SPAN (registered trademark) 40], sorbitan monostearate [SPAN (registered trademark) 60], sorbitan tristearate [SPAN (registered trademark) 65], glyceryl monooleate, sorbitan monooleate [SPAN (registered trademark) 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ (registered trademark) 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL (registered trademark)), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR (registered trademark)), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ (registered trademark) 30]), poly(vinyl pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate,Potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC® F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, doxate sodium, and / or combinations thereof may be mentioned.

[0200] Binders may be starch (e.g., corn starch and starch paste), gelatin, saccharides (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl pyrrolidone), magnesium aluminum silicate (VEEGUM® and arabinogalactan of the genus Larix), alginate, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylate, wax, water, and combinations thereof, or any suitable binder.

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

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

[0203] Examples of oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macadamia nut, meadowfoam seed, mink, nutmeg, olive, orange, orange raffia, palm, palm kernel, shea butter, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, seabuckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, camellia, vetiver, walnut, and wheat germ oil, as well as 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, but are not limited thereto.

[0204] Formulation Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can include a lipid component and one or more additional components such as a therapeutic and / or prophylactic agent. Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be designed for one or more specific uses or targets. The components of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be selected based on a specific use or target and / or based on the effectiveness, toxicity, cost, ease of use, availability, or other characteristics of one or more components. Similarly, a particular formulation of a nanoparticle composition can be selected for a specific use or target, e.g., depending on the effectiveness and toxicity of a particular combination of components.

[0205] The lipid component of the nanoparticle composition can include, for example, a lipid according to formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid (an unsaturated lipid such as DOPE or DSPC, etc.), a PEG lipid, and a structural lipid. The components of the lipid component can be provided in specific fractions.

[0206] In some embodiments, the lipid component of the nanoparticle composition comprises a lipid according to formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a PEG lipid, and a structural lipid. In certain embodiments, the lipid component of the nanoparticle composition comprises from about 30 mol% to about 60 mol% of a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), from about 0 mol% to about 30 mol% of a phospholipid, from about 18.5 mol% to about 48.5 mol% of a structural lipid, and from about 0 mol% to about 10 mol% of a PEG lipid, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises from about 35 mol% to about 55 mol% of a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), from about 5 mol% to about 25 mol% of a phospholipid, from about 30 mol% to about 40 mol% of a structural lipid, and from about 0 mol% to about 10 mol% of a PEG lipid. In a particular embodiment, the lipid component comprises about 50 mol% of said compound, about 10 mol% of a phospholipid, about 38.5 mol% of a structural lipid, and about 1.5 mol% of a PEG lipid. In another particular embodiment, the lipid component comprises about 40 mol% of said compound, about 20 mol% of a phospholipid, about 38.5 mol% of a structural lipid, and about 1.5 mol% of a PEG lipid. In some embodiments, the phospholipid can be DOPE or DSPC. In other embodiments, the PEG lipid is PEG-1 or PEG 2k-DMG can be, and / or the structural lipid can be cholesterol.

[0207] In some embodiments, empty lipid nanoparticles (empty LNPs) comprise a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a structural lipid, and a PEG lipid.

[0208] In some embodiments, loaded lipid nanoparticles (loaded LNPs) comprise a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents.

[0209] In some embodiments, empty LNPs or loaded LNPs comprise a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c) in an amount of about 40% to about 60%.

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

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

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

[0213] In some embodiments, the empty LNP or the filled LNP contains about 40 mol% to about 60 mol% of a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), about 0 mol% to about 20 mol% of a phospholipid, about 30 mol% to about 50 mol% of a structural lipid, and about 0 mol% to about 5 mol% of a PEG lipid.

[0214] In some embodiments, the empty LNP or the filled LNP contains about 40 mol% to about 60 mol% of a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), about 0 mol% to about 20 mol% of DSPC, about 30 mol% to about 50 mol% of cholesterol, and about 0 mol% to about 5 mol% of PEG 2k-DMG and, are included. In some embodiments, the empty LNP or the filled LNP comprises about 40 mol% to about 60 mol% of the compound of Table 1, about 0 mol% to about 20 mol% of DSPC, about 30 mol% to about 50 mol% of cholesterol, and about 0 mol% to about 5 mol% of PEG 2k -DMG and, are included.

[0215] In some embodiments, the empty LNP or the filled LNP comprises about 40 mol% to about 60 mol% of the compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), about 0 mol% to about 20 mol% of DSPC, about 30 mol% to about 50 mol% of cholesterol, and about 0 mol% to about 5 mol% of PEG-1. In some embodiments, the empty LNP or the filled LNP comprises about 40 mol% to about 60 mol% of the compound of Table 1, about 0 mol% to about 20 mol% of DSPC, about 30 mol% to about 50 mol% of cholesterol, and about 0 mol% to about 5 mol% of PEG-1.

[0216] In some embodiments, the empty LNP or the filled LNP comprises the compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a structural lipid, and a PEG lipid, the phospholipid is DSPC, and the structural lipid is cholesterol. In some embodiments, the empty LNP or the filled LNP comprises the compound of Table 1, a phospholipid, a structural lipid, and a PEG lipid, the phospholipid is DSPC, and the structural lipid is cholesterol.

[0217] In some embodiments, the empty LNP or the filled LNP comprises a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a structural lipid, and a PEG lipid, the structural lipid is cholesterol, and the PEG lipid is PEG 2k -DMG. In some embodiments, the empty LNP or the filled LNP comprises a compound of Table 1, a phospholipid, a structural lipid, and a PEG lipid, the structural lipid is cholesterol, and the PEG lipid is PEG 2k -DMG.

[0218] In some embodiments, the empty LNP or the filled LNP comprises a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a structural lipid, and a PEG lipid, the structural lipid is cholesterol, and the PEG lipid is PEG-1. In some embodiments, the empty LNP or the filled LNP comprises a compound of Table 1, a phospholipid, a structural lipid, and a PEG lipid, the structural lipid is cholesterol, and the PEG lipid is PEG-1.

[0219] In some embodiments, the empty LNP or the filled LNP comprises a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a structural lipid, and a PEG lipid, the phospholipid is DSPC, and the PEG lipid is PEG2k -DMG. In some embodiments, the empty LNP or the filled LNP comprises a compound of Table 1, a phospholipid, a structural lipid, and a PEG lipid, the structural lipid is DSPC, and the PEG lipid is PEG 2k -DMG.

[0220] In some embodiments, the empty LNP or the filled LNP comprises a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a structural lipid, and a PEG lipid, the phospholipid is DSPC, and the PEG lipid is PEG-1. In some embodiments, the empty LNP or the filled LNP comprises a compound of Table 1, a phospholipid, a structural lipid, and a PEG lipid, the phospholipid is DSPC, and the PEG lipid is PEG-1.

[0221] In some embodiments, the empty LNP or the filled LNP comprises a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a structural lipid, and a PEG lipid, the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG 2k -DMG. In some embodiments, the empty LNP or the filled LNP comprises a compound of Table 1, a phospholipid, a structural lipid, and a PEG lipid, the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG 2k -DMG.

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

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

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

[0225] Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be designed for one or more specific uses or targets. For example, the nanoparticle composition can be designed to deliver therapeutic and / or prophylactic agents such as RNA to specific cells, tissues, organs, or their systems or groups in the mammalian body. The physicochemical properties of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be modified to increase selectivity for a specific body target. For example, the particle size can be adjusted based on the pore sizes of different organs. The therapeutic and / or prophylactic agents included in the nanoparticle composition can also be selected based on the desired delivery target or targets. For example, the therapeutic and / or prophylactic agents can be selected for a specific indication, condition, disease, or disorder and / or for delivery to a specific cell, tissue, organ, or its system or group (e.g., local or specific delivery). In certain embodiments, the nanoparticle composition can include mRNA encoding a polypeptide of interest that is capable of being translated in a cell to produce the polypeptide of interest. Such a composition can be designed to be specifically delivered to a particular organ. In some embodiments, the composition can be designed to be specifically delivered to the mammalian liver.

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

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

[0228] In some embodiments, the nanoparticle composition comprises one or more RNAs, one or more RNAs, and lipids, and their amounts can be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. Generally, a lower N:P ratio is preferred. The one or more RNAs, lipids, and their amounts can be selected to have 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.

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

[0230] Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be characterized in various ways. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the nanoparticle composition. Zeta potential may be measured using dynamic light scattering or potentiometry (e.g., potentiometric titration). Also, dynamic light scattering may be used to determine the particle size. Also, instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may be used to measure multiple properties of the nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0231] The average size of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be in the range of tens of nm to 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, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 150 nm, from about 70 nm to about 130 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 150 nm, from about 80 nm to about 130 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, from about 90 nm to about 150 nm, from about 90 nm to about 130 nm, or from about 90 nm to about 100 nm. In certain embodiments, the average size of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be from about 70 nm to about 130 nm or from about 70 nm to about 100 nm. In specific embodiments, the average size can be about 80 nm. In other embodiments, the average size can be about 100 nm. In other embodiments, the average size can be about 120 nm.

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

[0233] The zeta potential of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can account for the surface charge of the nanoparticle composition. Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) with a relatively low positive or negative charge are generally desirable because higher charged species can interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of lipid nanoparticles (e.g., empty LNPs or 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.

[0234] The efficiency of encapsulation of a therapeutic and / or prophylactic agent describes the amount of the therapeutic and / or prophylactic agent that is encapsulated or associated with lipid nanoparticles (e.g., empty LNPs or loaded LNPs) after preparation, compared to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of the therapeutic and / or prophylactic agent in a solution containing lipid nanoparticles (e.g., empty LNPs or loaded LNPs) before and after the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) are broken down, 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. For the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) described herein, the encapsulation efficiency of the therapeutic and / or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%. In some embodiments, the encapsulation efficiency of the therapeutic and / or prophylactic agent is between 80% and 100%.

[0235] Pharmaceutical composition Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be formulated, in whole or in part, as a pharmaceutical composition. The pharmaceutical composition can comprise one or more lipid nanoparticles (e.g., empty LNPs or loaded LNPs). In one embodiment, the pharmaceutical composition comprises a population of lipid nanoparticles (e.g., empty LNPs or loaded LNPs). For example, the pharmaceutical composition can comprise one or more lipid nanoparticles (e.g., empty LNPs or loaded LNPs) comprising one or more different therapeutic and / or prophylactic agents. The pharmaceutical composition can further comprise one or more pharmaceutically acceptable excipients or adjunct components such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and drugs are available, for example, in Remington’s The Science and Practice of Pharmacy, 21 st Edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and adjunct components can be used in any pharmaceutical composition, except where any conventional excipient or adjunct component may be incompatible with one or more components of the nanoparticle composition. An excipient or adjunct component may be incompatible with the components of the nanoparticles (e.g., empty LNPs or loaded LNPs) if their combination with the components can produce any undesirable biological effects or other adverse effects.

[0236] In some embodiments, one or more excipients or adjunct components may constitute more than 50% of the total mass or volume of the pharmaceutical composition comprising the nanoparticle composition. For example, one or more excipients or adjunct components may constitute 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical regulations. 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 US Food and Drug Administration. In some embodiments, the excipient is of pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopeia (USP), the European Pharmacopeia (EP), the British Pharmacopeia, and / or the International Pharmacopeia.

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

[0238] In certain embodiments, the disclosed lipid nanoparticles (e.g., empty LNPs or loaded LNPs) and / or pharmaceutical compositions are refrigerated or frozen for storage and / or shipping (e.g., at a temperature of 4°C or less, from about -150°C to about 0°C, or from about -80°C to about 20°C such as, for example, about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C). For example, a pharmaceutical composition comprising any of the compounds of formulas (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), and (B-c) is a solution that is refrigerated for storage and / or shipping at, for example, about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C. In certain embodiments, the disclosure also relates to a method of increasing the stability of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) and / or pharmaceutical compositions comprising any of the compounds of formulas (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), and (B-c) by storing the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) and / or pharmaceutical compositions at a temperature of 4°C or less, such as from about -150°C to about 0°C or from about -80°C to about -20°C, for example, 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, the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) and / or pharmaceutical compositions disclosed herein are stable at a temperature of 4°C or lower (e.g., about 4°C to -20°C) for at least about 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months. In some embodiments, the formulation is stabilized at about 4°C for at least 4 weeks. In certain embodiments, the disclosed pharmaceutical composition comprises the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) disclosed herein and one or more pharmaceutically acceptable carriers selected from Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the disclosed pharmaceutical composition has 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 7.5 to 8, or 7 to 7.8). For example, the disclosed pharmaceutical composition comprises the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) disclosed herein, Tris, saline, and sucrose and has a pH of about 7.5 to 8, which is suitable for storage and / or shipment, for example, at about -20°C. For example, the disclosed pharmaceutical composition comprises the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) disclosed herein and PBS and has a pH of about 7 to 7.8, which is suitable for storage and / or shipment, for example, at 4°C or lower. As used in the context of the present disclosure, "stability," "stabilized," and "stable" refer to the resistance of the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size change, aggregation, change in encapsulation, etc.) when stresses such as shear force, freeze / thaw stress, etc. are applied under given manufacturing, preparation, transportation, storage, and / or use conditions.

[0239] In some embodiments, the disclosed pharmaceutical compositions comprise empty LNPs or loaded LNPs, cryoprotectants, buffers, or combinations thereof.

[0240] 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-propanediol), 1,3-propanediol, glycerol, (+ / -)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a non-detergent sulfobetaine (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), an osmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG200), PEG400, PEG600, PEG1000, PEG 2k- DMG, PEG3350, PEG4000, PEG8000, PEG10000, PEG20000, polyethylene glycol monomethyl ether 550 (mPEG550), mPEG600, mPEG2000, mPEG3350, mPEG4000, mPEG5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K15), pentaerythritol propoxylate, or polypropylene glycol P400), 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, myo-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.

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

[0242] Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) and / or pharmaceutical compositions comprising one or more lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be administered to any patient or subject, including a patient or subject who can benefit from the therapeutic effects provided by the delivery of a therapeutic and / or prophylactic agent to one or more specific cells, tissues, organs, or systems or groups thereof. The descriptions provided herein of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) and pharmaceutical compositions comprising lipid nanoparticles (e.g., empty LNPs or loaded LNPs) are primarily directed to compositions suitable for administration to humans, although it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammal. To prepare compositions suitable for administration to various animals, the modification of compositions suitable for administration to humans is well understood, and a veterinary pharmacologist of ordinary skill could design and / or perform such modifications with merely ordinary experimentation, if any. Subjects to which administration of the compositions is contemplated include, but are not limited to, humans, other primates, and other mammals, such as commercially relevant mammals including cows, pigs, horses, sheep, cats, dogs, mice, and / or rats. The subject lipid nanoparticles can also be used for in vitro and ex vivo use.

[0243] Pharmaceutical compositions comprising one or more lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be prepared by any method known in the field of pharmacology or developed in the future. Generally, such preparation methods include associating the active ingredient with an excipient and / or one or more other accessory components and then, if desired or necessary, dividing, shaping, and / or packaging the product into the desired single or multiple dosage units.

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

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

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

[0247] Injectable preparations, such as sterile aqueous or oily suspensions, may be formulated according to known techniques using suitable dispersing, wetting, and / or suspending agents. Sterile injectable preparations may be, for example, solutions, suspensions, and / or emulsions in sterile injectable solutions, suspensions, and / or emulsions in a non-toxic parenterally acceptable diluent and / or solvent, such as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile fixed oils have conventionally been used as a solvent or suspending medium. For this purpose, any bland fixed oil containing synthetic monoglycerides or diglycerides can be used. Fatty acids, such as oleic acid, can be used in the preparation of injectables.

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

[0249] To prolong the effect of the active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material with low water solubility. The rate of drug absorption then depends on its dissolution rate, which in turn can depend on crystal size and crystal form. Alternatively, delayed absorption of parenterally administered drug forms is achieved by dissolving or suspending the drug in an oily vehicle. Injectable depot forms are prepared by forming a microencapsulation matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide. Depending on the drug-to-polymer ratio and the nature of the specific polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable preparations are prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues.

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

[0251] Solid dosage forms for oral administration include capsules, tablets, pills, films, powders, and granules. The active ingredient of such solid dosage forms is mixed with at least one inert pharmaceutically acceptable excipient, such as sodium citrate or dicalcium phosphate, and / or filler or bulking agent (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid), binder (e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia), humectant (e.g., glycerol), disintegrant (e.g., agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarder (e.g., paraffin), absorption enhancer (e.g., quaternary ammonium compounds), wetting agent (e.g., cetyl alcohol and glycerol monostearate), absorbent (e.g., kaolin and bentonite clay, silicates), and lubricant (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may contain a buffering agent.

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

[0253] Dosage forms for topical and / or transdermal administration of the composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. Generally, the active ingredient is mixed under sterile conditions with pharmaceutically acceptable excipients and / or any necessary preservatives and / or buffering agents as required. In addition, the present disclosure contemplates the use of transdermal patches which in many cases have the additional advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared, for example, by dissolving and / or dispensing the compound in a suitable medium. Alternatively or additionally, the rate can be controlled either by providing a rate controlling membrane and / or by dispersing the compound in a polymeric matrix and / or gel.

[0254] Devices suitable for use in intradermal delivery of the pharmaceutical compositions described herein include short needle devices. The intradermal composition can be administered by a device that limits the effective penetration length of the needle into the skin. Jet injection devices that deliver a liquid composition to the dermis are suitable, either via a liquid jet injector and / or via a needle that generates a jet that penetrates the stratum corneum and reaches the dermis. Ballistic delivery powder / particle delivery devices that use compressed gas to accelerate a vaccine in powder form through the outer layer of the skin to the dermis are suitable. Alternatively or additionally, conventional syringes can be used in the classical Mantoux method of intradermal administration.

[0255] Formulations suitable for topical administration include, but are not limited to, liquids and / or semi-liquid preparations such as paints, lotions, oil-in-water and / or water-in-oil emulsions (e.g., creams, ointments and / or pastes), and / or solutions, and / or suspensions. Formulations that can be topically administered may contain, for example, from about 1% to about 10% (weight / weight) of the active ingredient, although the concentration of the active ingredient can be on the order of the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further contain one or more of the additional components described herein.

[0256] The pharmaceutical composition can be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may contain dry particles containing the active ingredient. Conveniently, such a composition can be administered in the form of dry powder using a device that includes a dry powder reservoir where the flow of the propellant can be directed to disperse the powder, and / or using a self-propelled solvent / powder dispensing container such as a device containing the active ingredient dissolved and / or suspended in a low-boiling propellant within a sealed container. The dry powder composition may contain a solid fine powder diluent such as sugar and is conveniently provided in unit dosage form.

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

[0258] The pharmaceutical composition formulated for pulmonary delivery can provide the active ingredient in the form of droplets of a solution and / or suspension. Such a formulation may be prepared, packaged, and / or sold as an aqueous and / or diluted alcohol solution and / or suspension, optionally sterilized, containing the active ingredient, and may be conveniently administered using any spraying and / or nebulizing device. Such a formulation may further contain one or more additional components including, but not limited to, flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants, and / or preservatives such as methyl hydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range of about 1 nm to about 200 nm.

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

[0260] Formulations suitable for nasal administration may contain, for example, from about 0.1% (w / w) to about 100% (w / w) of the active ingredient and may contain one or more of the additional ingredients described herein. The pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for buccal administration. Such formulations can be, for example, in the form of tablets and / or lozenges made using conventional methods, can contain, for example, from 0.1 to 20% (w / w) of the active ingredient, with the remainder being an orally dissolvable and / or degradable composition and optionally containing one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may contain a powder and / or an aerosolized and / or nebulized solution and / or suspension containing the active ingredient. Such powdered, aerosolized, and / or nebulized formulations may have an average particle and / or droplet size in the range of about 0.1 nm to about 200 nm when dispersed and may further contain one or more of any additional ingredients described herein.

[0261] The pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for ophthalmic administration. Such formulations can be, for example, in the form of eye drops containing a 0.1 / 1.0% (w / w) solution and / or a suspension in an aqueous or oily liquid excipient of the active ingredient. Such eye drops may further contain a buffering agent, a salt, and / or one or more other ingredients of any of the additional ingredients described herein. Other useful ophthalmically administrable formulations include those containing the active ingredient in microcrystalline form and / or in liposomal preparations. Ear drops and / or eye drops are contemplated as being within the scope of the present disclosure.

[0262] mRNA therapy mRNA as a drug modality has the potential to deliver secreted proteins, as well as intracellular and transmembrane proteins. mRNA as a drug modality has the potential to translocate and deliver into cells targets that are inaccessible when delivered in protein form, i.e., targets to which standard biologics do not have the ability to cross the cell membrane. One of the major challenges in realizing mRNA-based therapies is the identification of an optimal delivery vehicle. mRNA requires a delivery vehicle that can provide protection from endonucleases and exonucleases, as well as shield the cargo from the immune sentinels, due to its size, chemical instability, and potential immunogenicity. Lipid nanoparticles (LNPs) have been identified as the lead option in this regard.

[0263] An important performance criterion for lipid nanoparticle delivery systems is the ability to maximize cell uptake and enable efficient release of mRNA from endosomes. In one embodiment, the LNPs of the subject matter disclosed herein that contain the novel lipids exhibit improvements in at least one of cell uptake and endosomal release. At the same time, the LNPs need to provide a stable drug product and be safely administrable at therapeutically relevant levels. LNPs are typically multi-component systems consisting of amino lipids, phospholipids, cholesterol, and PEG lipids. Each component is necessary in terms of efficient delivery of the nucleic acid cargo and stability of the particles. An important component thought to drive cell uptake, endosomal escape, and tolerability is the amino lipid. Cholesterol and PEG lipids contribute to the stability of the drug product both in vivo and during storage, while phospholipids provide additional fusogenicity to the LNPs and thus help drive endosomal escape and make the nucleic acid bioavailable in the cytoplasm of the cell.

[0264] For oligonucleotide delivery, several series of amino lipids, including the amino lipid MC3 (DLin-MC3-DMA), have been developed over the past few decades. MC3-based LNPs have been shown to be effective for mRNA delivery. This class of LNPs is rapidly opsonized by apolipoprotein E (ApoE) upon intravenous delivery, enabling cellular uptake by the low-density lipoprotein receptor (LDLr). However, there remains a concern that the long tissue half-life of MC3 may contribute to unfavorable side effects that hinder its use for long-term therapies. Additionally, extensive literature evidence suggests that long-term administration of lipid nanoparticles can result in several toxic side effects, including complement activation-related pseudoallergy (CARPA) and liver injury. Therefore, there is a need for a class of LNPs with increased delivery efficiency along with metabolic and toxicity profiles that would enable long-term administration in humans to unlock the potential of mRNA and other nucleic acid-, nucleotide- or peptide-based therapies in humans.

[0265] The ability to treat a wide range of diseases requires the flexibility to administer safely over long periods at varying dose levels. Through systematic optimization of the amino lipid structure, the disclosed compounds have been identified as compounds with a good balance of chemical stability, improved delivery efficiency due to enhanced endosomal escape, rapid in vivo metabolism, and a clean toxicity profile. This combination of features provides drug candidates that can be administered over the long term without activating the immune system. Initial rodent screening led to the identification of lead lipids with good delivery efficiency and pharmacokinetics. Advanced LNPs were further profiled in non-human primates for delivery efficiency after single and repeated dosing. Finally, the optimized LNPs were evaluated in 1-month repeated-dose toxicity studies in rats and non-human primates. Without wishing to be bound by theory, the novel ionizable lipids of the present disclosure have improved cell delivery, improved protein expression, and improved biodegradability characteristics and can result in an increase in mRNA expression in cells of 2-fold, 5-fold, 10-fold, 15-fold, or more than 20-fold compared to LNPs lacking the inventive lipids. In another embodiment, LNPs containing the lipids of the invention can result in specific (e.g., preferential) delivery to certain cell types or types of cells compared to other cell types, thereby resulting in an increase in mRNA expression in certain cells or tissues of 2-fold, 5-fold, 10-fold, 15-fold, or more than 20-fold compared to LNPs lacking the inventive lipids. These improvements over the prior art enable the safe and effective use of mRNA-based therapies in acute and chronic diseases.

[0266] Method In some embodiments, the disclosure provides a method of delivering a therapeutic and / or prophylactic agent to a cell (e.g., a mammalian cell). The method includes contacting the disclosed filled LNP or pharmaceutical composition with the cell, whereby the therapeutic and / or prophylactic agent is 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 a subject a lipid nanoparticle comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents, whereby the therapeutic agent and / or prophylactic agent is delivered to the cell.

[0267] In some embodiments, the disclosure provides a method of delivering a therapeutic and / or prophylactic agent to a cell in a subject, the method including administering to the subject a lipid nanoparticle comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), 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 disclosure provides a method of delivering a therapeutic and / or prophylactic agent to a cell in a subject, the method including administering to the subject a lipid nanoparticle comprising a compound of formula (A-c), 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).

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

[0269] In some aspects, the disclosure provides a method for delivering (e.g., specifically delivering) a therapeutic agent and / or a prophylactic agent to a mammalian organ or tissue (e.g., liver, kidney, spleen, or lung). The method comprises contacting a cell with a filled LNP or pharmaceutical composition of the disclosure, whereby the therapeutic agent and / or the prophylactic agent is delivered to the target organ or tissue. In some embodiments, the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents, whereby the therapeutic agent and / or the prophylactic agent is delivered to the target organ or tissue.

[0270] In some embodiments, the disclosure provides a method for specifically delivering a therapeutic agent and / or a prophylactic agent to a target organ, the method comprising administering to the subject a lipid nanoparticle comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), DSPC, cholesterol, 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 disclosure provides a method for specifically delivering a therapeutic agent and / or a prophylactic agent to a target organ, the method comprising a compound of formula (A-c), DSPC, cholesterol, PEG 2k -DMG, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA), and administering to the subject a lipid nanoparticle comprising the same.

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

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

[0273] In some embodiments, the disclosure provides methods for improved delivery of therapeutic and / or prophylactic agents to a target tissue, the methods comprising administering to a subject lipid nanoparticles comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), DSPC, cholesterol, 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 disclosure provides methods for improved delivery of therapeutic and / or prophylactic agents to a target tissue, the methods comprising administering to a subject lipid nanoparticles comprising a compound of formula (A-c), DSPC, cholesterol, PEG 2k -DMG, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

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

[0275] In some aspects, the disclosure provides a method for producing a polypeptide of interest in a cell (e.g., a mammalian cell). The method comprises contacting the cell with a filled LNP or pharmaceutical composition of the disclosure, the filled LNP or pharmaceutical composition comprising mRNA, whereby the mRNA can be translated intracellularly to produce the polypeptide. In some embodiments, the cell is within a subject and the contacting comprises administering the cell to the subject. In some embodiments, the method comprises administering to a subject a lipid nanoparticle comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a structural lipid, a PEG lipid, and mRNA, whereby the mRNA can be translated intracellularly to produce the polypeptide.

[0276] In some embodiments, the disclosure provides a method for producing a polypeptide of interest in a cell, the method comprising administering to a subject lipid nanoparticles comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), DSPC, cholesterol, PEG 2k -DMG, and mRNA. For example, in some embodiments, the disclosure provides a method for producing a polypeptide of interest in a cell, the method comprising administering to a subject lipid nanoparticles comprising a compound of Table 1, DSPC, cholesterol, PEG 2k -DMG, and mRNA. For example, in some embodiments, the disclosure provides a method for producing a polypeptide of interest in a cell, the method comprising administering to a subject lipid nanoparticles comprising a compound of formula (A-c), DSPC, cholesterol, PEG 2k -DMG, and mRNA. The disclosure provides a method comprising the step of administering to a subject lipid nanoparticles.

[0277] In some embodiments, the disclosure provides a method for producing a polypeptide of interest in a cell, the method comprising administering to a subject lipid nanoparticles comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), DSPC, cholesterol, PEG-1, and mRNA. For example, in some embodiments, the disclosure provides a method for producing a polypeptide of interest in a cell, the method comprising administering to a subject lipid nanoparticles comprising a compound of formula (A-c), DSPC, cholesterol, PEG-1, and mRNA. For example, in some embodiments, the disclosure provides a method for producing a polypeptide of interest in a cell, the method comprising administering to a subject lipid nanoparticles comprising a compound of Table 1, DSPC, cholesterol, PEG-1, and mRNA.

[0278] In some embodiments, the disclosure provides a method of treating a disease or disorder in a mammal (e.g., a human) in need thereof. The method comprises administering to the mammal a therapeutically effective amount of the disclosed filled LNP or pharmaceutical composition. In some embodiments, the method comprises administering to the subject a lipid nanoparticle comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents, whereby the therapeutic and / or prophylactic agent is delivered to cells. In some embodiments, the disease or disorder is characterized by a 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.

[0279] In some embodiments, the disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject a lipid nanoparticle comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), 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 disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject a lipid nanoparticle comprising a compound of formula (A-c), DSPC, cholesterol, and PEG 2k- Administering to a subject a lipid nanoparticle comprising -DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA). For example, in some embodiments, the disclosure provides a method of treating a disease or disorder in a subject, the method comprising a compound of Table 1, DSPC, cholesterol, PEG 2k - Administering to a subject a lipid nanoparticle comprising -DMG and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA).

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

[0281] In yet another aspect, the disclosure provides a method of reducing immunogenicity, comprising introducing into a cell a filled LNP or pharmaceutical composition of the disclosure, wherein the filled LNP or pharmaceutical composition reduces induction of a cellular immune response in the cell to the filled LNP or pharmaceutical composition as compared to induction of a cellular immune response 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 administering to the subject a lipid nanoparticle comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA), wherein the lipid nanoparticle comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c) reduces induction of a cellular immune response in the cell to the lipid nanoparticle comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c) as compared to induction of a cellular immune response in the cell by a reference composition. For example, the cellular immune response is a innate immune response, an adaptive immune response, or both.

[0282] In some embodiments, the disclosure provides a method of reducing immunogenicity in a subject, the method comprising administering to the subject a lipid nanoparticle comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), DSPC, cholesterol, 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 disclosure provides a method of reducing immunogenicity in a subject, the method comprising a compound of formula (A-c), DSPC, cholesterol, PEG 2k -DMG, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA), and administering to the subject a lipid nanoparticle comprising the same. For example, in some embodiments, the disclosure provides a method of reducing immunogenicity in a subject, the method comprising a compound of Table 1, DSPC, cholesterol, PEG 2k -DMG, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA), and administering to the subject a lipid nanoparticle comprising the same.

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

[0284] The disclosure also includes a method of synthesizing a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), and a method of making a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) comprising a lipid component comprising a compound of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c).

[0285] Method for producing a polypeptide in a cell The present disclosure provides a method for producing a polypeptide of interest in mammalian cells. The method for producing a polypeptide involves contacting a cell with a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) comprising mRNA encoding the polypeptide of interest. When the nanoparticle composition is contacted with the cell, the mRNA can be taken up into the cell and translated to produce the polypeptide of interest.

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

[0287] The step of contacting a cell with a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) comprising mRNA can involve transfection or can cause transfection. The phospholipids contained in the lipid component of the lipid nanoparticle (e.g., an empty LNP or a loaded LNP) can promote transfection and / or increase transfection efficiency, for example, by interacting and / or fusing with the cell membrane or intracellular membranes. Transfection can enable translation of the mRNA within the cell.

[0288] In some embodiments, the lipid nanoparticles described herein (e.g., empty LNPs or loaded LNPs) can be used therapeutically. For example, the mRNA contained in the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can encode a therapeutic polypeptide (e.g., in a translatable region), and when it contacts and / or enters a cell (e.g., transfection), it can produce the therapeutic polypeptide. In other embodiments, the mRNA contained in the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can encode a polypeptide that can improve or increase the immunity of a subject. For example, the mRNA can encode granulocyte colony-stimulating factor or trastuzumab.

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

[0290] In some embodiments, contacting a cell with a lipid nanoparticle comprising mRNA (e.g., an empty LNP or a loaded LNP) can reduce the innate immune response of the cell to exogenous nucleic acids. The cell can be contacted with a first lipid nanoparticle (e.g., an empty LNP or a loaded LNP) comprising a first amount of a first exogenous mRNA comprising a translatable region, and the level of the innate immune response of the cell to the first exogenous mRNA can be determined. Thereafter, the cell can be contacted with a second composition comprising a second amount of the first exogenous mRNA, the second amount being less than the first amount of the first exogenous mRNA as compared to the first amount. Alternatively, the second composition can comprise a first amount of a second exogenous mRNA different from the first exogenous mRNA. The step of contacting the cell with the first and second compositions can be repeated one or more times. In addition, the efficiency of polypeptide production (e.g., translation) within the cell can optionally be determined, and the cell can be repeatedly re - contacted with the first and / or second compositions until the target protein production efficiency is achieved.

[0291] Method for delivering a therapeutic agent to cells and organs The present disclosure provides a method for delivering a therapeutic agent and / or a prophylactic agent to mammalian cells or organs. Delivering a therapeutic agent and / or a prophylactic agent to a cell involves administering to a subject a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) comprising the therapeutic agent and / or the prophylactic agent, and the administration of the composition involves contacting the composition with the cell. For example, a protein, a cytotoxic agent, a radioactive ion, a chemotherapeutic agent, or a nucleic acid (RNA, e.g., mRNA, etc.) can be delivered to a cell or an organ. When the therapeutic agent and / or the prophylactic agent is mRNA, upon contacting the nanoparticle composition with the cell, the translatable mRNA can be translated intracellularly to produce the polypeptide of interest. However, mRNA that is substantially non - translatable can also be delivered to the cell. Substantially non - translatable mRNA can be useful as a vaccine and / or can sequester the translation components of the cell to reduce the expression of other species within the cell.

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

[0293] In some embodiments, specific delivery of the mRNA contained in the disclosed loaded LNPs (i.e., lipid nanoparticles formulated with the disclosed compounds) can result in a greater than 2-fold, 5-fold, 10-fold, 15-fold, or 20-fold increase in mRNA expression compared to delivery of the mRNA contained in LNPs formulated with another lipid (i.e., any of the lipids of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c) not included).

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

[0295] In some embodiments, the ligand can be a surface-bound antibody that can allow for modulation of cell targeting specificity. This is particularly useful as the specificity of the antibody can be enhanced against the epitope of interest at the desired targeting site. In some embodiments, multiple antibodies are expressed on the surface of the cell and each antibody can have a different specificity for the desired target. Such an approach can increase the affinity and specificity of the targeting interaction.

[0296] The ligand can be selected, for example, by one of ordinary skill in the biological arts based on the desired localization or function of the cell.

[0297] Target cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, nerve cells, heart 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, reticulocytes, white blood cells, granulocytes, and tumor cells.

[0298] In some embodiments, lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can target 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 vivo and are known to associate with receptors such as the low-density lipoprotein receptor (LDLR) found on the surface of hepatocytes. Thus, lipid nanoparticles (e.g., empty LNPs or loaded LNPs) containing a lipid component having a neutral or near-neutral charge administered to a subject can acquire apoE in the subject's body and then deliver a therapeutic agent and / or prophylactic agent (e.g., RNA) to hepatocytes containing LDLR in a targeted manner.

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

[0300] Diseases, disorders, and / or conditions characterized by a dysfunctional or abnormal protein or polypeptide activity to which the composition 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, heart and renal vascular diseases, and metabolic diseases. A plurality of diseases, disorders, and / or conditions can be characterized by a lack of protein activity (or a substantial decrease such that appropriate protein function does not occur). Such proteins may be absent or may essentially not function. The present disclosure provides a method for treating such diseases, disorders, and / or conditions in a subject by administering lipid nanoparticles (e.g., empty LNPs or loaded LNPs) comprising an RNA and a lipid component comprising a lipid, phospholipid (optionally unsaturated), PEG lipid, and structural lipid according to formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), or (B-c), wherein the RNA can be an mRNA encoding a polypeptide that antagonizes or overcomes the abnormal protein activity present in the cells of the subject.

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

[0302] The filled LNPs can be administered by any route. In some embodiments, a composition comprising a prophylactic, diagnostic, or imaging composition comprising one or more of the filled LNPs described herein is administered by one or more of various routes including oral, intravenous, intramuscular, intraarterial, subcutaneous, transdermal or intradermal, intradermal, intraperitoneal, mucosal, nasal, intratumoral, intranasal; inhalation; oral spray and / or powder, nasal spray, and / or aerosol, and / or through a portal catheter. In some embodiments, the composition can be administered intravenously, intramuscularly, intradermally, intraarterially, intratumorally, subcutaneously, or by any other parenteral route of administration, or by inhalation. However, the present disclosure encompasses delivery or administration of the compositions described herein by any suitable route taking into account possible advancements in the science of drug delivery. Generally, the most appropriate route of administration will depend on various factors including the nature of the filled LNPs comprising one or more therapeutic and / or prophylactic agents (e.g., its stability in various body environments such as the bloodstream and gastrointestinal tract), the condition of the patient (e.g., whether the patient is able to tolerate a particular route of administration), and the like.

[0303] In certain embodiments, the compositions according to the present disclosure can be administered at a dosage level sufficient to deliver a therapeutic and / or prophylactic agent (e.g., mRNA) of 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 0.0001 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, 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 to about 1 mg / kg, about 0.0001 mg / kg to about 0.25 mg / kg, about 0.001 mg / kg to about 0.25 mg / kg, about 0.005 mg / kg to about 0.25 mg / kg, about 0.01 mg / kg to about 0.25 mg / kg, about 0.05 mg / kg to about 0.25 mg / kg, or about 0.1 mg / kg to about 0.25 mg / kg. A dosage of 1 mg / kg (mpk) provides 1 mg of the therapeutic and / or prophylactic agent per 1 kg of the subject's body weight. In some embodiments, a dosage of about 0.001 mg / kg to about 10 mg / kg of the therapeutic and / or prophylactic agent in the filled LNP can be administered. In other embodiments, a dosage of about 0.005 mg / kg to about 2.5 mg / kg of the therapeutic and / or prophylactic agent can be administered.In certain embodiments, a dosage of about 0.1 mg / kg to about 1 mg / kg can be administered. In other embodiments, a dosage of about 0.05 mg / kg to about 0.25 mg / kg can be administered. The dosage can be administered one or more times per day in the same or different amounts to obtain the desired level of mRNA expression and / or therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage can be delivered, for example, three times a day, twice a day, once a day, every other day, every three days, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations). In some embodiments, a single dose can be administered, for example, before or after a surgical procedure, or in the case of an acute disease, disorder, or condition.

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

[0305] It will further be 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 expected that the agents used in combination will be used at levels that do not exceed the levels at which they are used individually. In some embodiments, the levels used in combination may be lower than the levels used individually.

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

[0307] Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be used in combination with a drug to increase the effectiveness and / or treatment time frame of a composition. Such drugs can be, for example, anti-inflammatory compounds, steroids (e.g., corticosteroids), statins, estradiol, BTK inhibitors, S1P1 agonists, glucocorticoid receptor modulators (GRMs), or antihistamines. In some embodiments, lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be used in combination with dexamethasone, methotrexate, acetaminophen, an H1 receptor blocker, or an H2 receptor blocker. In some embodiments, a method of treating a subject in need of treatment for a disease or disorder or delivering a therapeutic and / or prophylactic agent to a subject (e.g., a mammal) can involve pre-treating the subject with one or more drugs prior to administering the nanoparticle composition. For example, the subject can be pre-treated with a useful amount (e.g., 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, or any other useful amount) of dexamethasone, methotrexate, acetaminophen, an H1 receptor blocker, or an H2 receptor blocker. The pre-treatment can be performed 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 the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) and can be performed, for example, once, twice, or more at an increased dosage.

[0308] One of ordinary skill 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 forms set forth for carrying out the above invention, but is intended to be as set forth in the appended claims.

[0309] In the claims, articles such as "a", "an", and "the" can mean one or more unless the contrary is indicated or is not apparent from the context. A claim or description that includes "or" between one or more members of a group is considered satisfied if, unless the contrary is indicated or is not apparent from the context, one, two or more, or all of the members of the group are present in, used in, or otherwise associated with 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 associated with a given product or process. The disclosure includes embodiments in which two or more or all of the group members are present in, used in, or associated with a given product or process. As used herein, expressions such as "one or more of A, B, or C", "one or more of A, B, and C", "one or more of A, B, and C", "selected from A, B, and C", "selected from the group consisting of A, B, and C", etc. are used synonymously and all, unless otherwise specified, refer to a selection from the group consisting of A, B, and / or C, i.e., one or more of A, one or more of B, one or more of C, or any combination thereof.

[0310] It should be noted that the term "comprising" is intended to be open and allows the inclusion of additional elements or steps, but does not require them. Thus, when the term "comprising" is used in this specification, the terms "consisting essentially of" and "consisting of" are also included and disclosed. Throughout this specification, when a composition is described as having, comprising, or including a particular component, the composition is also contemplated to consist essentially of or consist of the recited components. Similarly, when a method or process is described as having, comprising, or including a particular process step, the process is also contemplated to consist essentially of or consist of the recited process steps. Further, it should be understood that the order or sequence of steps for performing a particular act is not important so long as the invention continues to be operable. Additionally, two or more steps or acts can be performed simultaneously.

[0311] When ranges are specified, the endpoints are included. Further, unless otherwise indicated, or not apparent from the context and the understanding of one of ordinary skill in the art, values expressed as ranges are to be assumed to cover any and all subranges within the disclosed ranges, between the recited endpoints, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

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

[0313] The compounds of the present disclosure can be prepared in a variety of ways using standard synthetic methods and procedures that are known to those skilled in the art or would be apparent to those skilled in the art in view of the teachings herein, using commercially available starting materials, compounds known in the literature, or readily prepared intermediates. Standard synthetic methods and procedures for the preparation of organic molecules as well as for functional group transformation and manipulation can be obtained from the relevant scientific literature or standard textbooks in the field. Without limitation to any one or several sources, Smith, M.B., March, J., March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 th edition, John Wiley & Sons: New York, 2001, Greene, T.W., Wuts, P.G.M., Protective Groups in Organic Synthesis, 3 rd edition, John Wiley & Sons: New York, 1999, R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989), L. Fieser and M. Fieser, Fieser and Fieser’s Reagents for Organic Synthesis, John Wiley and Sons (1994), and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) are useful and recognized reference textbooks of organic synthesis known to those skilled in the art. The following description of the synthetic methods is designed to illustrate, rather than limit, the general procedures for the preparation of the compounds of the present disclosure.

[0314] The compounds of the present disclosure having any of the formulas described herein can be prepared from commercially available starting materials, or starting materials that can be prepared using literature procedures, according to the procedures shown in Schemes 1, 2, and 3 below. The variable elements in the schemes (e.g., R 1 , R 2 , and R 3 , etc. are as defined herein). One of ordinary skill in the art will note that the order of certain steps in the reaction sequences and synthetic schemes described herein, such as the introduction and removal of protecting groups, may be changed.

[0315] One of ordinary skill in the art will recognize that certain groups may require protection from reaction conditions via the use of protecting groups. Also, protecting groups can be used to modify similar functional groups within a molecule. Lists of protecting groups, as well as methods for introducing and removing these groups, can be found in Greene, T.W., Wuts, P.G.M., Protective Groups in Organic Synthesis, 3 rd edition, John Wiley & Sons: New York, 1999.

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

[0317] For the hydroxyl moiety, TBS, benzyl, THP, Ac.

[0318] For carboxylic acids, benzyl ester, methyl ester, ethyl ester, allyl ester.

[0319] For amines, Fmoc, Cbz, BOC, DMB, Ac, Bn, Tr, Ts, trifluoroacetyl, phthalimide, benzylideneamine.

[0320] For diols, Ac(x2)TBS(x2), or when combined with acetonide.

[0321] For thiols, AC.

[0322] For benzimidazole, there are SEM, benzyl, PMB, DMB.

[0323] For aldehyde, there are di-alkyl acetals such as dimethoxyacetal or diethylacetyl.

[0324] In the reaction schemes described herein, multiple stereoisomers can be generated. If a specific stereoisomer is not indicated, it is understood to mean all possible stereoisomers that can be generated from the reaction. One skilled in the art will recognize that the reaction can be optimized to preferentially give one isomer or that a new scheme can be devised to produce a single isomer. If a mixture is produced, techniques such as preparative thin-layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC can be used to separate the isomers.

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

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

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

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

[0329] In certain embodiments, the disclosure also includes any of the compounds of formula (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), and (B-c), and a method for synthesizing the compound(s) and intermediate(s) for synthesizing the compound.

[0330] In some embodiments, the method for synthesizing the disclosed compound comprises reacting a compound of formula (X2):

Chemical formula

[0331] The method may also include reacting a compound of formula (X1):

Chemical formula

[0332] In some embodiments, the intermediate(s) may include those having any of formulae (X1) and (X2):

Chemical formula

[0333] In addition, it should be understood that any particular embodiment of the present disclosure corresponding to the prior art may be explicitly excluded from any one or more of the claims. Such embodiments are considered to be known to those skilled in the art, so they may be excluded even if the exclusion is not explicitly described in this specification.

[0334] Even if not explicitly described in the cited documents, all cited documents, such as references, publications, databases, database entries, and technical fields cited in this specification, are incorporated herein by reference. In case of conflict between the cited source and the description of this application, the description of this application shall prevail.

Examples

[0335] Example 1: Synthesis of the compounds in Table 1 A. General considerations All solvents and reagents used were commercially available and used as received unless otherwise stated. 1 The 1H NMR spectra were recorded at 300 K using a Bruker Ultrashield 300 MHz instrument in CDCl 3 3. Chemical shifts are reported 1Report as parts per million (ppm) compared to TMS (0.00) at H. 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. Analysis by reverse phase UPLC-MS (retention time, RT, in minutes) was used to determine that all final compounds were of greater than 85% purity using a Waters Acquity UPLC instrument equipped with DAD and ELSD, and a ZORBAX Rapid Resolution High Definition (RRHD) SB-C18 LC column, 2.1 mm, 50 mm, 1.8 μm, with a gradient of 65% to 100% acetonitrile in water containing 0.1% TFA over 5 minutes at 1.2 mL / min. The injection volume was 5 μL and the column temperature was 80 °C. Detection was based on electrospray ionization (ESI) in positive mode using a Waters SQD mass spectrometer (Milford, MA, USA) and an evaporative light scattering detector. LCMS method: Instrument information: HPLC / MS - Agilent1100 Column: Agela Technologies Durashell C18 3.5 μm, 100 Å, 4.6×50 mm Mobile phase A: water / 0.1% trifluoroacetic acid Mobile phase B: acetonitrile / 0.1% trifluoroacetic acid Flow rate: 1 mL / min Gradient: 70% to 100% B in 5 minutes, hold at 100% B for 10 minutes, 100% B to 70% B in 1 minute, then stop. Column temperature: ambient temperature Detector: ELSD

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

[0337] The following abbreviations are used in this specification: THF: Tetrahydrofuran MeCN: Acetonitrile LAH: Lithium aluminum hydride DCM: Dichloromethane DMAP: 4-Dimethylaminopyridine LDA: Lithium diisopropylamide rt: Room temperature DME: 1,2-Dimethoxyethane n-BuLi: n-Butyllithium CPME: Cyclopentyl methyl ether i-Pr 2 EtN: N,N-Diisopropylethylamine

[0338] Typical syntheses of Compounds 7, 12, and 13

Chemical formula

Chemical formula

[0339] A2. Compound 2b: Methyl 3 - isopropylnonanoate

Chemical Structure

[0340] A3. Compound 2c: Methyl 3 - propylnonanoate

Chemical Structure

[0341] Typical Procedure B: LAH Reduction B1. Compound 3a: 3 - butylnonan - 1 - ol

Chemical Structure

[0342] B2. Compound 3b: 3 - isopropylnonan - 1 - ol

Chemical Structure

[0343] B3. Compound 3c: 3 - propylnonan - 1 - ol

Chemical Structure

[0344] Typical procedure C for the esterification of 8 - bromooctanoic acid, 4 C1. Compound 5a: 3 - butylnonyl 8 - bromooctanoate

Chemical Structure

[0345] C2. Compound 5b: 3 - isopropylnonyl 8 - bromooctanoate

Chemical formula

[0346] C3. Compound 5c: 3 - propylnonyl 8 - bromooctanoate

Chemical formula

[0347] Typical procedure D: N-alkylation of heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate, 6 D1. Compound 7: 3-butylnonyl 8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoate (86 g-nBu) [Chemical formula] In a 500 mL round-bottom flask connected to a condenser, heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate 6 (601 mg, 1.36 mmol), 3-butylnonyl 8-bromooctanoate 5a (606 mg, 1.49 mmol), potassium carbonate (676 mg, 4.9 mmol), and potassium iodide (248.4 mg, 1.49 mmol) were mixed in cyclopentyl methyl ether (30 mL) and acetonitrile (30 mL), and the reaction mixture was heated to 85 °C for 18 h. MS showed a clean conversion. The mixture was cooled to room temperature and diluted with hexane. The mixture was filtered through a pad of celite. After washing with hexane, the filtrate was concentrated to give a brown oil, which was purified by flash chromatography (SiO 2 : hexane / diethyl ether 0 - 100%) to give 7 as a colorless oil (588 mg, 56%). HPLC / ELSD: RT = 7.07 min, MS (CI): m / z (MH + ) C 48 H 95 NO 5 corresponding to 766.7. 1 1H NMR (300 MHz, CDCl 3) δ: ppm 4.85 (quintet, 1H, J = 6.1 Hz); 4.07 (t, 2H, J = 6.9 Hz); 3.50 (t, 2H, J = 5.5 Hz); 2.98 (bs, 1H); 2.55 (t, 2H, J = 5.2 Hz); 2.41 (t, 4H, J = 7.4 Hz); 2.26 (t, 4H, J = 7.4 Hz); 1.65 - 1.48 (m, 19H); 1.26 (br. m, 48H); 0.88 - 0.84 (m, 12H).

[0348] D2. Compound 12: Heptadec - 9 - yl 8 - ((2 - hydroxyethyl)(8 - ((3 - isopropylnonyl)oxy)-8 - oxooctyl)amino)octanoate (86 - g - iPr)

Chemical Structure

[0349] D3. Compound 13: Heptadecan-9-yl 8-((2-hydroxyethyl)(8-oxo-8-((3-propylnonyl)oxy)octyl)amino)octanoate (86-g-nPr) [Chemical formula] The same as procedure D1, but using 3-propylnonyl 8-bromooctanoate, 5c. Yield = 510 mg (68%). HPLC / ELSD: RT = 7.01 min, MS(CI): m / z(MH + )C 47 H 93 NO 5 is 752.6 for it. 1 1H NMR (300 M Hz, CDCl 3 ) δ: ppm 4.85 (quintet, 1H, J = 6.3 Hz); 4.07 (t, 2H, J = 7.1 Hz); 3.50 (t, 2H, J = 5.5 Hz); 2.98 (bs, 1H); 2.55 (t, 2H, J = 5.2 Hz); 2.41 (t, 4H, J = 7.4 Hz); 2.26 (t, 4H, J = 7.4 Hz); 1.65 - 1.48 (m, 17H); 1.26 (br. m, 48H); 0. 88 - 0.84 (m, 12H).

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

[0351] D4. Compound 8: Heptadec - 9 - yl 8 - ((2 - hydroxyethyl)(8 - oxo - 8 - ((2 - propylnonyl)oxy)octyl)amino)octanoate (86 - b - nPr)

Chemical Structure

[0352] Synthesis of intermediates: Intermediate AA: Ethyl 3 - propylhex - 2 - enoate

Chemical Structure

[0353] Intermediate AB: Ethyl 3-propylhexanoate

Chemical Structure

[0354] Intermediate AC: 3-propylhexan-1-ol

Chemical Structure

[0355] Intermediate AD: Ethyl 3 - butylhepta - 2 - enoate

Chemical Structure

[0356] Intermediate AE: Ethyl 3 - butylheptanoate

Chemical Structure

[0357] Intermediate AF: 3-Butylheptan-1-ol [Chemical formula] At 0 °C under N 2 atmosphere, a solution of ethyl 3-butylheptanoate (4.00 g, 18.7 mmol) in dry ether (15 mL) was added dropwise to a mixture of lithium aluminum hydride (850 mg, 22.4 mmol) in dry ether (23 mL). The mixture was stirred at room temperature for 2.5 h and then cooled to 0 °C. Water (1 mL per gram of LiAlH 4 ) was added dropwise to the solution, followed by 15% sodium hydroxide (1 mL per gram of LiAlH 4 ), and water (1 mL per gram of LiAlH 43 mL per gram was slowly added. 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 give 3-butylheptan-1-ol (3.19 g, 18.5 mmol, 99%) as a clear oil. 1 HNMR (300 MHz, CDCl 3 ) δ: ppm 3.66 (t,2H, J = 6.0 Hz);1.53 (q, 2H, J = 6.0 Hz);1.46-1.36 (m, 1H);1.35-1.21 (m, 12H);1.18(br. s, 1H);0.89 (br. t, 6H, J = 6.0 Hz).

[0358] Intermediate AG: Ethyl 3-pentyloct-2-enoate

Chemical Structure

[0359] Intermediate AH: Ethyl 3 - pentyl octanoate

Chemical formula

[0360] Intermediate AI: 3 - pentyl octan - 1 - ol

Chemical formula

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

[0362] Intermediate AK: 6 - allylundecane

Chemical Structure

[0363] Intermediate AL: 4 - pentylnonan - 1 - ol

Chemical Structure

[0364] Intermediate AM: 3-Propylhexyl 8-bromooctanoate

Chem.

[0365] Intermediate AN: 3-Butylheptyl 8-bromooctanoate

Chem.

[0366] Intermediate AO: 3-pentyloctyl 8-bromooctanoate

Chemical Structure

[0367] Intermediate AP: 4-Pentyldecyl 8-bromooctanoate

Chemical Structure

[0368] Intermediate AQ: pentadec-8-yl 8-bromooctanoate

Chemical formula

[0369] Intermediate AR: Tridecan-7-yl 8-bromooctanoate

Chemical Structure

[0370] Intermediate AS: Undecan-6-yl 8-bromooctanoate

Chemical formula

[0371] Intermediate AT: Nonan-5-yl 8-bromooctanoate

Chemical Structure

[0372] Intermediate AU: 3-propylhexyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate

Chemical Structure

[0373] Intermediate AV: 3-propylhexyl 8-((2-hydroxyethyl)amino)octanoate

Chemical Structure

[0374] Intermediate AW: 3-pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate [Chemical Structure Diagram] A solution of tert-butyl N-(3-aminopropyl)carbamate (15.5 g, 88.8 mmol) in EtOH (38 mL) was added with 3-pentyloctyl 8-bromooctanoate (6.00 g, 14.8 mmol) in EtOH (36 mL) over 20 minutes. The reaction mixture was heated to 60 °C and stirred at this temperature for 16 hours. Upon cooling, the solvent was evaporated, the residue was diluted with ethyl acetate, and washed with saturated NaHCO 3 aqueous solution and brine (5×) until no white precipitate was observed in the aqueous layer. The organic layer was separated, washed with brine, dried (MgSO 4 ), filtered, and concentrated. The residue was purified by flash chromatography (0 - 5 - 10 - 25 - 50 - 100% in dichloromethane (mixture of 1% NH 4 OH in dichloromethane, 20% MeOH)) to afford 3-pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate (4.23 g, 8.49 mmol, 57%) as a clear oil. 1 1H NMR (300 MHz, CDCl 3 ) δ: ppm 5.17 (br. s, 1H); 4.07 ( t, 2H, J = 6.0 Hz); 3.19 (br. q, 2H, J = 6.0 Hz); 2.66 (t, 2H, J = 6.0 Hz); 2.56 (t, 2H, J = 6 .0 Hz); 2.28 (t, 2H, J = 6.0 Hz); 1.70 - 1.52 (m, 6H); 1.51 - 1.39 (m, 3H); 1.44 (s, 9H); 1.36 - 1.19 (m, 22H); 0.88 (t, 6H, J = 6.0 Hz).

[0375] Intermediate AX: 4-pentylnonyl 8-((2-hydroxyethyl)amino)octanoate

Chemical Structure

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

Chemical Structure

[0377] Intermediate AZ: Heptadecan-9-yl 8-((3-hydroxypropyl)amino)octanoate [Chemical Structure] To a round-bottom flask equipped with a magnetic stir bar, heptadecan-9-yl 8-bromooctanoate (1.00 g, 2.17 mmol), propanolamine (4.97 mL, 65.0 mmol), and ethyl alcohol (2 mL) were added. The resulting mixture was stirred at 40 °C for 16 h. The reaction was diluted with dichloromethane, washed with water (2×), and the layers were separated. The organic layer was dried (MgSO 4 )), filtered, and concentrated. The crude material was purified by silica gel chromatography (0 - 5 - 10 - 25 - 50 - 100% in dichloromethane (mixture of 1% NH 4The crude material was purified by (a mixture of OH, 20% MeOH)) to obtain heptadecane-9-yl 8-((3-hydroxypropyl)amino)octanoate (723 mg, 1.59 mmol, 73%) as a clear oil. UPLC / ELSD: RT = 2.06 min, MS(ES): m / z(MH + )C 28 H 57 NO 3 was 456.17 for.

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

Chemical formula

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

Chemical formula

[0380] Intermediate BC: 3-pentyloctyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate [Chemical formula] 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 cyclopentylmethyl ether (9 mL) and acetonitrile (9 mL), potassium carbonate (1.66 g, 12.0 mmol) and potassium iodide (366 mg, 2.21 mmol) were added. 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, and dried (MgSO 4) Filtered and concentrated. The crude residue was purified by silica gel chromatography (0 - 5 - 10 - 25 - 50 - 100% in dichloromethane (mixture of 1% NH in dichloromethane, 20% MeOH)) to give 3 - pentyloctyl 8 - ((3 - ((tert - butoxycarbonyl)amino)propyl)(8 - oxo - 8 - ((3 - propylhexyl)oxy)octyl)amino)octanoate (698 mg, 0.91 mmol, 45%) as a golden oil. UPLC / ELSD: RT = 2.82 min, MS(ES): m / z(MH 4 )) C + H 46 H 90 N 2 O 6 corresponding to 767.59.

[0381] Intermediate BD: 3 - butylheptyl 8 - ((3 - ((tert - butoxycarbonyl)amino)propyl)(8 - oxo - 8 - ((3 - propylhexyl)oxy)octyl)amino)octanoate

Chemical Structure

[0382] Intermediate BE: Bis(3-propylhexyl) 8,8'-((3-((tert-butoxycarbonyl)amino)propyl)azanediyl)dioctanoate [Chemical formula] 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) were 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 4 ), filtered, and concentrated. The crude residue was purified by silica gel chromatography (0 - 5 - 10 - 25 - 50 - 100% in dichloromethane (a mixture of 1% NH 4 OH, 20% MeOH)) to give bis(3-propylhexyl) 8,8'-((3-((tert-butoxycarbonyl)amino)propyl)azanediyl)dioctanoate (730 mg, 1.03 mmol, 45%) as a clear viscous oil. UPLC / ELSD: RT = 2.58 min, MS(ES): m / z(MH +)C 42 H 82 N 2 O 6 is 711.59 with respect to

[0383] Intermediate BF: 3-Butylheptyl 8-((3-((tert-Butoxycarbonyl)amino)propyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate [Chemical formula] 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 cyclopentylmethyl ether (9 mL) and acetonitrile (9 mL) were added potassium carbonate (1.66 g, 12.0 mmol) and potassium iodide (366 mg, 2.21 mmol). The reaction mixture 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 4 ), filtered, and concentrated. The crude residue was purified by silica gel chromatography (0-5-10-25-50-100% in dichloromethane (mixture of 1% NH 4 OH in dichloromethane, 20% MeOH)) to give 3-butylheptyl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate (896 mg, 1.13 mmol, 56%) as a clear oil. UPLC / ELSD: RT = 2.95 min, MS (ES): m / z (MH + )C 48 H 94 N 2 O 6 is 795.59 with respect to

[0384] Intermediate BG: 3-Pentyloctyl 8-((3-((tert-Butoxycarbonyl)amino)propyl)(8-oxo-8-(undecan-6-yloxy)octyl)amino)octanoate

Chem.

[0385] Intermediate BH: Nonan-5-yl 8-((3-((tert-Butoxycarbonyl)amino)propyl)(8-oxo-8-((3-pentyloctyl)oxy)octyl)amino)octanoate

Chem.

[0386] Intermediate BI: pentadecane - 8 - yl 8 - ((3 - ((tert - butoxycarbonyl)amino)propyl)(8 - oxo - 8 - ((3 - propylhexyl)oxy)octyl)amino)octanoate

Chemical Structure

[0387] Intermediate BJ: 3 - propylhexyl 8 - ((3 - ((tert - butoxycarbonyl)amino)propyl)(8 - oxo - 8 - (tridecane - 7 - yloxy)octyl)amino)octanoate [Chemical Structure] 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 a solution of potassium iodide (0.588 g, 3.54 mmol), potassium carbonate (1.73 g, 12.5 mmol), and tridecane - 7 - yl 8 - bromooctanoate (1.26 g, 3.11 mmol) in CMPE (9 mL). The reaction mixture was stirred at 77 °C for 18 h. The reaction mixture was cooled to room temperature, filtered, and then the filtrate was evaporated under vacuum. The residue was purified by silica gel chromatography [0 - 70% in dichloromethane (a mixture of 1% NH 4 OH in dichloromethane, 20% MeOH)] to give 3 - propylhexyl 8 - ({3 - [(tert - butoxycarbonyl)amino]propyl}[8 - oxo - 8 - (tridecane - 7 - yloxy)octyl]amino)octanoate (1.32 g, 55.1%) as a yellowish oil. UPLC / ELSD: RT = 2.70 min, Measured value: 767.34. 1 1H NMR (300 MHz, CDCl 3 ) δ: ppm 5.67 (br. s, 1H); 4.89 (p, 1H); 4.10 ( t, 2H); 3.20 (q, 2H); 2.61 - 2.43 (m, 2H); 2.43 -2.35 (m, 4H); 2.30 (dt, 4H); 1.71 - 1.49 (m, 14H); 1.49 - 1.40 (m, 12H); 1.40 - 1.19 (m, 36H); 1.01 - 0.83 (m, 12H).

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

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

[0390] Intermediate BM: 3 - pentyloctyl 8 - ((3 - aminopropyl)(8 - oxo - 8 - ((3 - propylhexyl)oxy)octyl)amino)octanoate [Chemical Structure] To a solution of 3 - pentyloctyl 8 - ((3 - ((tert - butoxycarbonyl)amino)propyl)(8 - oxo - 8 - ((3 - propylhexyl)oxy)octyl)amino)octanoate (698 mg, 0.91 mmol) in methylene chloride (18 mL) was added trifluoroacetic acid (1.39 mL, 18.2 mmol). The reaction mixture was stirred at room temperature for 4 hours. The reaction was quenched with saturated NaHCO 3 aqueous solution and extracted with dichloromethane. The organic layer was separated, washed with brine, dried (MgSO 4 ), filtered, and concentrated. The crude material was purified by silica gel chromatography (0 - 5 - 10 - 25 - 50 - 100% in dichloromethane (a mixture of 1% NH 4 OH and 20% MeOH in dichloromethane)) to give 3 - pentyloctyl 8 - ((3 - aminopropyl)(8 - oxo - 8 - ((3 - propylhexyl)oxy)octyl)amino)octanoate (378 mg, 0.57 mmol, 62%) as a clear oil. UPLC / ELSD: RT = 2.26 min, MS(ES): m / z(MH + ) C 41 H 82 N 2 O 4 corresponding to 667.56.

[0391] Intermediate BN: 3-Butylheptyl 8-((3-aminopropyl)(8-oxo-8-((3-propylhexyl)oxy)octyl)amino)octanoate

Chem.

Claims

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

2. Formula (B): 【Chemistry 6】 or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' ａ But, R' 分岐状 or R' 環式 and R' 分岐状 but, 【Chemistry 7】 and R' 環式 but, 【Chemistry 8】 and 【Chemistry 9】 indicates the attachment point, R ａα and R ａβ are each H, and R ａγ and R ａδ Each independently represents H, C ２－１２ Alkyl, and C ２－１２ alkenyl; R ａγ and R ａδ At least one of ２－１２ Alkyl and C ２－１２ alkenyl, R ｂα , R ｂβ , R ｂγ , and R ｂδ Each independently represents H, C ２－３０ Alkyl, and C ５－２０ alkenyl; R ｂα , R ｂβ , R ｂγ , and R ｂδ At least one of ２－３０ Alkyl and C ５－２０ alkenyl, R ４ But -(CH ２ ) ２ OH, -(CH ２ ) ３ OH, -(CH ２ ) ４ OH, -(CH ２ ) ５ OH, and 【Chemistry 10】 is selected from the group consisting of 【Chemistry 11】 indicates the attachment point, R １０ But N(R) ２ and each R is independently １－６ Alkyl, C ２－３ alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R ５ However, independently, OH, C １－３ Alkyl, C ２－３ alkenyl, and H; Each R ６ However, independently, OH, C １～３ Alkyl, C ２～３ alkenyl, and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C １－１２ Alkyl or C ２－１２ alkenyl, Y ａ But, C ３－６ is a carbocyclic ring, R ＊ " ａ But, C １－１５ Alkyl and C ２－１５ alkenyl, l is selected from the group consisting of 1, 2, 3, 4, and 5; s is 2 or 3; The compound of formula (B) above, or an N-oxide thereof, or a salt or isomer thereof, wherein m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

3. Formula (1-1): 【Chemistry 12】 or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' ａ But, R' 分岐状 or R' 環式 and R' 分岐状 but, 【Chemistry 13】 and R' 環式 but, 【Chemistry 14】 and R' ｂ but, 【Chemistry 15】 and 【Chemistry 16】 indicates the attachment point, R ａγ and R ｂγ Each independently, C ２－１２ Alkyl or C ２－１２ alkenyl, R ２ and R ３ Each independently, C １－１４ Alkyl and C ２－１４ alkenyl, R ４ But -(CH ２ ) ２ OH, Each R' is independently C １－１２ Alkyl or C ２－１２ alkenyl, Y ａ But, C ３－６ is a carbocyclic ring, R ＊ " ａ But, C １－１５ Alkyl and C ２－１５ alkenyl, The compound of the formula (1-1) above, or an N-oxide thereof, or a salt or isomer thereof, wherein s is 2 or 3.

4. The structure: 【Chemistry 17】 2. A compound according to any one of the preceding claims, having one of the following:

5. R ａγ But, C ２－６ 2. A compound according to any one of the preceding claims which is alkyl.

6. R ｂγ But, C ２－６ 2. A compound according to any one of the preceding claims which is alkyl.

7. R ａγ and R ｂγ Each independently, C ２－６ 2. A compound according to any one of the preceding claims which is alkyl.

8. R ｂγ But, C ４－６ 2. A compound according to any one of the preceding claims which is alkyl.

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

10. Y ａ 4. A compound according to any one of the preceding claims, wherein is cyclohexyl or cyclopentyl.

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

12. Each R ’ But independently, C ２－５ 2. A compound according to any one of the preceding claims which is alkyl.

13. A compound selected from the following: 【Table 1】

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

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

16. The phospholipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), 2. The empty LNP of any one of the preceding claims, which is selected from the group consisting of sphingomyelin, and mixtures thereof.

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

18. 2. 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. The PEG lipid is ２ｋ -DMG and PEG-1: 【Chemistry 18】 5. The empty LNP of any one of the preceding claims, selected from:

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

21. 2. The loaded LNP of any one of the preceding claims, wherein the one or more therapeutic and / or prophylactic agents are nucleic acids.

22. 2. The loaded LNP of any one of the preceding claims, wherein the nucleic acid is RNA, and the RNA is selected from the group consisting of short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecules, microRNA (miRNA), antagomir, antisense RNA, ribozymes, dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

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

24. 13. A pharmaceutical composition comprising the loaded LNPs of any one of the preceding claims and a pharma- ceutically acceptable carrier.

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

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

27. A method for producing a polypeptide of interest in cells in a subject, the method comprising administering to the subject a loaded LNP according to any one of the preceding claims.

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

29. 10. 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. 13. The method according to any one of the preceding claims, wherein the administration is performed parenterally, intramuscularly, intradermally, subcutaneously and / or intravenously.

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