Novel ionized lipids and lipid nanoparticles and methods of using them

Abstract

Novel ionized lipids and lipid nanoparticles and methods of using them are provided. The present disclosure relates to novel ionizable lipids and lipid compositions that can be used to deliver therapeutic agents, as well as pharmaceutical compositions comprising these ionizable lipids.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 323,948, filed March 25, 2022, which is incorporated herein by reference in its entirety.

[0002] Lipid nanoparticles ("LNPs") formed from ionizable amine-containing lipids can function as therapeutic cargo vehicles for intracellular delivery of bioactive agents, such as coding RNA (i.e., messenger RNA (mRNA), guide RNA) and non-coding RNA (i.e., antisense, siRNA). LNPs can facilitate delivery of oligonucleotide agents across cell membranes and can be used to introduce components and compositions into living cells.

[0003] Bioactive agents that are particularly difficult to deliver into cells include proteins, nucleic acid-based drugs, and their derivatives, especially drugs containing relatively large oligonucleotides such as mRNA or guide RNA. Compositions for delivering promising mRNA therapeutics or editing technologies into cells, such as the delivery of components of the CRISPR / Cas9 system, are of particular interest.

[0004] With the recent emergence of pandemic diseases, messenger RNA therapy has become an increasingly important option for the treatment of various diseases, including viral infectious diseases and diseases associated with the deficiency of one or more proteins. Compositions that can stabilize and / or deliver RNA components and have properties useful for in vitro and in vivo delivery have also been of particular interest.

[0005] Thus, there continues to be a need in the art for novel lipid compounds for the development of lipid nanoparticles or other lipid delivery mechanisms for the delivery of therapeutic agents. The present invention addresses that need. Summary of the Invention

[0006] Disclosed herein are novel ionizable lipids that can be combined with at least one other lipid component, such as a neutral lipid, cholesterol, or a polymer-conjugated lipid, to form lipid nanoparticle compositions that can be used to facilitate intracellular delivery of therapeutic nucleic acids in vitro and / or in vivo.

[0007] This specification discloses ionizable amine-containing lipids useful for forming lipid nanoparticle compositions.Such LNP compositions can have advantageous properties for the delivery of nucleic acid cargo, such as the delivery of coding RNA and non-coding RNA to cells.Also provided is a method for treating various diseases or pathologies caused by the deficiency of infectious agents and / or proteins using the lipid nanoparticles disclosed herein.

[0008] For example, ionizable lipids of various formulas are disclosed below, including formulas (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), (IIIA)-(IIIIE), (IIIC-1), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), and (VC-1)-(VC-6).

[0009] One aspect of the present invention is a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein: [ka] is a cyclic or heterocyclic moiety, Y is alkyl, hydroxy, hydroxyalkyl, or [ka] and A is absent, -O-, -N(R 7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -SS-, or a divalent heterocycle; X and Z each independently represent absent, —O—, —CO—, —N(R 7 )-, -O-alkylene-; -Alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, or -S-, Each R 7 are independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 70 , R 80 , R 90 , R 100 , R 110 , and R 120 are each independently H, C-C optionally interrupted by a heteroatom or substituted by OH, SH, or halogen. 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl; l and m are each an integer of 1 to 10, t1 is an integer from 0 to 10, and W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterosilyl or heteroaryl.

[0010] In some embodiments, a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein [ka] is a cyclic or heterocyclic moiety, Y is alkyl, hydroxy, hydroxyalkyl, [ka] and A is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -SS-, or a divalent heterocycle; X and Z each independently represent absent, —O—, —N(R 7 )-, -O-alkylene-; -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, or -S-, Each R 7 are independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 70 , R 80 , R 90 , R 100 , R 110 , and R 120 are each independently H, C-C optionally interrupted by a heteroatom or substituted by OH, SH, or halogen. 16Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl; l and m are each an integer of 1 to 10, t is 0, 1, 2, or 3; t1 is an integer from 0 to 10, and W is hydroxyl, hydroxyalkyl, or one of the following moieties: [ka] During the ceremony Each Q is independently selected from absent, -O-, -C(O)-, -C(S)-, -C(O)O-, -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, or N + (R 7 ) 3-alkylene-Q-; Each R 8 are independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl, or two R 8 may form a ring together with the nitrogen atom, q is 0, 1, 2, 3, 4, or 5; and p is 0, 1, 2, 3, 4, or 5.

[0011] In some embodiments, Y is hydroxyl, [ka] is.

[0012] In some embodiments, compounds of formula (IA-1) or (IA-2): [ka] or a stereoisomer of any of the foregoing, During the ceremony [ka] is a cyclic or heterocyclic moiety, A is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, N(R 7 )C(O)NH—, —S—, —SS—, or a divalent heterocycle; X is absent, -O-, -C(O), -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, or -S-, Z is absent, -O-, -N(R 7 )-, -O-alkylene-; -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, or -S-, Each R 7 are independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 100 , R 110 , and R 120 are each independently H, C-C optionally interrupted by a heteroatom or substituted by OH, SH, or halogen. 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl; R 90 is C1-C 15 Branched or unbranched alkyl, C1-C 15 branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl; and t is 0, 1, 2, or 3; t1 is an integer from 0 to 10, l is an integer from 1 to 10, m is an integer from 1 to 10, and W is hydroxyl, hydroxyalkyl, or one of the following moieties: [ka] During the ceremony Q is -O-, -C(O)-, -C(S)-, -C(O)O-, -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and Each R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, or N + (R 7 ) 3-alkylene-Q-; Each R 8 are independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl, or two R 8 may form a ring together with the nitrogen atom q is 0, 1, 2, 3, 4, or 5; and p is 0, 1, 2, 3, 4, or 5.

[0013] In some embodiments, [ka] is the expression [ka] wherein: G1, G2, G3, G4, G5, G6, and G7 are each independently C(R')(R''), O, or N, provided that no more than two of G1-G7 are O or N; R' and R'' are each independently absent, H, alkyl, or two R's from two adjacent G's together form a second 5-7 membered cyclic or heterocyclic ring; and n1 and n2 each independently represent 0 or 1.

[0014] In some embodiments, [ka] is selected from pyrrolidine, piperidine, piperazine, cyclohexane, cyclopentane, tetrahydrofuran, tetrahydropyran, morpholine, and dioxane.

[0015] In some embodiments, [ka] is a 5-7 membered monocyclic ring. [ka] is a 5-7 membered monocyclic cycloalkane ring. [ka] is a 5- to 7-membered monocyclic heterocycle.

[0016] In some embodiments, [ka] is a bicyclic or tricyclic ring, ie, contains two or more rings, including fused rings.

[0017] In some embodiments, X is absent, —O—, or —C(O)—.

[0018] In some embodiments, Z is —O—, —C(O)O—, or —OC(O)—.

[0019] In some embodiments, R 30 , R 40 , R 50 , and R 60 are each H or C1-C4 branched or unbranched alkyl.

[0020] In some embodiments, R 30 , R 40、 R 50 , and R 60 are each H.

[0021] In some embodiments, R 70 and R 80 are H and R 90 is C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl, cycloalkyl, or substituted cycloalkyl. 90 is C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 90 is C1-C 15 In some embodiments, R is a branched or unbranched alkyl. 90 is C1-C 12 It may be branched or unbranched alkyl.

[0022] In some embodiments, R 70 is H and R 80 and R 90 are each independently C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl, or a cycloalkyl or substituted cycloalkyl. 80 and R 90 are each independently C1-C 15Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 80 and R 90 are each independently C1-C 15 In some embodiments, R is a branched or unbranched alkyl. 80 and R 90 are each independently C1-C 12 In some embodiments, R is a branched or unbranched alkyl. 80 and R 90 are each independently a C1-C8 branched or unbranched alkyl.

[0023] In some embodiments, R 100 is H and R 110 and R 120 are each independently C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl, or a cycloalkyl or substituted cycloalkyl. 110 and R 120 are each independently C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 110 and R 120 are each independently C1-C 15 In some embodiments, R is a branched or unbranched alkyl. 110 and R 120 are each independently C1-C 12 In some embodiments, R is a branched or unbranched alkyl. 110 and R 120 are each independently a C1-C8 branched or unbranched alkyl.

[0024] In some embodiments, l is 3 to 10, 3 to 7, or 4 to 7. In some embodiments, l is 4, 5, 6, 7, 8, 9, or 10. In some embodiments, l is 4, 5, 6, or 7.

[0025] In some embodiments, m is 4 to 10, 5 to 8, 1 to 7, 3 to 7, or 1 to 5. In some embodiments, m is 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 3, 4, or 5. In some embodiments, m is 5, 6, 7, or 8.

[0026] In some embodiments, M is —OC(O)—, —C(O)O—, —N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(OR 13 )-O-, -C(O)O(CH2) r- , -C(O)N(R 7 )(CH2) r -, or -C(OR 13 )-O-(CH2) r wherein each R 7 are independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, and R 13 is a branched or unbranched C3-C 10 alkyl, and r is 1, 2, 3, 4, or 5.

[0027] Another aspect of the present invention relates to lipid compositions comprising a lipid compound of any formula disclosed herein, e.g., any one of Formulas (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), (IIIA)-(IIIIE), (IIIC-1), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), and (VC-1)-(VC-6), wherein the lipid composition is a lipid nanoparticle (LNP). In some embodiments, the lipid composition further comprises a second lipid. In some embodiments, the lipid composition comprises a ratio of about 1:1 between the compound and the second lipid. In some embodiments, the second lipid is a cationic lipid, an anionic lipid, an ionizable lipid, or a zwitterionic lipid.

[0028] Also disclosed herein are pharmaceutical compositions comprising a pharmaceutically acceptable excipient and the lipid composition described herein, wherein the lipid composition comprises one or more lipid compounds selected from ionizable lipids of formulas (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), (IIIA)-(IIIIE), (IIIC-1), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), and (VC-1)-(VC-6). The pharmaceutical composition may further comprise a therapeutic agent. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Such compositions may be useful for forming lipid nanoparticles for delivery of therapeutic agents.

[0029] Another aspect of the present disclosure provides a method of delivering a therapeutic agent to a subject (e.g., a patient) in need thereof, comprising administering to the subject (e.g., a patient) a pharmaceutical composition comprising a lipid nanoparticle composition comprising a lipid compound of Formulas (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), (IIIA)-(IIIIE), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), and (VC-1)-(VC-6), a pharmaceutically acceptable salt thereof, and / or a stereoisomer of any of the foregoing, and a therapeutic agent. In some embodiments, the method further comprises preparing a lipid nanoparticle composition comprising a lipid compound of formula (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), and (IIIA)-(IIIIC), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), and (VC-1)-(VC-6), a pharmaceutically acceptable salt thereof, and / or a stereoisomer of any of the foregoing, and a therapeutic agent.

[0030] Another aspect of the present disclosure provides extrahepatic (e.g., to the pancreas, spleen, or lung) delivery of a therapeutic agent to a subject, comprising administering to the subject a pharmaceutical composition comprising a lipid nanoparticle composition comprising a lipid compound of Formulas (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), (IIIA)-(IIIIE), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), and (VC-1)-(VC-6), a pharmaceutically acceptable salt thereof, and / or a stereoisomer of any of the foregoing, and a therapeutic agent. In some embodiments, the total therapeutic agent administered to a subject has a spleen-to-liver ratio of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the total therapeutic agent administered to a subject has a spleen-to-liver ratio of at least 1. In some embodiments, the total therapeutic agent administered to a subject has a spleen-to-liver ratio of at least 5.

[0031] These and other aspects of the present disclosure will become evident upon reference to the following detailed description. [Brief explanation of the drawings]

[0032] [Figure 1] Figure 1 shows the spleen:liver ratio of mean radiance (p / s / cm2 / sr) for various exemplary lipid nanoparticle compositions containing exemplary lipid compounds (LNP 2230, LNP 2231) compared to lipid nanoparticle compositions containing C12-200 and MC3, respectively, based on EPO levels determined by in vivo bioluminescence imaging for each lipid nanoparticle composition, as described in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0033] definition As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0035] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0036] Unless the context requires otherwise, throughout this specification and claims, the term "comprise" and variations thereof, such as "comprises" and "comprising," are to be interpreted in an open and inclusive sense, i.e., "including but not limited to."

[0037] The phrase "inducing the expression of a desired protein" refers to the ability of a nucleic acid to increase the expression of a desired protein.To determine the level of protein expression, a test sample (e.g., a sample of cells in culture that express the desired protein) or a test mammalian (e.g., a mammalian, such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, is contacted with a nucleic acid (e.g., a nucleic acid combined with a lipid of the present disclosure).The expression of the desired protein in the test sample or test animal is compared with the expression of the desired protein in a control sample (e.g., a sample of cells in culture that express the desired protein) or a control mammalian (e.g., a mammalian, such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, which has not been contacted with or administered with a nucleic acid.If the desired protein is present in the control sample or control mammal, the expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In some embodiments, induction of desired protein expression is achieved when the ratio of the desired protein expression level in a test sample or test mammal to the desired protein expression level in a control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. If the desired protein is not present in a control sample or control mammal, induction of desired protein expression is achieved when any measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will understand suitable assays for determining protein expression levels in a sample, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.

[0038] The phrase "inhibit the expression of a target gene" refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene.To determine the degree of gene silencing, a test sample (e.g., a sample of cells in culture that express a target gene) or a test mammalian (e.g., a mammalian, such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, is contacted with the nucleic acid that silences, reduces, or inhibits the expression of a target gene.The expression of the target gene in the test sample or test animal is compared with the expression of the target gene in a control sample (e.g., a sample of cells in culture that express a target gene) or a control mammalian (e.g., a mammalian, such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, which is not contacted with or administered with nucleic acid.The expression of the target gene in the control sample or control mammalian can be assigned a value of 100%. In some embodiments, silencing, inhibition, or reduction of expression of a target gene is achieved when the level of target gene expression in a test sample or test mammal relative to the level of target gene expression in a control sample or control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other words, the nucleic acid can silence, reduce or inhibit the expression of the target gene in the test sample or test mammal by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the level of target gene expression in the control sample or control mammal that is not contacted with or administered with the nucleic acid. Suitable assays for determining the level of target gene expression include, but are not limited to, testing protein or mRNA levels using techniques known to those skilled in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

[0039] An "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to achieve the desired effect, e.g., increase or inhibit expression of a target sequence relative to the normal expression level detected in the absence of the nucleic acid. Increased expression of a target sequence is achieved when any measurable level of expression product not present in the absence of the nucleic acid is detected. When the expression product is present at a certain level before contact with the nucleic acid, increased expression is achieved when the fold increase over the value obtained using a nucleic acid, such as mRNA, relative to the control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000, or more. Inhibition of expression of a target gene or target sequence is achieved when the value obtained using a nucleic acid, such as an antisense oligonucleotide, relative to a control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of a target gene or target sequence include, for example, examination of protein or RNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of an appropriate reporter protein, and phenotypic assays known to those skilled in the art.

[0040] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. DNA may be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, polyvalent RNA, Dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include synthetic, natural, and non-natural nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-0-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single-nucleotide polymorphisms, and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via the phosphate group.

[0041] "Bases" include purines and pyrimidines (which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs), and synthetic derivatives of purines and pyrimidines (which include, but are not limited to, modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides).

[0042] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises partial or full-length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

[0043] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0044] The term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, that are generally characterized by poor solubility in water but solubility in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0045] "Steroids" are compounds with the following carbon skeleton: [ka] A non-limiting example of a steroid is cholesterol.

[0046] As used herein, the term "compound" is meant to include all isomers and isotopes of the depicted structure, all pharmaceutically acceptable salts, solvates, or hydrates thereof, and all crystalline forms (e.g., crystalline polymorphs), mixtures of crystalline forms, or anhydrates or hydrates thereof.

[0047] "Isotopes" refer to atoms having the same atomic number but different mass numbers, resulting from different numbers of neutrons in their nuclei. For example, isotopes of hydrogen include tritium (3H) and deuterium (2H).

[0048] "Isomers" The compounds described herein, or pharmaceutically acceptable salts thereof, may include all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, and the like. For example, compounds may contain one or more stereocenters and thus give rise to geometric isomers (e.g., double bonds giving rise to geometric E / Z isomers), enantiomers, diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers), and other stereoisomeric configurations that can be defined in terms of absolute stereochemistry, such as (R)- or (S)-, such as sugar isomers, or (D)- or (L)-, such as amino acids. The present disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R) and (S), or (D) and (L) isomers can be prepared using chiral synthesis or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of a racemate (or racemate of a salt or derivative), for example, using chiral high-pressure liquid chromatography (HPLC). Enantiomeric and stereoisomeric mixtures of compounds and means for resolving them into their component enantiomers or stereoisomers are well known. When compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0049] The terms "crystalline polymorph," "polymorph," or "crystalline form" refer to crystalline structures in which a compound (or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, density hardness, crystal shape, optical and electrical properties, stability, and solubility. Depending on the recrystallization solvent, crystallization rate, storage temperature, and other factors, one crystalline form may predominate. Crystalline polymorphs of a compound can be prepared by crystallization under different conditions. Crystallization of the compounds disclosed herein may produce solvates.

[0050] As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of an ionizable lipid compound of the present disclosure with one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, etc. Alternatively, the solvent may be an organic solvent.

[0051] As used herein, "ionizable lipid" refers to a lipid that can be charged. In some embodiments, the ionizable lipid comprises one or more positively charged amine groups. In some embodiments, the ionizable lipid can be ionized so that it can exist in a positively charged form or a neutral form depending on the pH. The ionization of the ionizable lipid affects the surface charge of lipid nanoparticles comprising the ionizable lipid under different pH conditions. The surface charge of lipid nanoparticles can, in turn, affect their plasma protein absorption, blood clearance, and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)), as well as their ability to form endosomolytic non-bilayer structures (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)), which can affect the intracellular delivery of nucleic acids.

[0052] The term "polymer-conjugated lipid" refers to a molecule that contains both a lipid portion and a polymer portion. A non-limiting example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include, for example, l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG). As used herein, the terms "PEG lipid" and "PEGylated lipid" are interchangeable and refer to a lipid that contains a polyethylene glycol component.

[0053] The term "neutral lipid" refers to any lipid that exists in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-5n-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramides, and steroids, such as sterols, and their derivatives. Neutral lipids may be synthetic or naturally occurring.

[0054] As used herein, "phospholipid" refers to a lipid containing a phosphate moiety and one or more carbon chains, such as an unsaturated fatty acid chain. A phospholipid may contain one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). Certain phospholipids may facilitate fusion with a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or an intracellular membrane). The fusion of a phospholipid with a membrane may allow one or more components of a lipid-containing composition to pass through the membrane, for example, allowing delivery of one or more components to a cell.

[0055] As used herein, the term "liposome" refers to a composition comprising an outer lipid membrane (e.g., a single lipid bilayer, known as a unilamellar liposome, or multiple lipid bilayers, known as a multilamellar liposome) enclosing an internal aqueous space that may contain cargo. See, e.g., Cullis et al., Biochim. Biophys Acta, 559:399-420 (1987), which is incorporated herein by reference in its entirety. Unilamellar liposomes generally have diameters ranging from about 20 to about 400 nanometers (nm), about 50 to about 300 nm, about 100 to about 200 nm, or about 300 to about 400 nm. Multilamellar liposomes typically have diameters ranging from about 1 to about 10 μm and may contain two to several hundred concentric lipid bilayers alternating with layers of aqueous phase.

[0056] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) and comprising one or more compounds of Formula (I) disclosed herein. In some embodiments, lipid nanoparticles comprising one or more compounds of Formula (I), pharmaceutically acceptable salts thereof, and / or any stereoisomers thereof, are included in compositions that can be used to deliver therapeutic agents, such as nucleic acids (e.g., mRNA), to a desired target site (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, lipid nanoparticles comprise one or more compounds of Formula (I), pharmaceutically acceptable salts thereof, and / or any stereoisomers thereof, and nucleic acids. In some embodiments, lipid nanoparticles comprise one or more compounds of Formula (I), pharmaceutically acceptable salts thereof, and / or any stereoisomers thereof, and nucleic acids, as well as one or more other lipids selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, therapeutic agents such as nucleic acids may be encapsulated within the lipid portion of the lipid nanoparticle or within the aqueous space covered by some or all of the lipid portion of the lipid nanoparticle, thereby protecting them from enzymatic degradation or other undesirable effects elicited by the host organism's or cellular machinery, such as a harmful immune response.

[0057] In some embodiments, the lipid nanoparticles have an average particle size of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 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, and are substantially non-toxic. In some embodiments, when the nucleic acid is present in the lipid nanoparticle, it is resistant to degradation by nucleases in aqueous solution. Lipid nanoparticles containing nucleic acids and methods for their preparation are described, for example, in U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031, and WO 2013 / 016058 and WO 2013 / 086373, 8,569,256, 5,965,542 and U.S. Patent Publication Nos. 2016 / 0199485, 2016 / 0009637, 2015 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, 2014 / 0308304, 2014 / 0200257, 2013 / 086373, 2013 / 0338210, 2013 / 0323269, 2013 / 0245107, 2013 / 0195920, and 2013 / 0123338 , No. 2013 / 0022649, No. 2013 / 0017223, No. 2012 / 0295832, No. 2012 / 0183581, No. 2012 / 0172411, No. 2012 / 0027803, No. 2012 / 0058188, No. 2011 / 0311583, 2011 / 0311582, 2011 / 0262527, 2011 / 0216622, 2011 / 0117125, 2011 / 0091525, 2011 / 0076335, 2011 / 00600 32, 2010 / 0130588, 2007 / 0042031, 2006 / 0240093, 2006 / 0083780, 2006 / 0008910, 2005 / 0175682, 2005 / 017054, 2005 / 0118253, 2005 / 0064595, 2004 / 0142025, 2007 / 0042031, 1999 / 009076, and International Publication Nos. 99 / 39741, 2017 / 117528, and 2019 / 117529. and WO 2017 / 004143, WO 2017 / 075531, WO 2015 / 199952, WO 2014 / 008334, WO 2013 / 086373, WO 2013 / 086322, WO 2013 / 016058, WO 2013 / 086373, WO 2011 / 141705, and WO 2001 / 07548, the entire disclosures of which are incorporated herein by reference for all purposes.

[0058] As used herein, the term "size" refers to the hydrodynamic diameter of a lipid nanoparticle population. Measurement of the size of a lipid nanoformulation can be used to indicate the size and population distribution (polydispersity index, PDI) of the composition.

[0059] As used herein, "polydispersity index" is the ratio between the weight average molar mass and the number average molar mass, Mn, which describes the homogeneity of the particle size distribution of a system. For example, a small value less than 0.3 indicates a narrow particle size distribution.

[0060] The polydispersity index can be used to indicate the homogeneity of a lipid composition (e.g., liposomes or LNPs), e.g., the particle size distribution of the liposomes or LNPs. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The lipid composition may have a polydispersity index of about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the lipid composition may be about 0.10 to about 0.20.

[0061] As used herein, the term "apparent pKa" refers to the pH at which a lipid nanoformulation (e.g., LNP) is 50% protonated. This can be used as an indicator of the pH range at which a lipid nanoformulation (e.g., LNP) becomes protonated and initiates the endosomal escape process in nucleotide delivery.

[0062] As used herein, the term "zeta potential" refers to the electrokinetic potential of lipids, for example, in lipid nanoformulations (e.g., LNP compositions). Zeta potential can describe the surface charge of LNP compositions. Zeta potential is useful for predicting organ tropism and potential interactions with serum proteins.

[0063] The zeta potential of a lipid composition (e.g., liposome or LNP) can be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential can describe the surface charge of the liposome or LNP. Lipid compositions (e.g., liposomes or LNPs) with a relatively low positive or negative charge are generally desirable because more highly charged species may have undesirable interactions with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the liposome or LNP may 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.

[0064] As used herein, "encapsulated" by a lipid refers to a therapeutic agent, such as a nucleic acid (e.g., mRNA), being fully or partially encapsulated by the lipid nanoparticle. In some embodiments, the nucleic acid (e.g., mRNA) is fully encapsulated in the lipid nanoparticle.

[0065] As used herein, "encapsulation efficiency" or "encapsulation efficiency" refers to the percentage of encapsulated cargo (e.g., therapeutic and / or prophylactic agent) successfully incorporated into (e.g., encapsulated in, or otherwise associated with) a lipid composition (e.g., an LNP or liposome) relative to the initial total amount of therapeutic and / or prophylactic agent provided. For example, if 97 mg of therapeutic and / or prophylactic agent are encapsulated in the lipid composition out of a total of 100 mg of therapeutic and / or prophylactic agent initially provided, the encapsulation efficiency may be given as 97%. Encapsulation efficiency can indicate the efficiency of encapsulated cargo (e.g., nucleic acid molecules) loaded into a lipid composition using a particular formulation method and recipe.

[0066] The efficiency of encapsulation of cargo, such as proteins and / or nucleic acids, is expressed as the amount of protein and / or nucleic acid encapsulated or associated with a lipid composition (e.g., liposomes or LNPs) after preparation relative to the initial amount provided. High encapsulation efficiencies are desirable (e.g., at least 70%, 80%, 90%, approaching 95%, or 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing liposomes and LNPs with that after the liposomes and LNPs are disrupted with one or more organic solvents or detergents. Anion exchange resins can also be used to measure the amount of free protein or nucleic acid (e.g., RNA) in solution. Fluorescence can also be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in solution. For the liposomes or LNPs described herein, the encapsulation efficiency of proteins and / or nucleic acids may 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 may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0067] "Serum stability" in relation to nucleic acid-lipid nanoparticles means that the nucleic acid is not significantly degraded after exposure to serum or nuclease assays that would significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0068] Some administration techniques can result in systemic delivery of certain drugs, but not others. "Systemic delivery" means that a useful, e.g., therapeutic, amount of drug is delivered to most of the body. Systemic delivery of lipid nanoparticles can be achieved by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0069] As used herein, "local delivery" refers to the direct delivery of a drug to a target site within an organism.For example, a drug can be delivered locally by direct injection into a disease site such as a tumor, other target site such as an inflammation site, or target organ such as the liver, heart, pancreas, or kidney.Local delivery can also include topical application or local injection techniques such as intramuscular, subcutaneous, or intradermal injection.Local delivery does not interfere with systemic pharmacological effects.

[0070] As used herein, "administration method" can include both systemic and local delivery. "Systemic delivery" means that a useful, e.g., therapeutic, amount of an agent is delivered to most of the body. Systemic delivery of liposomes or LNPs can be achieved by any means known in the art, including, for example, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery. As used herein, "local delivery" refers to the delivery of an agent directly to a target site within an organism. For example, an agent can be delivered locally by direct injection into a disease site such as a tumor, another target site such as a site of inflammation, or a target organ such as the liver, heart, pancreas, or kidney. Local delivery can also include topical application or local injection techniques such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not interfere with systemic pharmacological effects. As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues typically linked by peptide bonds, which can be naturally (e.g., isolated or purified) or synthetically produced.

[0071] "Nucleic acid" is meant to define an oligonucleotide or polynucleotide sequence. Non-limiting examples of oligonucleotides or polynucleotides are DNA, plasmid DNA, self-amplifying RNA, mRNA, siRNA, and tRNA. The term also encompasses RNA / DNA hybrids. Nucleotides are typically linked in nucleic acids by phosphodiester bonds, but the term "nucleic acid" also encompasses nucleic acid analogs with other types of bonds or backbones (e.g., phosphoramide, phosphorothioate, phosphorodithioate, O-methylphosphoramidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptide nucleic acid (PNA) bonds or backbones, among others). Nucleic acids may be single-stranded, double-stranded, or contain portions of both single-stranded and double-stranded sequences. Nucleic acids can contain any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including, for example, adenine, thymine, cytosine, guanine, uracil, and modified or non-standard bases, including, for example, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

[0072] As used herein, "RNA" refers to a ribonucleic acid, which may be natural or non-natural. For example, RNA may contain modifications and / or non-natural components, such as one or more nucleic acid bases, nucleosides, nucleotides, or linkers. RNA may contain a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, e.g., in vivo translation of an mRNA within a mammalian cell, can produce the encoded polypeptide. The RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), mRNA, and mixtures thereof.

[0073] "Alkyl" refers to an alkyl group consisting solely of carbon and hydrogen atoms, e.g., 1 to 24 carbon atoms (C1-C 24 alkyl), 4 to 20 carbon atoms (C4-C 20 alkyl), 6 to 16 carbon atoms (C6-C 16 alkyl), 6 to 9 carbon atoms (C6-C9 alkyl), 1 to 15 carbon atoms (C1-C 15 alkyl), 1 to 12 carbon atoms (C1-C 12 "C-C alkyl" refers to a straight or branched hydrocarbon chain radical having 1 to 8 carbon atoms (C-C alkyl), 1 to 8 carbon atoms (C-C alkyl), or 1 to 6 carbon atoms (C-C alkyl) and attached to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Unless stated otherwise in the specification, alkyl groups are optionally substituted.

[0074] An "alkylene" or "alkylene chain" is an alkylene group that connects the rest of the molecule to a radical group and consists solely of carbon and hydrogen, e.g., 1 to 24 carbon atoms (C-C 24 Alkylene, 1 to 15 carbon atoms (C1-C 15 Alkylene, 1 to 12 carbon atoms (C1-C 12 "Alkylene" refers to a straight or branched divalent hydrocarbon chain having 1 to 8 carbon atoms (C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), or 1 to 2 carbon atoms (C1-C2 alkylene), for example, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain.

[0075] The term "alkenyl" refers to a straight or branched hydrocarbon chain having one or more double bonds. Unless otherwise indicated, "alkenyl" generally refers to a C2-C8 alkenyl (e.g., a C2-C6 alkenyl, a C2-C4 alkenyl, or a C2-C3 alkenyl). Typical examples of alkenyl include, but are not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl, and 3-octenyl groups.

[0076] The term "alkynyl" refers to a straight or branched hydrocarbon chain containing 2 to 8 carbon atoms and characterized by one or more triple bonds. Unless otherwise indicated, "alkynyl" generally refers to C2-C8 alkynyl (e.g., C2-C6 alkynyl, C2-C4 alkynyl, or C2-C3 alkynyl). Some examples of typical alkynyls are ethynyl, 2-propynyl, and 3-methylbutynyl, as well as propargyl. 2 and sp 3The carbons can optionally serve as the point of attachment for the alkenyl and alkynyl groups, respectively.

[0077] As used herein, the term "cycloalkyl" or "cyclyl" includes saturated and partially unsaturated, but not aromatic, cyclic hydrocarbon groups having 3 to 12 carbons, e.g., 3 to 8 carbons, and e.g., 3 to 6 carbons, where the cycloalkyl group may further be optionally substituted. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0078] The term "heteroaryl" or "heteroa" refers to an aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic), and 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. The term also includes groups in which a heteroaromatic ring is fused to one or more aryl, cycloalkyl, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Examples of heteroaryl groups include pyrrolyl, pyridyl, pyridazinyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, pyrazinyl, indolizinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, isothiazolyl, thiadiazolyl, purinyl, naphthyridinyl, pteridinyl, isoindolyl, benzyl, benzo ... Examples include inzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one.

[0079] The term "heterocyclyl," "heterocycle," "heterocyclic radical," or "heterocycle" refers to a 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms in the monocyclic ring, 1 to 6 heteroatoms in the bicyclic ring, or 1 to 9 heteroatoms in the tricyclic ring, selected from O, N, or S (e.g., carbon atoms and 1 to 3, 1 to 6, or 1 to 9 heteroatoms of N, O, or S, respectively, in the monocyclic, bicyclic, or tricyclic ring), and 0, 1, 2, or 3 atoms in each ring may be substituted by substituents. As used herein, it generally can include both non-aromatic and aromatic rings (e.g., generally covered by heteroaryl). The term also includes groups in which a heterocycle is fused to one or more aryl, cycloalkyl, or heterocyclyl rings. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl).

[0080] Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, quinuclidinyl, and the like.

[0081] Examples of heterocyclyl groups also include pyrrolyl, pyridyl, pyridazinyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, pyrazinyl, indolizinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, isothiazolyl, thiadiazolyl, purinyl, naphthyridinyl, pteridinyl, isoindolyl, benzothienyl, benzophenone, benzothienyl ...thienyl, thienyl, thienyl, thienyl, thienyl, thienyl, thienyl, th Typical heteroaryl groups include benzoyl, benzofuranyl, dibenzofuranyl, indazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one.

[0082] Divalent alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl radicals are formed by removing a hydrogen atom from an alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl radical, respectively (or by removing two hydrogen atoms from an alkane, alkene, arene, heteroarene, cycloalkane, or heterocycle, respectively).

[0083] For example, the term "divalent heterocycle" or "divalent heterocycle" refers to a divalent form of a heterocycle, i.e., a divalent or divalent radical formed by removing a hydrogen atom from a heterocycle radical (or by removing two hydrogen atoms from a heterocycle). For example, a divalent or divalent form of a heterocycle is formed by removing a hydrogen atom from each of two different atoms of the heterocycle. An example is 1,2,3,triazole. [ka] The divalent or bivalent form of is formed by removing a hydrogen atom from each of two different atoms (from a carbon atom or a nitrogen atom) of the triazole ring, [ka] It may have the structure:

[0084] The term "alkoxy" refers to an --O-alkyl radical.

[0085] The term "aminoalkyl" refers to an alkyl substituted with an amino. The term "alkylamino" refers to an amino substituted with an alkyl.

[0086] The term "aminocarbonyl" refers to a -C(O)-amino radical.

[0087] As used herein, the term "substituted" refers to any of the above groups (e.g., alkyl, hydroxyalkyl, alkylene, cycloalkyl, cycloalkylene, amino, aminocarbonyl, heterocyclyl, or heteroaryl) where one or more hydrogen atoms have been replaced with, but are not limited to, a halogen atom, such as F, CI, Br, or I; an oxo group (=O); a hydroxyl group (-OH); an alkoxy, alkoxyalkyl, aralkoxy, C-C 12Alkyl groups such as alkyl groups; cycloalkyl groups; alkenyl, alkynyl, aryl, aralkylheterocyclyl, heterocyclyl, heteroaryl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, aryloxy, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, aralkoxycarbonyl, sulfonyl, alkylaminolactam, alkylaminoheteroaryl, alkylaminoheterosil, and aminosulfonamido are also substituted with bonds to non-hydrogen atoms. Exemplary substituents also include -(C=O)OR; -O(C=O)R; -C(=O)R; -OR; -S(O) x R;-S-SR;-C(=O)SR;-SC(=O)R;-NRR';-R'C(=O)R;-C(=O)RR';-RC(=O)R'R";-OC(=O)RR';-RC(=O)OR';-R'S(O) X R”R;-R'S(O) X R; and -S(O) x R, R', and R" are independently in each occurrence H, C1-C 15 alkyl or cycloalkyl, heterocyclyl, or optionally substituted heteroaryl, where x is 0, 1, or 2. In some embodiments, the substituent is C-C 12In some embodiments, the substituent is an alkyl group. In some embodiments, the substituent is a cycloalkyl group. In some embodiments, the substituent is a halo group, such as fluoro. In some embodiments, the substituent is an oxo group. In some embodiments, the substituent is a hydroxyl group. In some embodiments, the substituent is a hydroxyalkylene group (-R-OH). In some embodiments, the substituent is an alkoxy group (-OR). In some embodiments, the substituent is a carboxyl group. In some embodiments, the substituent is an amino group (-NRR'). Suitable substituents also include divalent substituents on saturated carbon atoms, including, but not limited to: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2))2-3O-, or -S(C(R * 2))2-3S-, where R * is selected from hydrogen, substituted or unsubstituted C alkyl, or an unsubstituted 5-6 membered saturated or partially unsaturated ring, or an aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0088] "Halo" or "halogen" refers to any radical of fluorine, chlorine, bromine, or iodine.

[0089] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described circumstance event may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur. For example, "optionally substituted alkyl" means that the alkyl radical may be substituted or unsubstituted, and that the description includes both substituted and unsubstituted alkyl radicals.

[0090] The present disclosure is also intended to encompass pharmaceutically acceptable compounds of any formula specified herein that are isotopically labeled by replacing one or more atoms with atoms having a different atomic mass or mass number. Examples of isotopes that may be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as: 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 C1, 123 I, and 125 I. These isotopically labeled compounds may be useful in aiding in the determination or measurement of compound efficacy, for example, by characterizing the site or mode of action, or binding affinity to a pharmacologically important site of action. Certain isotopically labeled compounds of structure (I), (IA) or (IB), for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotope tritium, i.e., 3 H, and carbon-14, i.e., 14 C may be useful for this purpose in view of its ease of incorporation and rapid means of detection.

[0091] Deuterium, i.e., 2 Substitution with heavy isotopes such as H may offer certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be useful in some circumstances.

[0092] 11 C. 18 F, 15 O and 13Substitution with positron-emitting isotopes, such as N, can be useful in positron emission topography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art, or by methods similar to those described in the Preparations and Examples set forth below, substituting an appropriate isotopically labeled reagent for the previously used non-labeled reagent.

[0093] The present disclosure is also intended to encompass in vivo metabolic products of the disclosed compounds. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily due to enzymatic processes. Accordingly, embodiments of the present disclosure include compounds produced by a process comprising administering an ionizable lipid of the present disclosure to a mammal for a period of time sufficient to yield its metabolic products. Such products are typically identified by administering a detectable dose of a radiolabeled compound of the present disclosure to an animal, such as a rat, mouse, guinea pig, monkey, or human, allowing sufficient time for metabolism to occur, and isolating the conversion products from urine, blood, or other biological samples.

[0094] A "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, flow agent, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent that is approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine.

[0095] "Pharmaceutically acceptable salts" include both acid and base addition salts.

[0096] "Pharmaceutically acceptable acid addition salts" are salts that retain the biological effectiveness and properties of the free base, which are not biologically or otherwise undesirable, and include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid It refers to salts formed with organic acids such as glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0097] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acid and are not biologically or otherwise undesirable. These salts are prepared from the addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Non-limiting examples of inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resin salts. Non-limiting examples of organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0098] The crystallization of the ionized lipid disclosed herein can produce a solvate. As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of the ionized lipid of the present disclosure with one or more solvent molecules. The solvent can be water, in which case the solvate can be a hydrate. Alternatively, the solvent can be an organic solvent. Thus, the compounds of the present disclosure can exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., as well as corresponding solvate forms. The solvates of the compounds of the present disclosure can be true solvates, while in other cases, the compounds of the present disclosure can simply retain incidental water or be a mixture of water and some incidental solvent.

[0099] A "pharmaceutical composition" refers to a composition that may contain an ionizable lipid of the present disclosure and a vehicle generally accepted in the art for the delivery of a biologically active compound to a mammal, e.g., a human. Such a vehicle includes a pharmaceutically acceptable carrier, diluent, or excipient thereof.

[0100] "Effective amount" or "therapeutically effective amount" refers to the amount of ionized lipid of the present disclosure that is sufficient to treat a mammal, such as a human, when administered to the mammal. The amount of lipid nanoparticles of the present disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the condition and its severity, the method of administration, and the age of the mammal being treated, but can be routinely determined by those skilled in the art taking into account their own knowledge and this disclosure.

[0101] As used herein, "treating" or "treatment" includes treatment of a disease or condition of interest in a mammal, such as a human, having the disease or condition of interest; (i) preventing the onset of a disease or condition in a mammal, particularly where such mammal is predisposed to the condition but has not yet been diagnosed as having the condition; (ii) preventing the disease or condition, i.e., arresting its development; (iii) alleviating the disease or condition, i.e., causing regression of the disease or condition; or (iv) Relieving symptoms resulting from a disease or condition, including alleviating pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably or may differ in that a particular illness or condition may not have a known causative agent (so that its etiology remains unknown) and therefore is not yet recognized as a disease, but only as an undesirable condition or syndrome, with some specific set of symptoms identified by physicians.

[0102] The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may contain one or more stereocenters and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined in terms of absolute stereochemistry as (R)- or (S)-amino acids, or as (D)- or (L)-. The present disclosure is intended to encompass all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R) and (S), or (D) and (L) isomers can be prepared using chiral syntheses or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). When the compounds described herein contain an olefinic double bond or other center of geometric asymmetry, unless otherwise specified, it is intended that the compounds include both E and Z geometric isomers, as well as all tautomeric forms.

[0103] "Stereoisomer" refers to a compound made up of the same atoms joined by the same bonds, but having different, incompatible three-dimensional structures. The present disclosure contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0104] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the present disclosure. However, it will be understood by those skilled in the art that the present disclosure may be practiced without these details.

[0105] Ionized lipid compounds One aspect of the present invention is a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein: [ka] is a cyclic or heterocyclic moiety, Y is alkyl, hydroxy, hydroxyalkyl, or [ka] and A is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -SS-, or a divalent heterocycle; X and Z each independently represent absent, —O—, —CO—, —N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, or -S-, Each R 7 are independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 70 , R 80 , R 90 , R 100 , R 110 , and R 120 are each independently H, C-C optionally interrupted by a heteroatom or substituted by OH, SH, or halogen. 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl; l and m are each an integer of 1 to 10, t1 is an integer from 0 to 10, and W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterosilyl or heteroaryl.

[0106] In some embodiments, a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein [ka] is a cyclic or heterocyclic moiety, Y is alkyl, hydroxy, hydroxyalkyl, or [ka] and A is absent, -O-, -N(R 7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -SSS-, X and Z each independently represent absent, —O—, —N(R 7 )-, -O-alkylene-; -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, or -S-, Each R 7 are independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 70 , R 80 , R 90 , R 100 , R 110 , and R 120 are each independently H, C-C optionally interrupted by a heteroatom or substituted by OH, SH, or halogen. 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl; l and m are each an integer of 1 to 10, t is 0, 1, 2, or 3; and W is hydroxyl, hydroxyalkyl, or one of the following moieties: [ka] During the ceremony Q is -O- or -N(R 7 ) and R 6are independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl; Each R 8 are independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl, or two R 8 may form a ring, q is 0, 1, 2, 3, 4, or 5; and p is 0, 1, 2, 3, 4, or 5.

[0107] In some embodiments, in any of the formulas described herein, Y is hydroxyl, [ka] is.

[0108] In some embodiments, R 70 and R 80 are H and R 90 is C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl, cycloalkyl, or substituted cycloalkyl. 90 is C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 90 is C1-C 15 In some embodiments, R is a branched or unbranched alkyl. 90 is C1-C 12 It may be branched or unbranched alkyl.

[0109] In some embodiments, R 70 is H and R 80 and R 90 are each independently C1-C 15 Branched or unbranched alkyl, C1-C 15In some embodiments, R is a branched or unbranched alkenyl, or a cycloalkyl or substituted cycloalkyl. 80 and R 90 are each independently C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 80 and R 90 are each independently C1-C 15 In some embodiments, R is a branched or unbranched alkyl. 80 and R 90 are each independently C1-C 12 In some embodiments, R is a branched or unbranched alkyl. 80 and R 90 are each independently a C1-C8 branched or unbranched alkyl.

[0110] In some embodiments, R 100 is H and R 110 and R 120 are each independently C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl, or a cycloalkyl or substituted cycloalkyl. 110 and R 120 are each independently C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 110 and R 120 are each independently C1-C 15 In some embodiments, R is a branched or unbranched alkyl. 110 and R 120 are each independently C1-C 12 In some embodiments, R is a branched or unbranched alkyl. 110 and R 120 are each independently a C1-C8 branched or unbranched alkyl.

[0111] In some embodiments, compounds of formula (IA-1) or (IA-2): [ka] or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein: [ka] is a cyclic or heterocyclic moiety, A is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -SS-, or a divalent heterocycle; X is absent, -O-, -CO-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O, -N(R 7 )C(O)-, -C(O)N(R 7 )-, or -S-, Z is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, or -S-, Each R 7 are independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 100 , R 110 , and R 120 are each independently H, C-C optionally interrupted by a heteroatom or substituted by OH, SH, or halogen. 16 Branched or unbranched alkyl, or C1-C16 branched or unbranched alkenyl; R 90 is C1-C 15 Branched or unbranched alkyl, C1-C 15 branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl; t is 0, 1, 2, or 3; t1 is an integer from 0 to 10, l is an integer from 1 to 10, m is an integer from 1 to 10, and W is hydroxyl, hydroxyalkyl, or one of the following moieties: [ka] During the ceremony Each Q is independently selected from absent, -O-, -C(O)-, -C(S)-, -C(O)O-, -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and Each R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, or N + (R 7 ) 3-alkylene-Q-; Each R 8 are independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl, or two R 8 may form a ring together with the nitrogen atom, each q is independently 0, 1, 2, 3, 4, or 5; and Each p is independently 0, 1, 2, 3, 4, or 5.

[0112] Embodiments regarding the various variables in formulas (I) and (IA-1) and (IA-2) are discussed further below.

[0113] In some embodiments, in any of the formulas described herein: [ka] is the expression [ka] wherein: G1, G2, G3, G4, G5, and G6 are each independently C(R')(R''), O, or N, provided that no more than two of G1-G6 are O or N; R' and R'' are each independently absent, H, alkyl, or two R's from two adjacent G's together form a second 5-7 membered cyclic or heterocyclic ring; and n1 and n2 each independently represent 0 or 1.

[0114] In some embodiments, in any of the formulas described herein: [ka] teeth, It is selected from pyrrolidine, piperidine, piperazine, cyclohexane, cyclopentane, tetrahydrofuran, tetrahydropyran, morpholine, and dioxane.

[0115] In any of the formulas described herein, in some embodiments, [ka] is a 5-7 membered monocyclic ring. [ka] is a 5-7 membered monocyclic cycloalkane ring. [ka] is a 5- to 7-membered monocyclic heterocycle.

[0116] In any of the formulas described herein, in some embodiments, [ka] is a bicyclic or tricyclic ring, ie, contains two or more rings, including fused rings.

[0117] In any of the formulas described herein, in some embodiments, [ka] teeth, [ka] is selected from the group consisting of:

[0118] In some embodiments, [ka] teeth, [ka] is selected from the group consisting of:

[0119] In some embodiments, [ka] teeth, [ka] In one embodiment, [ka] teeth, [ka] It has the following structure.

[0120] In some embodiments, [ka] teeth, [ka] In one embodiment, [ka] teeth, [ka] In one embodiment, [ka] teeth, [ka] In one embodiment, [ka] teeth, [ka] It has the following structure.

[0121] In some embodiments, [ka] teeth, [ka] In one embodiment, [ka] teeth, [ka] It has the following structure.

[0122] In some embodiments, [ka] teeth, [ka] In one embodiment, [ka] teeth, [ka] It has the following structure.

[0123] In some embodiments, [ka] teeth, [ka] In one embodiment, [ka] teeth, [ka] It has the following structure.

[0124] In some embodiments, [ka] teeth, [ka] In one embodiment, [ka] teeth, [ka] It has the following structure.

[0125] In some embodiments, [ka] teeth, [ka] In one embodiment, [ka] teeth, [ka] It has the following structure.

[0126] In some embodiments, in any of the formulas described herein, Y, [ka] For A, there is no -O-, -N(R 7 )-, N(R 7 )C(O)-, [ka] -OC(O)-, or -C(O)O-, wherein R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, amino, aminoalkyl, thiol, thiolalkyl, or N + (R 7 ) 3-alkylene-Q-, and R 7 is H or C1-C3 alkyl. In one embodiment, A is absent. In one embodiment, A is -O-. In one embodiment, A is -N(R 7 )- and R 7 is H or C1-C3 alkyl. In one embodiment, A is -OC(O)- or -C(O)O-. In one embodiment, A is [ka] is.

[0127] In some embodiments, in any of the formulas described herein, t1 is 0, 1, 2, 3, or 4 and t is 0, 1, or 2.

[0128] In some embodiments, in any of the formulas described herein, W is hydroxyl, hydroxyalkyl, or one of the following moieties: [ka] During the ceremony, Each Q is independently selected from absent, -O-, -C(O)-, -C(S)-, -C(O)O-, -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 )- and Each R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, or N + (R 7 ) 3-alkylene-Q-; Each R 8 are independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl, or two R 8 may form a ring together with the nitrogen atom, each q is independently 0, 1, 2, 3, 4, or 5; and Each p is independently 0, 1, 2, 3, 4, or 5.

[0129] In some embodiments, in any of the formulas described herein, W is OH, [ka] and During the ceremony, q is 0, Each R8 are independently H, C1-C3 alkyl, or hydroxyalkyl, or two R 8 together with the nitrogen atom form a 5-membered ring optionally substituted with one or more alkyl groups, Each R 6 are independently H, hydroxyl, C1-C3 alkyl, or -Q-alkylene-N + (R 7 )3, Each Q is independently selected from absent, —O—, —C(O)—, —N(R 7 )-, -C(R 7 )2-, -C(O)O-, -C(O)N(R 7 )-, or -C(S)N(R 7 )- and Each R 7 is independently H, C1-C3 alkyl, or hydroxyalkyl.

[0130] In some embodiments, in any of the formulas described herein, W is OH, [ka] is.

[0131] In some embodiments, W is OH.

[0132] In some embodiments, W is [ka] where q is 0 and each R 8 is independently H, C1-C3 alkyl, or hydroxyalkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0133] In some embodiments, W is [ka] where each R 8 are independently H, C1-C3 alkyl, or hydroxyalkyl, and Q is -N(R 7 )-, -C(R 7 )2-, -C(O)O-, -C(O)N(R 7 )-, or -C(S)N(R 7 )- and each R 7 is independently H, C1-C3 alkyl, or hydroxyalkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0134] In some embodiments, W is [ka] where each R 6 are independently H, C1-C3 alkyl, hydroxyl, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, C1-C3 alkyl, or -Q-alkylene-N + (R 7 )3. In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0135] In some embodiments, W is [ka] wherein each Q is independently absent, -N(R 7 )-, -C(R 7 )2-, -C(O)O-, -C(O)N(R 7 )-, or -C(S)N(R 7 )- and each R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, or N + (R 7 ) 3-alkylene-Q-, and each R 7 is independently H, C1-C3 alkyl, hydroxy, or hydroxyalkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0136] In some embodiments, W is [ka] wherein each Q is independently absent, -N(R 7 )-, -C(R 7 )2-, -C(O)O-, -C(O)N(R 7 )-, or -C(S)N(R 7 )- and each R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, or N+ (R 7 ) 3-alkylene-Q-, and each R 7 are independently H, C1-C3 alkyl, hydroxy, or hydroxyalkyl.

[0137] In some embodiments, W is [ka] wherein each Q is independently absent, -N(R 7 )-, -C(R 7 )2-, -C(O)O-, -C(O)N(R 7 )-, or -C(S)N(R 7 )- and each R 6 are independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, or N + (R 7 ) 3-alkylene-Q-, and each R 7 are independently H, C1-C3 alkyl, hydroxy, or hydroxyalkyl.

[0138] In some embodiments, [ka] In the formula, q is 0 or 1, and each R 7 is independently H, C1-C3 alkyl. In one embodiment, W is [ka] is.

[0139] In some embodiments, W is [ka] wherein each Q is independently -N(R 7 )-, -C(R 7 )2-, -C(O)O-, -C(O)N(R 7 )-, or -C(S)N(R7 )- and each R 6 is independently H or C1-C3 alkyl, and each Q is independently -O-, -C(O)-, -N(R 7 ) and R 7 is H, C1-C3 alkyl, hydroxy, or hydroxyalkyl. In one embodiment, W is [ka] In one embodiment, W is [ka] and in one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0140] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0141] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] and in one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0142] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0143] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0144] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] In one embodiment, W is [ka] is.

[0145] In some embodiments, in any of the formulas described herein, Y or [ka] teeth, OH, [ka] where each R c is independently H or C1-C3 alkyl, and each t1 is independently 1, 2, 3, or 4.

[0146] In some embodiments, in any of the formulas described herein, Y or [ka] teeth, OH, [ka] is.

[0147] In some embodiments, in any of the formulas described herein, X is absent, —O—, or —C(O)—.

[0148] In some embodiments, in any of the formulas described herein, Z is —O—, —C(O)O—, or —OC(O)—.

[0149] In some embodiments, in any of the formulas described herein, R 30 , R 40 , R 50 , and R 60 are each H or C1-C4 branched or unbranched alkyl.

[0150] In some embodiments, in any of the formulas described herein, R 30 , R 40、R50 , and R 60 are each H.

[0151] In any of the formulas described herein, in some embodiments, R 70 and R 80 are H and R 90 is C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl, cycloalkyl, or substituted cycloalkyl. 90 is C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 90 is C1-C 15 In some embodiments, R is a branched or unbranched alkyl. 90 is C1-C 12 In some embodiments, R is a branched or unbranched alkyl. 90 is a C1-C8 branched or unbranched alkyl.

[0152] In any of the formulas described herein, in some embodiments, R 70 is H and R 80 and R 90 are each independently H, C1-C 15 Branched or unbranched alkyl, C1-C 15 branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl, but R 80 and R 90At least one of R is not H. 80 and R 90 are each independently H, C1-C 15 Branched or unbranched alkyl, or C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 80 and R 90 are each independently H or C1-C 15 In some embodiments, R 80 and R 90 are each independently H or C1-C 12 In some embodiments, R 80 and R 90 are each independently H or C1-C8 branched or unbranched alkyl.

[0153] In any of the formulas described herein, in some embodiments, R 100 is H and R 110 and R 120 are each independently H, C1-C 15 Branched or unbranched alkyl, C1-C 15 branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl, but R 110 and R 120 At least one of R is not H. 110 and R 120 are each independently H or C1-C 15 Branched or unbranched alkyl, or C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 110 and R 120 are each independently H or C1-C 15 In some embodiments, R 110 and R 120 are each independently H or C1-C 12 In some embodiments, R 110 and R 120are each independently H or C1-C8 branched or unbranched alkyl.

[0154] In any of the formulas described herein, in some embodiments, l is 3 to 10, 3 to 7, or 4 to 7. In some embodiments, l is 4, 5, 6, 7, 8, 9, or 10. In some embodiments, l is 3, 4, 5, 6, or 7. In some embodiments, l is 4, 5, 6, or 7.

[0155] In any of the formulas described herein, in some embodiments, m is 4 to 10, 5 to 8, 1 to 7, 3 to 7, or 1 to 5. In some embodiments, m is 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 1, 2, 3, 4, or 5. In some embodiments, m is 3, 4, or 5. In some embodiments, m is 5, 6, 7, or 8.

[0156] In any of the formulas described herein, in some embodiments, R 70 is H. In some embodiments, R 100 is H.

[0157] In any of the formulas described herein, in some embodiments, [ka] are independently selected from: [ka] wherein t is 0, 1, 2, 3, 4, or 5.

[0158] In any of the formulas described herein, in some embodiments, M is —OC(O)—, —C(O)O—, —N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(OR 13 )-O-, -C(O)O(CH2) r -, -C(O)N(R7 )(CH2) r -, or -C(OR 13 )-O-(CH2) r- where each R 7 are independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, and R 13 is a branched or unbranched C3-C 10 alkyl, and r is 1, 2, 3, 4, or 5. In some embodiments, M is —OC(O)— or —C(O)O—.

[0159] In any of the formulas described herein, in some embodiments, X is absent, —O—, or —C(O)—; Z is -O-, -C(O)O-, or -OC(O)-; M is -OC(O)- or -C(O)O-; Y or [ka] is OH, [ka] Each R c is independently H or C1-C3 alkyl, each t1 is independently 1, 2, 3, or 4, and R 30 , R 40 , R 50 , and R 60 are each H or C1-C4 branched or unbranched alkyl, and R 70 is H and R 80 and R 90 are each independently H or C1-C 12 is a branched or unbranched alkyl, and R 100 is H and R 110 and R 120 are each independently H or C1-C 12 Branched or unbranched alkyl, but R 80 and R 90 At least one of is not H, and R 110and R 120 At least one of is not H, l is 3 to 7, and m is 1 to 5.

[0160] In some embodiments, the disclosure herein provides a compound of the formula: [ka] All variables in this formula are defined and exemplified as described in the previous embodiments.

[0161] In some embodiments, the disclosure herein provides a compound of the formula: [ka] and a lipid compound having the formula: During the ceremony, each m1 independently represents an integer from 3 to 6; each l1 is independently an integer from 4 to 8; m2 and l2 each independently represent an integer of 0 to 3, R 80 and R 90 are each independently an unsubstituted C5-C8 alkyl, or R 80 is H or unsubstituted C1-C4 alkyl, and R 90 is unsubstituted C5-C 11 is alkyl, and R 110 and R 120 are each independently an unsubstituted C5-C8 alkyl, or R 110 is H or unsubstituted C1-C4 alkyl, and R 120 is unsubstituted C5-C 11 All other variables in these formulas are defined and exemplified as described in the embodiments above.

[0162] In some embodiments, in these formulas, R 80 is H or unsubstituted C1-C2 alkyl, and R 90 is unsubstituted C6-C 10 alkyl, and R110 and R 120 are each independently unsubstituted C5-C8 alkyl. In some embodiments, R 80 , R 90 , R 110 , and R 120 are each independently unsubstituted C5-C8 alkyl.

[0163] In some embodiments, the disclosure herein provides a compound of the formula: [ka] TIFF2025510229000148.tif65151. All variables in these formulas are defined and exemplified as described in the embodiments above. In some embodiments, in these formulas, R 80 is H or unsubstituted C1-C2 alkyl, and R 90 is unsubstituted C6-C 10 alkyl, and R 110 and R 120 are each independently unsubstituted C5-C8 alkyl. In some embodiments, R 80 , R 90 , R 110 , and R 120 are each independently unsubstituted C5-C8 alkyl. [ka] teeth, OH, [ka] is.

[0164] In some embodiments, the disclosure herein provides a compound of the formula: [ka] All variables in this formula are defined and exemplified as described in the previous embodiments.

[0165] In some embodiments, the disclosure herein provides a compound of the formula: [ka] and a lipid compound having the formula: each m1 independently represents an integer from 3 to 6; each l1 is independently an integer from 4 to 8; m2 and l2 each independently represent an integer of 0 to 3, R 80 and R 90 are each independently an unsubstituted C5-C8 alkyl, or R 80 is H or unsubstituted C1-C4 alkyl, and R 90 is unsubstituted C5-C 11 is alkyl, and R 110 and R 120 are each independently an unsubstituted C5-C8 alkyl, or R 110 is H or unsubstituted C1-C4 alkyl, and R 120 is unsubstituted C5-C 11 All other variables in these formulas are defined and exemplified as described in the embodiments above. In some embodiments, in these formulas, R 80 is H or unsubstituted C1-C2 alkyl, and R 90 is unsubstituted C6-C 10 alkyl, and R 110 and R 120 are each independently unsubstituted C5-C8 alkyl. In some embodiments, R 80 , R 90 , R 110 , and R 120 are each independently unsubstituted C5-C8 alkyl.

[0166] In some embodiments, the disclosure herein provides a compound of the formula: [ka] The lipid compound includes a lipid compound having the formula:

[0167] All variables in these formulas are defined and exemplified as described in the embodiments above. In some embodiments, in these formulas, R 80 is H or unsubstituted C1-C2 alkyl, and R 90 is unsubstituted C6-C 10 alkyl, and R 110 and R 120 are each independently unsubstituted C5-C8 alkyl. In some embodiments, R 80 , R 90 , R 110 , and R 120 are each independently unsubstituted C5-C8 alkyl. [ka] teeth, OH, [ka] is.

[0168] In some embodiments, the present disclosure provides a compound of formula (IIA): [ka] and pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: A is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -SSS-, X is absent, -O-, -CO-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O, -NHC(O)-, -C(O)N(R 7 )-, or -S-, Z is absent, -O-, -N(R7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or -S-; Each R 7 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 100 , R 110 , and R 120 are each independently H, C-C optionally interrupted by a heteroatom or substituted by OH, SH, or halogen. 16 Branched or unbranched alkyl, or C1-C 16 branched or unbranched alkenyl; R 90 is C1-C 15 Branched or unbranched alkyl, C1-C 15 branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl; t is 0, 1, 2, or 3; l is an integer from 1 to 10, m is an integer from 1 to 10, and W is hydroxyl, hydroxyalkyl, or one of the following moieties: [ka] During the ceremony Q is -O- or -N(R 7 ) and R 6 are independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl; Each R 8 are independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl, or two R8 may form a ring, q is 0, 1, 2, 3, 4, or 5; and p is 0, 1, 2, 3, 4, or 5.

[0169] In some embodiments, the present disclosure provides a compound of formula (IIIA): [ka] With respect to the ionizable lipid of formula (IIIA), its pharmaceutically acceptable salts, and any stereoisomers of the foregoing, the definitions of the variables in (IIIA) are the same as those in (IIA).

[0170] In some embodiments, the present disclosure provides a compound of formula (IIB): [ka] and pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: A is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -SSS-, X is absent, -O-, -CO-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O, -NHC(O)-, -C(O)N(R 7 )-, or -S-, Z is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or -S-; Each R 7 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 70 , R 80 , R 90 , R 100 , R 110 , and R 120 are each independently H, C-C optionally interrupted by a heteroatom or substituted by OH, SH, or halogen. 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl; t is 0, 1, 2, or 3; l is an integer from 1 to 10, m is an integer from 1 to 10, and W is hydroxyl, hydroxyalkyl, or one of the following moieties: [ka] During the ceremony Q is -O- or -N(R 7 ) and R 6 are independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl; Each R 8 are independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl, or two R 8 may form a ring, q is 0, 1, 2, 3, 4, or 5; and p is 0, 1, 2, 3, 4, or 5.

[0171] In some embodiments, the present disclosure provides a compound of formula (IIIB): [ka] With respect to the ionizable lipid of formula (IIIB), its pharmaceutically acceptable salts, and any stereoisomers of the foregoing, the definitions of the variables in (IIIB) are the same as those in (IIB).

[0172] In some embodiments, the present disclosure provides a compound of formula (IIC): [ka] and pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: A is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R')-, N(R 7 )C(O)N(R 7 )-, -S-, -SS-, R 30 , R 40 , R 50 , R 60 , R 100 , R 110 , and R 120 are each independently H, C-C optionally interrupted by a heteroatom or substituted by OH, SH, or halogen. 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl; R 90 is C1-C 15 Branched or unbranched alkyl, C1-C 15 branched or unbranched alkenyl, cycloalkyl or substituted cycloalkyl; Each R 7 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; t is 0, 1, 2, or 3; l is an integer from 1 to 10, m is an integer from 1 to 10, and W is hydroxyl, hydroxyalkyl, or one of the following moieties: [ka] During the ceremony Q is -O- or -N(R 7 )- and R 6 are independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl; Each R 8 are independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl, or two R 8 may form a ring, q is 0, 1, 2, 3, 4, or 5; and p is 0, 1, 2, 3, 4, or 5.

[0173] In some embodiments, the present disclosure provides a compound of formula (IIIC): [ka] With respect to the ionizable lipid of formula (IIIC), its pharmaceutically acceptable salts, and any stereoisomers of the foregoing, the definitions of the variables in (IIIC) are the same as those in (IIA).

[0174] In some embodiments, the present disclosure provides a compound of formula (IIID): [ka] With respect to the ionizable lipid of formula (I), its pharmaceutically acceptable salts, and any stereoisomers of the foregoing, the definitions of the variables in (IID) are the same as defined above.

[0175] In some embodiments, the present disclosure provides a compound of formula (IIIE): [ka] With respect to the ionizable lipid of formula (I), its pharmaceutically acceptable salts, and any stereoisomers of the foregoing, the definitions of the variables in (IID) are the same as defined above.

[0176] Embodiments regarding the various variables in formula (IIA), (IIB), (IIC), (IIIA), (IIIB), (IIIC), (IIID), or (IIIE) are further discussed below.

[0177] In some embodiments, X is absent, —O—, or —C(O)—. In one embodiment, X is absent. In one embodiment, X is —O—. In one embodiment, X is —C(O)—.

[0178] In some embodiments, Z is -O-, -C(O)O-, or -OC(O)-. In one embodiment, Z is -O-. In one embodiment, Z is -C(O)O- or -C(O)O-.

[0179] In some embodiments, R 30 , R 40 , R 50 , and R 60 are each H or C1-C4 branched or unbranched alkyl.

[0180] In some embodiments, R 30 , R 40、 R 50 , and R 60 are each H.

[0181] In some embodiments, R 70 and R 80 are H and R 90 is C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 90 is C1-C 15 In some embodiments, R is a branched or unbranched alkyl. 90 is C1-C 12It may be branched or unbranched alkyl.

[0182] In some embodiments, R 70 is H and R 80 and R 90 are each independently C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 80 and R 90 are each independently C1-C 15 In some embodiments, R is a branched or unbranched alkyl. 80 and R 90 are each independently C1-C 12 In some embodiments, R is a branched or unbranched alkyl. 80 and R 90 are each independently a C1-C8 branched or unbranched alkyl.

[0183] In some embodiments, R 100 is H and R 110 and R 120 are each independently C1-C 15 Branched or unbranched alkyl, C1-C 15 In some embodiments, R is a branched or unbranched alkenyl. 110 and R 120 are each independently C1-C 15 In some embodiments, R is a branched or unbranched alkyl. 110 and R 120 are each independently C1-C 12 In some embodiments, R is a branched or unbranched alkyl. 110 and R 120 are each independently a C1-C8 branched or unbranched alkyl.

[0184] In some embodiments, l is 3 to 10, 3 to 7, or 4 to 7. In some embodiments, l is 4, 5, 6, 7, 8, 9, or 10. In some embodiments, l is 4, 5, 6, or 7.

[0185] In some embodiments, m is 4-10, 5-8, 1-7, 3-7, or 1-5.

[0186] In some embodiments, m is 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 3, 4, or 5. In some embodiments, m is 5, 6, 7, or 8.

[0187] In some embodiments, M is —OC(O)—, —C(O)O—, —N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(OR 13 )-O-, -C(O)O(CH2) r- , -C(O)N(R 7 )(CH2) r -, or -C(OR 13 )-O-(CH2) r - and each R 7 are independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, and R 13 is a branched or unbranched C3-C 10 alkyl, and r is 1, 2, 3, 4, or 5. In some embodiments, M is —OC(O)— or -C(O)O-.

[0188] In some embodiments, the pKa of the protonated form of the ionized lipid compounds described herein is about 4.5 to about 8.0, e.g., about 4.6 to about 7.8, about 4.6 to about 7.3, about 4.6 to about 6.8, about 4.6 to about 6.2, about 4.6 to about 6.0, about 4.6 to about 5.9, about 4.6 to about 5.8, about 4.6 to about 5.6, about 4.6 to about 5.5, about 5.7 to about 6.5, about 5.7 to about 6.4, or about 5.8 to about 6.2. In some embodiments, the pKa of the protonated form of the ionized lipid compound is about 4.6 to about 7.8. In some embodiments, the pKa of the protonated form of the ionized lipid compound is about 4.6 to about 5.6. In some embodiments, the pKa of the protonated form of the ionized lipid compound is about 5.5 to about 6.0. In some embodiments, the pKa of the protonated form of the ionizable lipid compound is about 6.1 to about 6.3. In some embodiments, the pKa of the protonated form of the ionizable lipid compound is about 4.7 to about 5.1.

[0189] Non-limiting examples of ionizable lipid compounds disclosed herein are listed in Table 1 below. [Table 1] TIFF2025510229000168.tif218164 TIFF2025510229000169.tif224164 TIFF2025510229000170.tif217164 TIFF2025510229000171.tif212164 TIFF2025510229000172.tif201164 TIFF2025510229000173.tif195163 TIFF2025510229000174.tif219164 TIFF2025510229000175.tif221163 TIFF2025510229000176.tif205164 TIFF2025510229000177.tif197164 TIFF2025510229000178.tif228164 TIFF2025510229000179.tif185163 TIFF2025510229000180.tif206156 TIFF2025510229000181.tif69156 Additional non-limiting examples of ionizable lipid compounds disclosed herein are listed in Table 2 below.

[0190] [Table 2] TIFF2025510229000183.tif203157 TIFF2025510229000184.tif233155 TIFF2025510229000185.tif236156 TIFF2025510229000186.tif202157 TIFF2025510229000187.tif154157 TIFF2025510229000188.tif208156 TIFF2025510229000189.tif217156 TIFF2025510229000190.tif195156 TIFF2025510229000191.tif215155 TIFF2025510229000192.tif206156 TIFF2025510229000193.tif198156 TIFF2025510229000194.tif180156 TIFF2025510229000195.tif191156 TIFF2025510229000196.tif208155 TIFF2025510229000197.tif122156

[0191] lipid composition The ionizable lipids disclosed herein can be used to form lipid compositions. Accordingly, another aspect of the present invention relates to lipid compositions comprising the lipid compounds described in the above aspect of the invention herein relating to novel ionizable lipid compounds.

[0192] All of the above descriptions and all of the embodiments discussed in the above aspects relating to lipid compounds, including compounds covered by formulae (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), (IIIA)-(IIIIE), (IIIC-1), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), and (VC-1)-(VC-6), are all applicable to these aspects of the invention relating to lipid compositions.

[0193] As described herein, suitable lipid compounds for use in the lipid compositions include all isomers and isotopes of the aforementioned compounds, as well as all pharmaceutically acceptable salts, solvates, or hydrates thereof, and all crystalline forms, mixtures of crystalline forms, and anhydrates or hydrates thereof.

[0194] In some embodiments, the lipid composition comprises one or more compounds described herein, or pharmaceutically acceptable salts thereof. In some embodiments, the lipid composition is a liposome or lipid nanoparticle (LNP). In one embodiment, the lipid composition is an LNP.

[0195] In addition to one or more compounds described herein, the lipid composition may further comprise a second lipid. In some embodiments, the present disclosure relates to a lipid composition comprising: (i) one or more lipid compounds selected from the ionizable lipids of formula (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), (IIIA)-(IIIIE), (IIIC-1), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), and (VC-1)-(VC-6), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing; and (ii) a second lipid. In some embodiments, the lipid composition comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of one or more lipid compounds.

[0196] In some embodiments, the second lipid is cationic, non-cationic (e.g., neutral, anionic, or zwitterionic), or ionizable. In some embodiments, the lipid composition comprises a lipid compound and a second lipid (e.g., a helper lipid) in a ratio of about 1:1.

[0197] In some embodiments, the second lipid is a cationic lipid, an anionic lipid, another ionizable lipid, or a zwitterionic lipid.

[0198] In some embodiments, the present disclosure relates to lipid nanoparticle compositions comprising (i) one or more ionizable lipid compounds described herein and (ii) one or more lipid components.

[0199] In some embodiments, one or more lipid components in the lipid composition comprise one or more helper lipids and one or more PEG-lipids. In some embodiments, the lipid components comprise one or more helper lipids, one or more PEG-lipids, and one or more neutral lipids. In some embodiments, the lipid composition may further comprise a sterol and a PEG-lipid. In some embodiments, the lipid composition may further comprise a sterol, a PEGylated lipid, a phospholipid, and / or a neutral lipid.

[0200] In some embodiments, one or more natural and / or synthetic lipid compounds may be used in preparing the lipid composition. The lipid composition may contain negatively charged lipids, positively charged lipids, or a combination thereof.

[0201] Non-ionized lipid components Charged and neutral lipids Examples of suitable negatively charged (anionic) lipids include, but are not limited to, dimyristoyl-, dipalmitoyl-, and distearoyl-phosphatidylglycerol; dimyristoyl-, dipalmitoyl-, and dipalmitoyl-phosphatidic acid; dimyristoyl-, dipalmitoyl-, and dipalmitoyl-phosphatidylethanolamine, and their unsaturated diacyl and mixed acyl chain counterparts, and cardiolipin.

[0202] Examples of positively charged (cationic) lipids include, but are not limited to, N,N'-dimethyl-N,N'-dioctacylammonium bromide (DDAB) and chloride (DDAC), N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl)cholesterol (DC-chol), 1,2-dioleoyloxy-3-[trimethylammonio]-propane (DOTAP), and 1,2-dioctadecyloxy-3-[trimethylammonio]-propane (DSTAP), and 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium chloride (DORI), and cationic lipids described, for example, in Martin et al., Current Pharmaceutical Design, pages 1-394, which is incorporated herein by reference in its entirety.

[0203] Additional exemplary cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), 1,2-dioleoyl-3-dimethylammonium chloride ... and mixtures thereof. The neutral lipids may include dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), and / or mixtures thereof.

[0204] In some embodiments, the lipid component comprises one or more neutral lipids. The neutral lipids may be one or more phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids may be assembled into one or more lipid bilayers. Generally, the phospholipid may comprise a phospholipid moiety and one or more fatty acid moieties. For example, the phospholipid may have the following formula: [ka] wherein Rp represents the phospholipid moiety, and R A and R B represents a fatty acid moiety, with or without unsaturation, which may be the same or different. The phospholipid moiety may be phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, or sphingomyelin. The fatty acid moiety may be lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, or docosahexaenoic acid. Non-natural species, including naturally occurring species with modifications and substitutions such as branching, oxidation, cyclization, and alkynes, are also contemplated. For example, the phospholipid may be functionalized with one or more alkynes (e.g., an alkenyl group in which one or more double bonds are replaced with a triple bond) or crosslinked to one or more alkynes. Under appropriate reaction conditions, when exposed to an azide, the alkyne group can undergo copper-catalyzed cycloaddition. Such reactions can be useful for functionalizing the lipid bilayer of lipid nanoparticles to facilitate membrane penetration or cell recognition, or for conjugating lipid nanoparticles to useful components such as targeting or imaging moieties (e.g., dyes).

[0205] In some embodiments, the neutral lipid is a phospholipid, such as distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero- 3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl The phosphatidylethanolamine may be phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a mixture thereof.

[0206] Additional non-limiting examples of neutral lipids include phospholipids, such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylethanolamine (DPPG ... Other examples of phospholipids include diacylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are C 10 -C 24 The acyl group may be derived from a fatty acid having a carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0207] Steroids and other non-ionized lipid components In some embodiments, the lipid component in the lipid composition comprises one or more steroids or analogs thereof.

[0208] In some embodiments, the lipid components in lipid composition comprise sterols such as cholesterol, sterol, and their derivatives.Non-limiting examples of cholesterol derivatives include polar analogs such as 5a-cholestanol, 5a-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5a-cholestan, cholestenone, 5a-cholestanone, 5a-cholestanone, and cholesteryl decanoate; and mixtures thereof.In some embodiments, cholesterol derivative is polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[0209] In some embodiments, the non-ionized lipid component comprises or consists of a mixture of one or more phospholipids and cholesterol or its derivatives. In some embodiments, the non-ionized lipid component present in the lipid composition comprises or consists of one or more phospholipids, for example, cholesterol-free lipid particle formulations. In some embodiments, the non-ionized lipid component present in the lipid composition comprises or consists of cholesterol or its derivatives, for example, phospholipid-free lipid particle formulations.

[0210] In some embodiments, the lipid component in the lipid composition (e.g., LNP composition) comprises a phytosterol or a combination of a phytosterol and cholesterol. In some embodiments, the phytosterol is selected from the group consisting of β-sitosterol, stigmasterol, β-sitostanol, campesterol, brassicasterol, and combinations thereof. In some embodiments, the phytosterol is selected from the group consisting of β-sitosterol, β-sitostanol, campesterol, brassicasterol, Compound S-140, Compound S-151, Compound S-156, Compound S-157, Compound S-159, Compound S-160, Compound S-164, Compound S-165, Compound S-170, Compound S-173, Compound S-175, and combinations thereof. In some embodiments, the phytosterol is selected from the group consisting of Compound S-140, Compound S-151, Compound S-156, Compound S-157, Compound S-159, Compound S-160, Compound S-164, Compound S-165, Compound S-170, Compound S-173, Compound S-175, and combinations thereof. In some embodiments, the phytosterol is a combination of Compound S-141, Compound S-140, Compound S-143, and Compound S-148. In some embodiments, the phytosterol comprises sitosterol or a salt or ester thereof. In some embodiments, the phytosterol comprises stigmasterol or a salt or ester thereof.

[0211] In some embodiments, the phytosterol is beta-sitosterol, [ka] a salt thereof, or an ester thereof.

[0212] In some embodiments, the lipid composition (eg, an LNP composition) comprises a phytosterol, or a salt or ester thereof, and cholesterol or a salt thereof.

[0213] In some embodiments, the target delivery cells for the lipid composition are cells described herein (e.g., hepatocytes or splenocytes), and the phytosterol or salt or ester thereof is selected from the group consisting of β-sitosterol, β-sitostanol, campesterol, and brassicasterol, and combinations thereof. In some embodiments, the phytosterol is β-sitosterol. In some embodiments, the phytosterol is β-sitostanol. In some embodiments, the phytosterol is campesterol. In some embodiments, the phytosterol is brassicasterol.

[0214] In some embodiments, the target delivery cells for the lipid composition are cells described herein (e.g., hepatocytes or splenocytes), and the phytosterol or its salt or ester is selected from the group consisting of β-sitosterol, stigmasterol, and combinations thereof. In some embodiments, the phytosterol is β-sitosterol. In some embodiments, the phytosterol is stigmasterol.

[0215] Other examples of non-ionizable lipid components include non-phosphorus-containing lipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin.

[0216] In some embodiments, the non-ionized lipid component is present at 10 mol% to 60 mol%, 20 mol% to 55 mol%, 20 mol% to 45 mol%, 20 mol% to 40 mol%, 25 mol% to 50 mol%, 25 mol% to 45 mol%, 30 mol% to 50 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 35 mol% to 45 mol%, 37 mol% to 42 mol%, or 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, or 45 mol% (or any fraction or range therein) of the total lipid present in the lipid composition.

[0217] In embodiments where the lipid composition contains a mixture of phospholipids and cholesterol or a cholesterol derivative, the mixture may be present at up to 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipid present in the lipid composition.

[0218] In some embodiments, the phospholipid component in the mixture may be present at 2 mol% to 20 mol%, 2 mol% to 15 mol%, 2 mol% to 12 mol%, 4 mol% to 15 mol%, or 4 mol% to 10 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid composition. In some embodiments, the phospholipid component in the mixture may be present at 5 mol% to 10 mol%, 5 mol% to 9 mol%, 5 mol% to 8 mol%, 6 mol% to 9 mol%, 6 mol% to 8 mol%, or 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, or 10 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid composition.

[0219] In some embodiments, the sterol component (e.g., cholesterol component) in the mixture may be present at 25 mol% to 45 mol%, 25 mol% to 40 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 27 mol% to 37 mol%, 25 mol% to 30 mol%, or 35 mol% to 40 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid composition. In some embodiments, the cholesterol component in the mixture may be present at 25 mol% to 35 mol%, 27 mol% to 35 mol%, 29 mol% to 35 mol%, 30 mol% to 35 mol%, 30 mol% to 34 mol%, 31 mol% to 33 mol%, or 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, or 35 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid composition.

[0220] In embodiments in which the lipid composition does not contain phospholipids, cholesterol or a derivative thereof may be present at up to 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipids present in the lipid composition.

[0221] In some embodiments, the sterol component (e.g., cholesterol or a derivative thereof) in a phospholipid-free lipid particle formulation may be present at 25 mol% to 45 mol%, 25 mol% to 40 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 31 mol% to 39 mol%, 32 mol% to 38 mol%, 33 mol% to 37 mol%, 35 mol% to 45 mol%, 30 mol% to 35 mol%, 35 mol% to 40 mol%, or 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, or 40 mol% (or any fraction or range therein) of the total lipid present in the lipid composition.

[0222] In some embodiments, the non-ionized lipid component may be present at 5 mol% to 90 mol%, 10 mol% to 85 mol%, 20 mol% to 80 mol%, 10 mol% (e.g., phospholipids only), or 60 mol% (e.g., phospholipids and cholesterol or derivatives thereof) (or any fraction or range therein) of the total lipid present in the lipid composition.

[0223] The percentage of non-ionizable lipid present in the lipid composition is a target amount, and the actual amount of non-ionizable lipid present in the particle can vary, for example, by ±5 mol %.

[0224] lipid conjugates The lipid compositions described herein may further comprise one or more lipid conjugates. The conjugated lipids may prevent particle aggregation. Non-limiting examples of conjugated lipids include PEG-lipid conjugates, cationic polymer-lipid conjugates, and mixtures thereof.

[0225] In some embodiments, lipid conjugate is PEG lipid or PEG-modified lipid (also called PEGylated lipid).PEG lipid is the lipid modified with polyethylene glycol.Examples of PEG lipid include but are not limited to PEG (PEG-DAA) that is bonded to dialkyloxypropyl, PEG (PEG-DAG) that is bonded to diacylglycerol, PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DEG), PEG (PEG-PE) that is bonded to phospholipids such as phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG (PEG-CER) that is bonded to ceramide, PEG that is bonded to cholesterol or its derivative, and their mixtures.

[0226] For example, the PEG lipid may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0227] In some 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.

[0228] In some 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-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-l,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA).

[0229] PEG is a linear water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified according to their molecular weight and include the following: monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM), as well as compounds containing terminal hydroxyl groups instead of terminal methoxy groups (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH2).

[0230] The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from 550 daltons to 10,000 daltons. In certain examples, the PEG moiety has an average molecular weight of 750 daltons to 5,000 daltons (e.g., 1,000 daltons to 5,000 daltons, 1,500 daltons to 3,000 daltons, 750 daltons to 3,000 daltons, 750 daltons to 2,000 daltons). In some embodiments, the PEG moiety has an average molecular weight of 2,000 daltons or 750 daltons.

[0231] In certain examples, PEG can be optionally substituted with alkyl, alkoxy, acyl or aryl group.PEG can be directly conjugated to lipid, or can be linked to lipid via linker moiety.For example, any linker moiety suitable for connecting PEG to lipid can be used, including non-ester-containing linker moiety and ester-containing linker moiety.In some embodiments, linker moiety is non-ester-containing linker moiety. Suitable non-ester containing linker moieties include, but are not limited to, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, disulfide, and combinations thereof (such as linkers containing both carbamate and amide linker moieties). In some embodiments, a carbamate linker is used to attach PEG to a lipid.

[0232] In some embodiments, PEG is attached to a lipid using an ester-containing linker moiety. Suitable ester-containing linker moieties include, for example, carbonate (-OC(O)O-), succinoyl, phosphate (-O-(O)POH-O-), sulfonate, and combinations thereof.

[0233] Phosphatidylethanolamines with various acyl chain groups of various chain lengths and degrees of saturation can be conjugated to PEG to form lipid conjugates. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art.

[0234] In some embodiments, the phosphatidylethanolamine contains saturated or unsaturated fatty acids with carbon chain lengths ranging from C10 to C20. Phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0235] The term "diacylglycerol" or "DAG" includes compounds having two fatty acyl chains, R1 and R2, each having 2 to 30 carbon atoms, independently attached to the 1- and 2-positions of glycerol by an ester bond. The acyl groups may be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C12), myristoyl (CM), palmitoyl (C16), stearoyl (C18), and icosyl (C20). In some embodiments, R1 and R2 are the same, i.e., both R1 and R2 are myristoyl (i.e., dimyristoyl) and both R1 and R2 are stearoyl (i.e., distearoyl).

[0236] The term "dialkyloxypropyl" or "DAA" includes compounds having two alkyl chains, R and R', where R and R' both independently have 2 to 30 carbons. The alkyl groups may be saturated or have varying degrees of unsaturation.

[0237] In some embodiments, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C10) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16) conjugate, or a PEG-distearyloxypropyl (C18) conjugate. In some embodiments, the PEG has an average molecular weight of 750 or 2,000 daltons. In some embodiments, the terminal hydroxyl group of the PEG is replaced with a methyl group.

[0238] In addition to the above, other hydrophilic polymers can be used instead of PEG. Examples of suitable polymers that can be used instead of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0239] In some embodiments, the PEG lipid has the formula [ka] or a salt thereof, wherein R 3PL1 -OR OPL1 and R OPL1 is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r PL1 is an integer between 1 and 100 inclusive, L 1is an optionally substituted C 1-10 alkylene and optionally substituted C 1-10 At least one methylene of the alkylene is independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R NPL1 ), S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), -C(O)O, OC(O), OC(O)O, OC(O)N(R NPL1 ), NR NPL1 C(O)O, or NR NPL1 C(O)N(R NPL1 ), D is a moiety obtained by click chemistry or cleavable under physiological conditions, m PL1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, A is a group having the formula: [ka] and L 2 Each instance of is independently a bond or an optionally substituted C 1-6 alkylene and optionally substituted C 1-6 One methylene unit of alkylene is O, N(R NPL1 ), S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), C(O)O, OC(O)O, -OC(O)N(R NPL1 ), NR NPL1 C(O)O, or NR NPL1 C(O)N(R NPL1 ), optionally replaced by R 2SL Each instance of is independently an optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl, and optionally R 2SLone or more of the methylene units in NPL1 ), O, S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), -NR NPL1 C(O)N(R NPL1 ), C(O)O, OC(O), OC(O)O, OC(O)N(R NPL1 ), NR NPL1 C(O)O, C(O)S, -SC(O), C(=NR NPL1 ), C(=NR NPL1 )N(R NPL1 ), NR NPL1 C(=NR NPL1 ), -NR NPL1 C(=NR NPL1 )N(R NPL1 ), C(S), C(S)N(R NPL1 ), NR NPL1 C(S), NR NPL1 C(S)N(R NPL1 ), S(O), OS(O), OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R NPL1 )S(O), S(O)N(R NPL1 ), -N(R NPL1 )S(O)N(R NPL1 ), OS(O)N(R NPL1 ), N(R NPL1 )S(O)O, S(O)2, N(R NPL1 )S(O)2, -S(O)2N(R NPL1 ), N(R NPL1 )S(O)2N(R NPL1 ), OS(O)2N(R NPL1 ), or N(R NPL1 )S(O)O, R NPL1 each instance of is independently substituted with hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; and p SL is either 1 or 2.

[0240] In some embodiments, the PEG lipid has the formula [ka] or a salt thereof, wherein r PL1 , L 1 ,D,m PL1 , and A is as defined above.

[0241] In some embodiments, the PEG lipid has the formula [ka] or a salt or isomer thereof, wherein R 3PEG -OR O and R O is hydrogen, C 1-6 alkyl, or oxygen protecting group; r PEG is an integer from 1 to 100 (for example, 40 to 50, for example, 45), R 5PEG is C 10-40 Alkyl (e.g., C 17 alkyl), C 10-40 Alkenyl, or C 10-40 alkynyl, and optionally R 5PEG One or more methylene groups in 3-10 Carbocyclylene, 4-10 membered heterocyclylene, C 6-10 Arylene, 4-10 membered heteroarylene, -N(R NPEG )-, -O-, -S-, -C(O)-, -C(O)N(R NPEG )-, -NR NPEG C(O)-, -NR NPEG C(O)N(R NPEG)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R NPEG )-, -NR NPEG C(O)O-, -C(O)S-, -SC(O)-, -C(=NR NPEG )-, -C(=NR NPEG )N(R NPEG )-, -NR NPEG C(=NR NPEG )-, -NR NPEG C(=NR NPEG )N(R NPEG )-, -C(S)-, -C(S)N(R NPEG )-, -NR NPEG C(S)-, -NR NPEG C(S)N(R NPEG )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R NPEG )S(O)-, -S(O)N(R NPEG )-, -N(R NPEG )S(O)N(R NPEG) -,-OS(O)N(R NPEG )-, -N(R NPEG )S(O)O-, -S(O)2-, -N(R NPEG )S(O)2-, -S(O)2N(R NPEG )-, -N(R NPEG )S(O)2N(R NPEG )-, -OS(O)2N(R NPEG )-, or -N(R NPEG )S(O)2O-, and R NPEG Each instance of is independently hydrogen, C 1-6 alkyl, or nitrogen protecting groups.

[0242] In some embodiments, the PEG lipid has the formula [ka] wherein r PEG is an integer from 1 to 100 (for example, 40 to 50, such as 45).

[0243] In some embodiments, the PEG lipid has the formula [ka] or a salt or isomer thereof, wherein s PL1 is an integer from 1 to 100 (for example, 40 to 50, such as 45).

[0244] In some embodiments, the PEG lipid has the formula [ka] or a pharmaceutically acceptable salt thereof, a tautomer or a stereoisomer thereof, wherein R 8 and R 9 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally being interrupted by one or more ester linkages (e.g., R 8 and R 9 are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms, and w has an average value in the range of 30 to 60 (eg, the average w is about 49).

[0245] In some embodiments, incorporating any of the above-mentioned PEG-lipids into lipid compositions can improve the pharmacokinetics and / or biodistribution of the lipid compositions.For example, incorporating any of the above-mentioned PEG-lipids into lipid compositions can reduce accelerated blood clearance (ABC) effect.

[0246] Other ionizable lipids In some embodiments, the lipid composition may include one or more additional ionizable lipids different from the ionizable lipids described herein. Examples of ionizable lipids include, but are not limited to: [ka] Acuitas Lipid 9, and Acuitas Lipid 10 (see WO 2017 / 004143 A1, which is incorporated by reference in its entirety).

[0247] In one embodiment, the additional ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), e.g., as described in Example 1 of U.S. Pat. No. 9,867,888, which is incorporated herein by reference in its entirety.

[0248] In one embodiment, the additional ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01), synthesized, for example, in Example 13 of WO 2015 / 095340, which is incorporated herein by reference in its entirety.

[0249] In one embodiment, the additional ionizable lipid is di((Z)-non-2-en-1-yl)9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., as synthesized in Example 7, 8, or 9 of U.S. Patent No. 2012 / 0027803, which is incorporated herein by reference in its entirety.

[0250] In one embodiment, the additional ionizable lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol)(C12-200), synthesized, for example, in Examples 14 and 16 of WO 2010 / 053572, which is incorporated herein by reference in its entirety.

[0251] In one embodiment, the additional ionizable lipid is the imidazole cholesterol ester (ICE) lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, e.g., structure (I) of WO 2020 / 106946, which is incorporated herein by reference in its entirety.

[0252] In one embodiment, the additional ionizable lipid is MC3(6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-l9-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), as described, for example, in Example 9 of WO 2019 / 051289 A9, which is incorporated herein by reference in its entirety.

[0253] In one embodiment, the additional ionizable lipid is the lipid ATX-002, described, for example, in Example 10 of WO 2019 / 051289 A9, which is incorporated by reference in its entirety.

[0254] In one embodiment, the additional ionizable lipid is (l3Z,l6Z)-A,A-dimethyl-3-nonyldocosa-l3,l6-dien-l-amine (compound 32), e.g., as described in Example 11 of WO 2019 / 051289 A9, which is incorporated herein by reference in its entirety.

[0255] In one embodiment, the additional ionizable lipid is, for example, compound 6 or compound 22, described in Example 12 of WO 2019 / 051289 A9, the entire contents of which are incorporated herein by reference.

[0256] Examples of additional ionizable lipids useful in the lipid compositions include those listed in Table 1 of WO 2019 / 051289, which is incorporated herein by reference.

[0257] Additional lipid ingredients Some non-limiting examples of additional lipid compounds that can be used (e.g., in combination with the ionizable lipid compounds and other lipid components described herein) to form lipid compositions include: [ka] JPEG2025510229000209.jpg68157

[0258] In some embodiments, the lipid composition further comprises a lipid of formula (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), or (ix).

[0259] In some embodiments, the lipid composition further comprises the following compound having the following structure: [ka] During the ceremony, X 1 , O, NR 1 , or a direct bond, and X 2 is C2-5 alkylene, and X 3 is C(=O) or a direct bond, R 1 is H or Me, and R 3 is C alkyl, and R 2 is C alkyl, or R 2 is the nitrogen atom to which it is attached and X 2 together with 1 to 3 carbon atoms of X 1 is NR 1 and R 1 and R 2 together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, or R 2 is R 3and together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y 1 is C2- 12 is alkylene, and Y 2 is selected from: [ka] n is 0 to 3, R 4 is C1- 15 is alkyl, Z 1 is C 1-6 is alkylene or a direct bond, and Z 2 teeth, [ka] (any orientation) or absent, except Z 1 If is a direct bond, Z 2 does not exist, R 5 is C 5-9 Alkyl or C 6-10 is alkoxy, and R 6 is C 5-9 Alkyl or C 6-10 is an alkoxy; W is methylene or a direct bond, and R 7 is H or Me, or a salt thereof, However, R 3 and R 2 is a C2 alkyl, X 1 is O and X 2 is a straight chain C3 alkylene, and X 3 is C(=O) and Y 1 is a linear C5 alkylene, and (Y 2 )nR 4 teeth [ka] and R 4 is a straight chain C5 alkyl, and Z 1is a C2 alkylene, and Z 2 is absent, W is methylene, and R 7 is H and R 5 and R 6 is not a C2 alkoxy.

[0260] In some embodiments, the lipid composition further comprises one or more compounds of formula (x).

[0261] Additional non-limiting examples of lipid compounds that may be further included in the lipid composition further include (e.g., in combination with the lipid compounds and other lipid components described herein): [ka] JPEG2025510229000215.jpg219160

[0262] In some embodiments, the lipid composition further comprises one or more compounds of formula (xi), (xii), (xiii), (xiv), (xv), (xvi), (xvii), (xviii) (e.g., (xviii)a, (xviii)b), or (xix).

[0263] In some embodiments, the lipid composition further comprises a lipid formed by one of the following reactions: [ka]

[0264] In some embodiments, the lipid composition has formula (xxi): [ka] and further comprising (e.g., in combination with lipid compounds and other lipid components described herein) a lipid having the formula: each n is independently an integer from 2 to 15; L1 and L3 each independently represent -OC(O)- * or -C(O)O- *And, * " indicates the point of attachment to R1 or R3, R1 and R3 each independently represent oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyl a straight or branched C9-C alkyl group optionally substituted by one or more substituents selected from the group consisting of alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 alkenyl, and R2 is selected from the group consisting of: [ka]

[0265] In some embodiments, the lipid composition further comprises one or more compounds of formula (xxi). In some embodiments, the compounds of formula (xxi) include those described in WO 2021 / 113777 (e.g., lipids of formula (1), such as the lipids in Table 1 of WO 2021 / 113777), which is incorporated by reference in its entirety.

[0266] In some embodiments, the lipid composition has formula (xxii): [ka] and further comprising (e.g., in combination with lipid compounds and other lipid components described herein) a lipid having the formula: each n is independently an integer from 1 to 15; R1 and R2 are each independently selected from the group consisting of: [ka] R3 is selected from the group consisting of: [ka]

[0267] In some embodiments, the lipid composition further comprises one or more compounds of formula (xxii). In some embodiments, compounds of formula (xxii) include those described in WO 2021 / 113777 (e.g., lipids of formula (2), such as the lipids in Table 2 of WO 2021 / 113777), which is incorporated by reference in its entirety.

[0268] In some embodiments, the lipid composition has formula (xxiii): [ka] and further comprising (e.g., in combination with lipid compounds and other lipid components described herein) a lipid having the formula X is -O-, -S-, or -OC(O)- * is selected from: * indicates the point of attachment to R1, R1 is selected from the group consisting of: [ka] R2 is selected from the group consisting of: [ka]

[0269] In some embodiments, the lipid composition further comprises one or more compounds of formula (xxiii). In some embodiments, compounds of formula (xxiii) include those described in WO 2021 / 113777 (e.g., lipids of formula (3), such as the lipids in Table 3 of WO 2021 / 113777), which is incorporated by reference in its entirety.

[0270] Examples of additional lipids that may be used in the lipid composition include, but are not limited to, one or more of the following formulas: X of U.S. Patent Application Publication No. 2016 / 0311759; I of U.S. Patent Application Publication No. 20150376115 or U.S. Patent Application Publication No. 2016 / 0376224; I, II, or III of U.S. Patent Application Publication No. 2016 / 0151284; I, IA, or IIA of U.S. Patent Application Publication No. 2017 / 0210967; Ic of U.S. Patent Application Publication No. 2015 / 0140070; A of U.S. Patent Application Publication No. 2013 / 0178541; U.S. Publication No. 2013 / 0303587 or U.S. Patent Application Publication No. 2013 / 0123338, I; U.S. Patent Application Publication No. 2015 / 0141678, I; U.S. Patent Application Publication No. 2015 / 0239926, II, III, IV, or V; U.S. Patent Application Publication No. 2017 / 0119904, I; WO 2017 / 117528, I or II; U.S. Patent Application Publication No. 2012 / 0149894, A; U.S. Patent Application Publication No. 2015 / 0057373, A; WO 2013 / 116126, A; U.S. Patent Application Publication No. 2013 / 0090 372, A; U.S. Patent Application Publication No. 2013 / 0274523, A; U.S. Patent Application Publication No. 2013 / 0274504, A; U.S. Patent Application Publication No. 2013 / 0053572, A; WO 2013 / 016058, A; WO 2012 / 162210, A; U.S. Patent Application Publication No. 2008 / 042973, I; U.S. Patent Application Publication No. 2012 / 01287670, I, II, III, or IV; U.S. Patent Application Publication No. 2014 / 0200257, I or II; U.S. Patent Application Publication No. 2015 / 0203446, I, II, or II I; I or III of U.S. Patent Application Publication No. 2015 / 0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of U.S. Patent Application Publication No. 2014 / 0308304; U.S. Patent Application Publication No. 2013 / 0338210; I, II, III, or IV of WO 2009 / 132131; A of U.S. Patent Application Publication No. 2012 / 01011478; I or XXXV of U.S. Patent Application Publication No. 2012 / 0027796; XIV or XVII of U.S. Patent Application Publication No. 2012 / 0058144;U.S. Patent Application Publication No. 2013 / 0323269; U.S. Patent Application Publication No. 2011 / 0117125, I; U.S. Patent Application Publication No. 2011 / 0256175, I, II, or III; U.S. Patent Application Publication No. 2012 / 0202871, I, II, III, IV, V, VI, VII, VIII, IX, X, XI, and XII; U.S. Patent Application Publication No. 2011 / 0076335, I, II, III, IV, V, VI, VII, VIII, X, and XII , XIII, XIV, XV, or XVI; I or II of U.S. Patent Application Publication No. 2006 / 008378; I of U.S. Patent Application Publication No. 2013 / 0123338; ​​I or XAYZ of U.S. Patent Application Publication No. 2015 / 0064242, XVI, XVII, or XVIII of U.S. Patent Application Publication No. 2013 / 0022649; I, II, or III of U.S. Patent Application Publication No. 2013 / 0116307; U.S. Patent Application Publication No. 2013 / 011 No. 6307, I, II, or III; U.S. Patent Application Publication No. 2010 / 0062967, I or II; U.S. Patent Application Publication No. 2013 / 0189351, IX, U.S. Patent Application Publication No. 2014 / 0039032, I; U.S. Patent Application Publication No. 2018 / 0028664, V; U.S. Patent Application Publication No. 2016 / 0317458, I; U.S. Patent Application Publication No. 2013 / 0195920, I; U.S. Patent Application No. 10,221,127, V, VI, or 1. 0; III-3 of WO 2018 / 081480; I-5 or I-8 of WO 2020 / 081938; 18 or 25 of U.S. Patent No. 9,867,888; A of U.S. Patent Application Publication No. 2019 / 0136231; II of WO 2020 / 219876; I of U.S. Patent Application Publication No. 2012 / 0027803, OF-02 of U.S. Patent Application Publication No. 2019 / 0240349; 23 of U.S. Patent No. 10,086,013; Miao cKK-E12 / A6 from WO 2010 / 053572; C12-200 from WO 2010 / 053572; 7C1 from Dahlman et al. (2017); 304-O13 or 503-O13 from Whitehead et al.; TS-P4C2 from U.S. Pat. No. 9,708,628; I from WO 2020 / 106946; I from WO 2020 / 106946;(1), (2), (3), or (4) of WO 2021 / 113777; and any one of Tables 1-16 of WO 2021 / 113777, all of which are incorporated herein by reference in their entireties.

[0271] In some embodiments, the lipid conjugate (e.g., a PEG lipid) is present at 0.1 mol% to 2 mol%, 0.5 mol% to 2 mol%, 1 mol% to 2 mol%, 0.6 mol% to 1.9 mol%, 0.7 mol% to 1.8 mol%, 0.8 mol% to 1.7 mol%, 0.9 mol% to 1.6 mol%, 0.9 mol% to 1.8 mol%, 1 mol% to 1.8 mol%, 1 mol% to 1.7 mol%, 1.2 mol% to 1.8 mol%, 1.2 mol% to 1.7 mol%, 1.3 mol% to 1.6 mol%, or 1.4 mol% to 1.5 mol% (or any fraction or range therein) of the total lipid present in the lipid composition. In some embodiments, the lipid conjugate (e.g., a PEG lipid) is present at 0 mol% to 20 mol%, 0.5 mol% to 20 mol%, 2 mol% to 20 mol%, 1.5 mol% to 18 mol%, 2 mol% to 15 mol%, 4 mol% to 15 mol%, 2 mol% to 12 mol%, 5 mol% to 12 mol%, or 2 mol% (or any fraction or range therein) of the total lipid present in the lipid composition.

[0272] In some embodiments, the lipid conjugate (e.g., PEG-lipid) lipid composition is present at 4 mol% to 10 mol%, 5 mol% to 10 mol%, 5 mol% to 9 mol%, 5 mol% to 8 mol%, 6 mol% to 9 mol%, 6 mol% to 8 mol%, or 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, or 10 mol% (or any fraction or range therein) of the total lipid present in the lipid composition.

[0273] The percentage of lipid conjugate (e.g., PEG lipid) present in the lipid composition of the present disclosure is a target amount, and the actual amount of lipid conjugate present in the composition can vary, for example, by ±2 mol%. Those skilled in the art will understand that the concentration of lipid conjugate can vary depending on the lipid conjugate used and the rate at which the lipid particles become fusogenic.

[0274] By controlling the composition and concentration of lipid conjugate, the rate at which lipid conjugate is exchanged from lipid composition can be controlled, and thus the rate at which lipid composition becomes fusogenic.In addition, other variables, including, for example, pH, temperature or ionic strength, can be used to change and / or control the rate at which lipid composition becomes fusogenic.Other methods that can be used to control the rate at which lipid composition becomes fusogenic will be clear to those skilled in the art after reading this disclosure.In addition, by controlling the composition and concentration of lipid conjugate, the size of lipid particles can be controlled.

[0275] In some embodiments, lipid compositions containing ionizable lipid compounds may contain 30-70% ionizable lipid compounds, 0-60% cholesterol, 0-30% phospholipids, and 1-10% polyethylene glycol (PEG). In some embodiments, lipid compositions contain 30-40% ionizable lipid compounds, 40-50% cholesterol, and 10-20% PEG lipids. In some embodiments, lipid compositions contain 50-75% ionizable lipid compounds, 20-40% cholesterol, 5-10% phospholipids, and 1-10% PEG lipids. Lipid compositions may contain 60-70% ionizable lipid compounds, 25-35% cholesterol, and 5-10% PEG lipids.

[0276] In some embodiments, the lipid component of the lipid composition includes about 30 mol% to about 60 mol% (e.g., about 35-55 mol%, or about 40-50 mol%) of an ionizable lipid compound described herein, about 0 mol% to about 30 mol% (e.g., 5-25 mol%, or 10-20 mol%) of phospholipids, about 15 mol% to about 50 mol% (e.g., 18.5-48.5 mol%, or 30-40 mol%) of sterols, and about 0 mol% to about 10 mol% (e.g., 1-5 mol%, or 1.5-2.5 mol%) of PEGylated lipids, wherein the total mol% of the lipid components does not exceed 100%.

[0277] In some embodiments, the lipid composition may contain up to 90% ionizable lipid compounds and 2-15% helper lipids.

[0278] In some embodiments, the lipid composition may be a lipid particle composition, e.g., containing 8-30% ionizable lipid compounds, 5-30% helper lipids, and 0-20% cholesterol. In some embodiments, the lipid nanoparticle composition contains 4-25% ionizable lipids, 4-25% helper lipids, 2-25% cholesterol, 10-35% cholesterol-PEG, and 5% cholesterol-amine. In some embodiments, the lipid nanoparticle composition contains 2-30% ionizable lipids, 2-30% helper lipids, 1-15% cholesterol, 2-35% cholesterol-PEG, and 1-20% cholesterol-amine. In some embodiments, the lipid nanoparticle composition contains up to 90% ionizable lipids and 2-10% helper lipids. In some embodiments, the lipid nanoparticle composition contains 100% ionizable lipids.

[0279] Other components of the LNP composition The lipid nanoparticle composition may contain one or more ingredients in addition to those described above. For example, the LNP composition may contain one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0280] The lipid nanoparticle composition may also include one or more permeability enhancer molecules, carbohydrates, polymers, surface modifiers, or other components.

[0281] Suitable carbohydrates can include monosaccharides (eg, glucose) and polysaccharides (eg, glycogen and its derivatives and analogs).

[0282] Polymers may be used to encapsulate or partially encapsulate the nanoparticle composition. The polymer may be biodegradable and / or biocompatible. Suitable polymers include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, 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-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), and poly(D,L-lactide-co-PPO-co-D,L-lactide).L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters, polyvinyl halides such as poly(vinyl acetate), poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, and polymers of acrylic acid, such as 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), and poly(octadecyl acrylate). Examples of suitable polyols include polyols and copolymers thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarates, polyoxymethylene, poloxamers, polyoxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol.

[0283] Suitable surface modifiers include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocysteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letostein, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexin, and erdosteine), and DNase (e.g., rhDNase). Surface modifiers may be disposed within the lipid nanoparticles and / or on the surface of the lipid nanoparticles (e.g., by coating, adsorption, covalent bonding, or other process).

[0284] Lipid nanoparticle composition can also comprise one or more functionalized lipids.For example, lipid can be functionalized with alkyne group, which can undergo cycloaddition reaction when exposed to azide under suitable reaction conditions.In particular, lipid bilayer can be functionalized in this manner with one or more groups useful for promoting membrane permeation, cell recognition or imaging.The surface of lipid nanoparticle can also be conjugated with one or more useful antibodies.Functional groups and conjugates useful for target cell delivery, imaging and membrane permeation are well known in the art.

[0285] The lipid nanoparticle composition may contain any substance useful in pharmaceutical compositions. For example, the lipid nanoparticle composition may contain one or more pharmaceutically acceptable excipients or subcomponents, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonicity agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and other species. Excipients such as waxes, butters, colorants, coating agents, flavors, and fragrances may also be included.

[0286] Suitable 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, dry starch, corn starch, powdered sugar, and / or combinations thereof. Granulating and dispersing agents may be selected from the non-limiting list consisting of potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0287] Suitable surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glycerin monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl ... cellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [ SPAN® 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone),Examples of suitable anti-inflammatory agents include diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC® F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0288] Suitable binders may be starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, breadwort gum, ghatti gum, mucilage of isopole shell, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; and combinations thereof, or any other suitable binder.

[0289] Suitable preservatives may include, but are not limited to, antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, 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, malic 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 preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE™, KATHON™, and / or EUXYL®.

[0290] Suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0291] Suitable oils include almond, apricot kernel, avocado, babassu, bergamot, blackcurrant seed, borage, juniper, chamomile, canola, caraway, carnauba, castor, cinnamon bark, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, linseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cucumber, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, and tawny. Ingredients include, but are not limited to, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savory, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, camellia, vetiver, walnut, and wheat germ oils, as well as butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, simethicone, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0292] In some embodiments, the lipid composition further comprises one or more cryoprotectants. Suitable cryoprotectants include polyols (e.g., diols or triols, such as propylene glycol (i.e., 1,2-propanediol), 1,3-propanediol, glycerol, (+ / -)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), non-surfactant sulfobetaines (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), osmolytes (e.g., L-proline or trimethylamine N-oxide dihydrate), polymers (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 600, PEG 700, PEG 800, PEG 900, PEG 1000, PEG 1100, PEG 1200, PEG 1300, PEG 1400, PEG 1500, PEG 1600, PEG 1700, PEG 1800, PEG 1900, PEG 2000, PEG 2100, PEG 2200, PEG 2300, PEG 2400, PEG 2500, PEG 2600, PEG 2700, PEG 2800, PEG 2900, PEG 3000, PEG 3100, PEG 3200, PEG 3300, PEG 3400, PEG 3500, PEG 3600, PEG 3700, PEG 3800, PEG 390 2k-DMG, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K15), pentaerythritol propoxylate, or polypropylene glycol P 400), organic solvents (e.g., dimethyl sulfoxide (DMSO) or ethanol), sugars (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, mesoerythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, or D-(+)-glucose monohydrate), or salts (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof), or any combination thereof.

[0293] 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.

[0294] In some embodiments, the composition further comprises one or more buffers. Suitable buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium 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, aminosulfonic acid buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof.

[0295] In some embodiments, the buffer is an acetate buffer, a citrate buffer, a phosphate buffer, a Tris buffer, or a combination thereof.

[0296] In some embodiments, the lipid composition further comprises one or more nucleic acids, ionizable lipids, amphiphiles, phospholipids, cholesterol, and / or PEG-conjugated cholesterol.

[0297] Pharmaceutical Composition Another aspect of the present disclosure also provides pharmaceutical compositions comprising the lipid composition described herein, which comprises one or more lipid compounds selected from the ionizable lipid compounds described herein (e.g., those having the structures of Formulae (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), (IIIA)-(IIIIE), (IIIC-1), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), and (VC-1)-(VC-6)), and a pharmaceutically acceptable excipient. The pharmaceutical composition may further comprise a therapeutic agent.

[0298] All statements above and all embodiments, and exemplary variables and compounds discussed in the above aspects relating to lipid compound aspects, including compounds covered by formulae (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), (IIIA)-(IIIIE), (IIIC-1), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), and (VC-1)-(VC-6), are all applicable to these aspects of the invention relating to pharmaceutical compositions.

[0299] All statements and all embodiments discussed in the above aspects relating to lipid composition aspects, including various other lipid components, are applicable to these aspects of the invention relating to pharmaceutical compositions.

[0300] In lipid compositions containing a therapeutic agent, the ratio of total lipid components to cargo (e.g., encapsulated therapeutic agent, such as a nucleic acid) can vary as desired. For example, the total lipid components to cargo ratio (mass or weight) can be from about 10:1 to about 30:1. In some embodiments, the total lipid components to cargo ratio (mass / mass ratio, w / w ratio) can be within the range of about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amount of total lipid components and cargo can be adjusted to provide a desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or greater. Generally, the overall lipid content of the lipid composition can range from about 5 mg / ml to about 30 mg / mL.

[0301] therapeutic agent nucleic acid molecule In some embodiments, the lipid composition further comprises one or more nucleic acid molecules, which may be a plasmid, an immunostimulatory oligonucleotide, an antisense oligonucleotide, an antagomir, an aptamer, a deoxyribozyme (DNAzyme), and a ribozyme.

[0302] In some embodiments, the lipid composition further comprises one or more of RNA and / or DNA.

[0303] In some embodiments, the nucleic acid molecule is DNA. In some embodiments, the DNA is linear, circular, single-stranded, or double-stranded.

[0304] In some embodiments, the nucleic acid molecule is RNA. In some embodiments, the RNA is mRNA, miRNA, siRNA, RNA aptamer, linear RNA, circular RNA, single-stranded RNA, double-stranded RNA, tRNA, microRNA (miRNA) or miRNA precursor, Dicer substrate small interfering RNA (dsiRNA), short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), guide RNA (gRNA), lncRNA, ncRNA, sncRNA, rRNA, snRNA, piRNA, snoRNA, snRNA, scaRNA, exRNA, scaRNA, Y RNA, or hnRNA. In some embodiments, the RNA is mRNA. In one embodiment, the mRNA is modified mRNA.

[0305] In some embodiments, the nucleic acid molecule is an enzymatic nucleic acid molecule.The term "enzymatic nucleic acid molecule" refers to a nucleic acid molecule that has complementarity in the substrate binding region to a specific gene target and also has the enzymatic activity of specifically cleaving target RNA.That is, the enzymatic nucleic acid molecule can intermolecularly cleave RNA, thereby inactivating the target RNA molecule.The term enzymatic nucleic acid is used interchangeably with terms such as ribozyme, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulable ribozyme, catalytic oligonucleotide, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme.All of these terms describe nucleic acid molecules with enzymatic activity.

[0306] In some embodiments, the nucleic acid molecule is an antisense nucleic acid. The term "antisense nucleic acid" refers to a non-enzymatic nucleic acid molecule that binds to a target RNA through RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid) interactions and alters the activity of the target RNA.

[0307] In some embodiments, the nucleic acid molecule may be a 2-5A antisense chimera. The term "2-5A antisense chimera" refers to an antisense oligonucleotide containing a 5'-phosphorylated 2'-5'-linked adenylate residue.

[0308] In some embodiments, the nucleic acid molecule may be a triplex-forming oligonucleotide. The term "triple-strand-forming oligonucleotide" refers to an oligonucleotide that can bind to double-stranded DNA in a sequence-specific manner to form a triple-stranded helix.

[0309] In some embodiments, the nucleic acid molecule may be a decoy RNA. The term "decoy RNA" refers to an RNA molecule or aptamer that is designed to preferentially bind to a specific ligand. Such binding can result in the inhibition or activation of the target molecule.

[0310] In some embodiments, the nucleic acid molecule (eg, RNA or DNA) encodes a therapeutic peptide or polypeptide operably linked to a promoter of the DNA. Therapeutic peptides or polypeptides can be, for example, transcription factors; chromatin remodeling factors; antigens; hormones; enzymes (such as nucleases, e.g., endonucleases, e.g., nuclease components of the CRISPR system, e.g., Cas9, dCas9, aCas9-nickase, Cpf / Cas12a); Crispr-binding enzymes, e.g., base editors or prime editors; mobile genetic element proteins (e.g., transportases, retrotranspotases, recombinases, integrases); gene writers; polymerases; methylases; demethylases; acetylases; deacetylases; kinases; phosphatases; ligases; deubiquitinases; integrases; recombinases; topoisomerases; gyrases; helicases; lysosomal acid hydrolases); antibodies; receptor ligands; receptors; coagulation factors; membrane proteins; mitochondrial proteins; nuclear proteins; antibodies or other protein scaffold binding agents such as centrins, darpins, or adnectins.

[0311] In some embodiments, the nucleic acid molecule is an RNA comprising a gRNA nucleic acid. In some embodiments, the gRNA nucleic acid is a gRNA. In some embodiments, the nucleic acid molecule is an RNA comprising a Class 2 Cas nuclease mRNA and a gRNA. In some embodiments, the gRNA nucleic acid is a dual guide RNA (dgRNA) or encodes a dual guide RNA (dgRNA). In some embodiments, the gRNA nucleic acid is a single guide RNA (sgRNA) or encodes a single guide RNA (sgRNA). In some embodiments, the gRNA is a modified gRNA. In some embodiments, the modified gRNA comprises a modification in one or more of the first five nucleotides at the 5' end. In some embodiments, the modified gRNA comprises a modification in one or more of the last five nucleotides at the 3' end.

[0312] In some embodiments, the nucleic acid molecule is RNA, including mRNA. In some embodiments, the RNA component comprises an RNA-guided DNA binding agent, such as a Cas nuclease mRNA (such as a class 2 Cas nuclease mRNA) or a Cas9 nuclease mRNA.

[0313] All nucleic acid molecules described herein can be chemically modified. Various modification strategies for nucleic acid molecules are well known to those skilled in the art. In some embodiments, the nucleic acid molecule comprises one or more modifications selected from the group consisting of pseudouridine, 5-bromouracil, 5-methylcytosine, peptide nucleic acid, heterologous nucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, florophore (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotide, biotin-conjugated nucleotide, fluorescent base analog, CpG island, methyl-7-guanosine, methylated nucleotide, inosine, thiouridine, pseudourdine, dihydrouridine, quosine, and wyosine. In some embodiments, the antisense oligonucleotide can be a locked nucleic acid oligonucleotide (LNA). The term "locked nucleic acid (LNA)" refers to an oligonucleotide containing one or more nucleotide components in which an extra methylene bridge locks the ribose moiety in either the C3'-endo (beta-D-LNA) or C2'-endo (alpha-L-LNA) configuration (Grunweller A, Hartmann RK, BioDrugs, 21(4):235-243 (2007)).

[0314] In some embodiments, the composition further comprises one or more template nucleic acid molecules.

[0315] Additional examples of nucleic acid molecules (including tumor suppressor genes, antisense oligonucleotides, siRNA, miRNA, or shRNA) can be found in U.S. Patent Application Publication No. 2007 / 0065499 and U.S. Patent No. 7,780,882, which are incorporated by reference in their entireties.

[0316] In some embodiments, a pharmaceutical composition may include multiple nucleic acid molecules that may be of the same or different types.

[0317] Nucleic acids for use in the embodiments of the present disclosure can be prepared according to any available technology. For mRNA, the main preparation method is, but is not limited to, enzymatic synthesis (also known as in vitro transcription), which is currently the most efficient method for producing long, sequence-specific mRNA. In vitro transcription describes the process of template-directed synthesis of RNA molecules from a modified DNA template consisting of an upstream bacteriophage promoter sequence (such as, but not limited to, those derived from T7, T3, and SP6 coliphages) linked to a downstream sequence encoding a gene of interest. Template DNA can be prepared for in vitro transcription from many sources using suitable techniques well known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LDin RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v.941 Conn GL (ed), New York, NY Humana Press, 2012).

[0318] RNA transcription occurs in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using various commercially available kits, including, but not limited to, RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription Kit (Life Technologies), and commercially available reagents containing RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are well known in the art (see, for example, Losick, R., 1972, In vitro transcription, Ann Rev Biochem v. 41 409-46; Kamaka, RT and Kraus, WL 2001. In vitro Transcription. Current Protocols in Cell Biology. 2: 11.6: 11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of which are incorporated herein by reference).

[0319] The desired in vitro transcribed mRNA can be purified from unwanted components of the transcription or related reaction, including unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc. Techniques for isolating mRNA transcripts are well known in the art. Well-known procedures include, but are not limited to, phenol / chloroform extraction or precipitation with any alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride.

[0320] Additional non-limiting examples of purification procedures that can be used include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10,889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LDin RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA synthesis Methods v.941 Conn GL (ed), New York, NY Humana Press, 2012).Purification can be carried out using various commercially available kits, including but not limited to SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0321] Furthermore, while reverse transcription can produce large amounts of mRNA, the product may contain numerous aberrant RNA impurities associated with undesired polymerase activity, which may need to be removed from the full-length mRNA preparation. These include short RNAs resulting from abortive transcription initiation, as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and self-complementary 3' extension. It has been demonstrated that these impurities, which have dsRNA structures, can result in undesirable immunostimulatory activity through interactions with various innate immune sensors in eukaryotic cells that recognize specific nucleic acid structures and function to elicit strong immune responses. This, in turn, can dramatically reduce mRNA translation, as protein synthesis is reduced during the innate cellular immune response. Therefore, additional techniques for removing these dsRNA impurities have been developed and are known in the art, including, but not limited to, scalable HPLC purification (see, for example, Kariko, K., Muramatsu, H., Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v.39 el42; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PHEd), 2013). HPLC-purified mRNA has been reported to be translated at much higher levels, especially in primary cells and in vivo.

[0322] A wide variety of modifications have been described in the art that can be used to alter specific properties of in vitro transcribed mRNA and improve its utility. These include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5' end of the mature molecule, which plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which in turn is involved in enhancing intracellular mRNA stability and the efficiency of mRNA translation. Therefore, the highest level of protein expression is achieved with capped mRNA transcripts. The 5'-cap contains a 5'-5'-triphosphate linkage between the 5'-most nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the terminal and penultimate 5'-nucleotides on the 2'-hydroxyl group.

[0323] Multiple distinct cap structures can be used to generate the 5'-cap of in vitro transcribed synthetic mRNA. 5'-capping of synthetic mRNA can be performed co-transcriptionally (i.e., capping during in vitro transcription) using chemical cap analogs. For example, the anti-reverse cap analog (ARC A) cap contains a 5'-5'-triphosphate guanine-guanine linkage, where one guanine contains an N7 methyl group and a 3'-0-methyl group. However, up to 20% of transcripts remain uncapped during this co-transcription process, and synthetic cap analogs are not identical to the 5'-cap structure of authentic cellular mRNA, potentially resulting in reduced translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped after transcription. These can produce more authentic 5'-cap structures that structurally or functionally mimic endogenous 5'-caps with enhanced cap-binding protein binding, increased half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' decapping. Numerous synthetic 5'-cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PHEd), 2013).

[0324] At the 3'-end, a long chain of adenine nucleotides (poly-A tail) is usually added to mRNA molecules during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved, freeing the 3' hydroxyl for poly-A polymerase to add a chain of adenine nucleotides to the RNA in a process called polyadenylation. The poly(A) tail has been widely shown to enhance both the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A) binding protein and the regulation of mRNA stability, Trends Bio Sci v.14 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v.265, 11-23; Dreyfus, M. and Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v1 1, 611-613).

[0325] Poly(A) tailing of in vitro transcribed mRNA can be achieved using various techniques, including, but not limited to, cloning a poly(T) tract into a DNA template or post-transcriptional addition using poly(A) polymerase. The former allows for in vitro transcription of mRNA with a poly(A) tail of a defined length depending on the size of the poly(T) tract, but requires additional template manipulation. The latter requires enzymatic addition of a poly(A) tail to in vitro transcribed mRNA using poly(A) polymerase, which catalyzes the incorporation of adenine residues into the 3' end of the RNA. This does not require additional DNA template manipulation, but results in mRNAs with poly(A) tails of heterogeneous lengths. 5'-capping and 3'-poly(A) tailing can be performed using various commercially available kits, including, but not limited to, Poly(A) Polymerase Tailing Kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra Kit, and Poly(A) Tailing Kit (Life Technologies), as well as commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0326] In addition to 5' cap and 3' polyadenylation, other modifications of in vitro transcription products have been reported to bring about benefits related to translation efficiency and stability.It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors in eukaryotes and induce strong innate immune responses.Since most nucleic acids from natural sources contain modified nucleosides, it has been shown that the ability to distinguish pathogenic DNA and RNA from self-DNA and RNA is at least partly based on structure and nucleoside modification.In contrast, in vitro synthesized RNA lacks these modifications, and therefore can cause immune stimulation, which in turn can inhibit the above-mentioned effective mRNA translation.Introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus alleviating this undesirable immunostimulatory activity and enhancing translational competence (e.g., Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: Implications for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology). v.969 (Rabinovich, PHEd), 2013; Kariko, K., Muramatsu, H., Welsh, FA, Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840). Modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored, and utilized using common methods and procedures known in the art. A variety of nucleoside modifications are available that can be incorporated to some extent into in vitro transcribed mRNA, alone or in combination with other modified nucleosides (see, for example, U.S. Patent Application Publication No. 2012 / 0251618).In vitro synthesis of nucleoside-modified mRNA has been reported to have reduced capacity to activate immunosensors while simultaneously enhancing translational capacity.

[0327] Other components of mRNA that can be modified to benefit translatability and stability include 5' and 3' untranslated regions (UTRs).Optimizing UTRs (favorable 5' and 3' UTRs can be obtained from cellular or viral RNA) has been shown to increase mRNA stability and translation efficiency of in vitro transcribed mRNA, either together or independently (see, for example, Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PHEd), 2013).

[0328] In addition to mRNA, other nucleic acid payloads may be used in the present disclosure.For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis, enzymatic and chemical cleavage of long-chain precursors, in vitro transcription as described above, etc.The synthesis method of DNA and RNA nucleotides is widely used and well known in the art (see, for example, Gait, MJ (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Ishington, DC: IRL Press, 1984, and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v.288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005; both are incorporated herein by reference).

[0329] For plasmid DNA, preparation for use in embodiments of the present disclosure generally utilizes, but is not limited to, in vitro propagation and isolation of the plasmid DNA in liquid cultures of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows bacteria containing the plasmid of interest to selectively grow in antibiotic-containing cultures. Methods for isolating plasmid DNA are widely used and well known in the art (see, e.g., Heilig, J., Elbing, K.L. and Brent, R., (2001), Large-Scale Preparation of Plasmid DNA, Current Protocols in Molecular Biology, 41:11:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstrom, S., Bjornestedt, R. and Schmidt, SR, (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture, Biotechnol. Bioeng., 99:557-566; and U.S. Pat. No. 6,197,553 Bl). Plasmid isolation can be performed using a variety of commercially available kits, including but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits, as well as commercially available reagents.

[0330] In some embodiments, the lipid nanoparticle compositions are useful for expressing proteins encoded by mRNA. In some embodiments, provided herein are methods for expressing proteins encoded by mRNA.

[0331] In some embodiments, the lipid composition has an N / P ratio of about 1:1 to about 30:1, e.g., about 3:1 to about 20:1, about 3:1 to about 15:1, about 3:1 to about 10:1, or about 3:1 to about 6:1. For example, the N / P ratio of the nucleic acid molecule-encapsulating lipid composition may be 6±1, or the N / P ratio of the nucleic acid molecule-encapsulating lipid composition may be 6±0.5. In some embodiments, the N / P ratio of the nucleic acid molecule-encapsulating lipid composition ranges from about 3:1 to about 15:1. In some embodiments, the N / P ratio of the nucleic acid molecule-encapsulating lipid composition is about 6. The N:P ratio refers to the molar ratio of amines (e.g., amines in ionizable lipids) present in the lipid composition or lipid nanoformulation to phosphate present in the nucleic acid molecule. This is a factor in efficient packaging and efficacy.

[0332] Other therapeutic agents The therapeutic agent may be a peptide or protein, a small molecule drug, encapsulated in the lipid composition. The pharmaceutical composition may contain two or more different therapeutic agents from a nucleic acid molecule, a peptide or protein, and a small molecule drug.

[0333] In some embodiments, the protein can be a peptide or polypeptide, such as, for example, a transcription factor; a chromatin remodeling factor; an antigen; a hormone; an enzyme (such as a nuclease, e.g., an endonuclease, e.g., a nuclease component of a CRISPR system, e.g., Cas9, dCas9, aCas9-nickase, Cpf / Cas12a); a Crispr-binding enzyme, e.g., a base editor or prime editor; a mobile genetic element protein (e.g., a transportase, retrotranspotase, recombinase, integrase); a gene writer; a polymerase; a methylase; a demethylase; an acetylase; a deacetylase; a kinase; a phosphatase; a ligase; a deubiquitinase; an integrase; a recombinase; a topoisomerase; a gyrase; a helicase; a lysosomal acid hydrolase; an antibody; a receptor ligand; a receptor; a coagulation factor; a membrane protein; a mitochondrial protein; a nuclear protein; an antibody or other protein scaffold binding agent such as a centrin, a darpin, or an adnectin.

[0334] In some embodiments, a pharmaceutical composition may include multiple protein molecules, which may be of the same or different types.

[0335] In some embodiments, the therapeutic agent is a small molecule drug, eg, a small molecule drug approved for human use by an appropriate regulatory agency.

[0336] In some embodiments, a pharmaceutical composition may include multiple small molecule drugs, which may be of the same or different type.

[0337] In some embodiments, the therapeutic agent is a vaccine. In some embodiments, the vaccine is an RNA vaccine, such as an RNA cancer vaccine or an RNA vaccine for an infectious disease (e.g., an influenza virus vaccine or a coronavirus vaccine (e.g., a COVID-19 vaccine).

[0338] Other ingredients The pharmaceutical composition may contain one or more pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients are selected based on the mode and route of administration. Suitable pharmaceutical carriers or excipients for use in pharmaceutical formulations are described in Remington: The Science and Practice of Pharmacy, 21 st Ed.,Gennaro,Ed.,Lippencott Williams & Wilkins(2005);Handbook of Pharmaceutical Excipients,6 th Edition, Rowe et al., Eds., Pharmaceutical Press (2009); and the USP / NF (United States Pharmacopeia and the National Formulary), which are incorporated herein by reference in their entireties.

[0339] In some embodiments, the pharmaceutically acceptable excipient comprises one or more of an antioxidant, binder, anti-adhesive, buffer, colorant, diluent (e.g., solid or liquid), disintegrant (e.g., coating disintegrates), dispersant, digestive agent, filler, emulsifier, flavoring agent, lubricant, pH adjuster, pigment, preservative, stabilizer, solubilizer, solvent, suspending agent, sweetener, or wetting agent, or a combination thereof.

[0340] Examples of suitable excipients include, but are not limited to, acacia, alginate, calcium phosphate, calcium carbonate, calcium silicate, carbopol gel, carboxymethylcellulose, carnauba wax, cellulose, crospovidone, dextrose, diacetylated monoglyceride, ethylcellulose, gelatin, glyceryl monostearate 40-50, acacia gum, gum arabic, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hypromellose phthalate, hypromellose, lactose, lecithin, magnesium stearate, kaolin, methacrylic acid copolymer type C, mannitol, methylcellulose ... The inactive ingredients may include cellulose, methyl hydroxybenzoate, microcrystalline cellulose, povidone, polyethylene glycol, polysorbate 80, polyvinylpyrrolidone, propyl hydroxybenzoate, sodium carboxymethylcellulose, sodium hydroxide, sodium stearyl fumarate, sodium starch glycolate, starch, sorbitan monooleate, sorbitol, sorbic acid, sucrose, talc, tragacanth, talc, triethyl citrate, titanium dioxide, yellow iron oxide, talc, an oil vehicle (e.g., peanut oil, liquid paraffin, mineral oil, olive oil, almond oil, glycerin, propylene glycol), or water.

[0341] When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material (e.g., normal saline), which acts as a vehicle, carrier, or medium for the active ingredient. As is known in the art, the type of diluent can vary depending on the intended route of administration.

[0342] Pharmaceutical compositions may contain pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, or or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0343] Suitable carriers or excipients for pharmaceutical compositions may also include substances that enhance the individual's body's ability to absorb LNPs or liposomes. Suitable carriers and / or excipients also include any substance that can be used to bulk formulations containing LNPs or liposomes to allow for convenient and accurate administration. In addition, carriers and / or excipients may be used in the manufacturing process to facilitate handling of LNPs or liposomes. Different carriers and / or excipients may be used depending on the route of administration and the form of the drug.

[0344] Carriers and / or excipients may also include vehicles and / or diluents. "Vehicle" generally refers to any of a variety of media that act as a solvent or carrier, and "diluent" refers to a diluent used to dilute the active ingredients of the composition. Suitable diluents include any substance that can reduce the viscosity of a pharmaceutical product. The type and amount of carriers and / or excipients are selected as a function of the pharmaceutical form selected. Suitable pharmaceutical forms include liquid systems such as solutions, injections, and suspensions; semi-solid systems such as colloids, gels, pastes, or creams; and solid systems such as powders, granules, tablets, capsules, pellets, microparticles, minitablets, microcapsules, micropellets, and suppositories.

[0345] Each of the above systems may be suitably formulated for normal, delayed, or accelerated release using techniques well known in the art.

[0346] Formulation, Dosage, and Route of Administration The pharmaceutical compositions described herein can be prepared according to standard techniques and the techniques described herein.For example, pharmaceutical compositions can be prepared by conventional methods, such as by conventional mixing, dissolving, granulating, sugar-coating, nudging, emulsifying, encapsulating, encapsulating or lyophilizing processes.The methods for preparing formulations well known in the art are well known in the art.See, for example, Remington: The Science and Practice of Pharmacy, 21 st See Ed., Gennaro, Ed., Lippencott Williams & Wilkins (2005), and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J.C. Johnlan, 1988-1999, Marcel Dekker, New York.

[0347] The therapeutic agent may be encapsulated within the lipid composition; for example, the therapeutic agent may be located completely or partially within the interior space of the LNP, within the lipid layer / membrane, or associated with the outer surface of the lipid layer / membrane. One purpose of incorporating a therapeutic agent into an LNP is to protect the therapeutic agent from the environment, which may contain enzymes, chemicals, or conditions that degrade the therapeutic agent and / or systems or receptors that cause rapid excretion of the therapeutic agent. Furthermore, incorporating a therapeutic agent into an LNP may facilitate uptake of the therapeutic agent and thus enhance therapeutic efficacy. In some embodiments, the ratio of lipid component to therapeutic agent (mass / mass ratio, w / w ratio) in the pharmaceutical composition can range from about 1:1 to about 25:1, 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0348] The lipid or pharmaceutical composition may contain about 5 to about 95% by weight of the therapeutic agent, based on the weight of the lipid or pharmaceutical composition. In some embodiments, the lipid or pharmaceutical composition contains about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% by weight of the therapeutic agent, based on the weight of the LNP or pharmaceutical composition. In some embodiments, the lipid composition or pharmaceutical composition comprises about 5 to 95%, about 5 to 90%, about 5 to 80%, about 5 to 70%, about 5 to 60%, about 5 to 50%, about 5 to 40%, about 5 to 30%, about 5 to 20%, about 5 to 10%, about 10 to 95%, about 10 to 90%, about 10 to 80%, about 10 to 70%, about 10 to 60%, about 10 to 50%, about 10 to 40%, about 10 to 30%, about 10 to 20%, about 20 to 95%, about 20 to 90%, about 20 to 80%, about 20 to 70%, about 20 to 60%, about 20 to 50%, about 20 to 40%, The therapeutic agent is contained in an amount of about 20-30%, about 30-95%, about 30-90%, about 30-80%, about 30-70%, about 30-60%, about 30-50%, about 30-40%, about 40-95%, about 40-90%, about 40-80%, about 40-70%, about 40-60%, about 40-50%, about 50-95%, about 50-90%, about 50-80%, about 50-70%, about 50-60%, about 60-95%, about 60-90%, about 60-80%, about 60-70%, about 70-95%, about 70-90%, about 70-80%, about 80-95%, about 80-90%, or about 90-95%.

[0349] The lipid composition or pharmaceutical composition may contain total lipids in an amount of about 5 to about 95% by weight, based on the weight of the lipid composition or pharmaceutical composition. In some embodiments, the lipid composition or pharmaceutical composition comprises about 5 to 95%, about 5 to 90%, about 5 to 80%, about 5 to 70%, about 5 to 60%, about 5 to 50%, about 5 to 40%, about 5 to 30%, about 5 to 20%, about 5 to 10%, about 10 to 95%, about 10 to 90%, about 10 to 80%, about 10 to 70%, about 10 to 60%, about 10 to 50%, about 10 to 40%, about 10 to 30%, about 10 to 20%, about 20 to 95%, about 20 to 90%, about 20 to 80%, about 20 to 70%, about 20 to 60%, about 20 to 50%, about 20 to 40%, The total lipid content is about 20-30%, about 30-95%, about 30-90%, about 30-80%, about 30-70%, about 30-60%, about 30-50%, about 30-40%, about 40-95%, about 40-90%, about 40-80%, about 40-70%, about 40-60%, about 40-50%, about 50-95%, about 50-90%, about 50-80%, about 50-70%, about 50-60%, about 60-95%, about 60-90%, about 60-80%, about 60-70%, about 70-95%, about 70-90%, about 70-80%, about 80-95%, about 80-90%, or about 90-95%.

[0350] The lipid composition or pharmaceutical composition of the present disclosure can be administered by various routes, for example, to achieve systemic delivery via intravenous, parenteral, intraperitoneal, intramuscular, intraductal or local route.In some embodiments, siRNA can be delivered intracellularly, for example, into the cells of target tissue such as lung or liver, or into inflamed tissue.In some embodiments, the present disclosure provides a method for delivering siRNA in vivo.Nucleic acid-lipid composition can be administered to subject intravenously, subcutaneously or intraperitoneally.

[0351] As used herein, the term "oral" refers to a route of administration other than enteral administration. Examples of parenteral administration include, but are not limited to, oral, epicutaneous, epidural, extraamniotic, intra-arterial, intra-articular, intracardiac, intracavernosal, intracerebral, intraventricular, intradermal, intralesional, intramuscular, intraocular, intraosseous, intraperitoneal, intrapulmonary, intrathecal, intrauterine, intravaginal, intravenous, intravesical, intravitreal, nasal, perivascular, subcutaneous, sublingual, transdermal, topical, transepithelial, or transmucosal. Parenteral administration may be by continuous infusion over a selected period of time.

[0352] The compositions and methods of the present disclosure can be administered to a subject by various mucosal administration methods, including oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal or cutaneous delivery, or topical delivery to the eye, ear, skin, or other mucosal surfaces. In some aspects of the present disclosure, the mucosal tissue layer comprises an epithelial cell layer. The epithelial cells can be pulmonary, tracheal, bronchial, alveolar, nasal, oral, epidermal, or gastrointestinal. The compositions of the present disclosure can be administered using conventional actuators, such as mechanical spray devices and pressurized, electrically operated, or other types of actuators.

[0353] The compositions of the present disclosure may be administered in aqueous solution as nasal or pulmonary sprays, or may be dispensed in spray form by various methods known to those skilled in the art. Pulmonary delivery of the compositions of the present disclosure is achieved, for example, by administering the composition in the form of drops, particles, or sprays, which may be aerosolized, atomized, or nebulized. The particles, spray, or aerosol of the composition may be in either liquid or solid form. A non-limiting example of a system for dispensing a liquid as a nasal spray is disclosed in U.S. Pat. No. 4,511,069. Such formulations may be conveniently prepared by dissolving a composition according to the present disclosure in water to form an aqueous solution and sterilizing the solution. The formulation may be placed in a multi-dose container in a sealed dispensing system, for example, as disclosed in U.S. Pat. No. 4,511,069. Other suitable nasal spray delivery systems are described in TRANSDERMAL SYSTEMIC MEDICATION, Y.W. Chien, ed., Elsevier Publishers, New York, 1985, and U.S. Pat. No. 4,778,810. Additional aerosol delivery forms can include, for example, compressed air jet nebulizers, ultrasonic nebulizers, and piezoelectric nebulizers that deliver bioactive agents dissolved or suspended in a pharmaceutical solvent, such as water, ethanol, or mixtures thereof.

[0354] The nasal and pulmonary spray solutions of the present disclosure typically contain a drug to be delivered and are optionally formulated with a surfactant, such as a non-ionic surfactant (e.g., polysorbate-80), and one or more buffers. In some embodiments of the present disclosure, the nasal spray solution further contains a propellant. The pH of the nasal spray solution may be between pH 6.8 and 7.2. The pharmaceutical solvent used may also be a slightly acidic aqueous buffer solution with a pH of between 4 and 6. Other ingredients, including preservatives, surfactants, dispersants, or gases, may be added to enhance or maintain chemical stability.

[0355] In some embodiments, the present disclosure is a pharmaceutical product comprising a solution containing a composition of the present disclosure and an actuator for a pulmonary, mucosal, or nasal spray or aerosol.

[0356] The dosage form of the composition of the present disclosure may be a liquid in the form of drops or an emulsion, or in the form of an aerosol.

[0357] The dosage form of the composition of the present disclosure may be a solid that can be reconstituted in a liquid before administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet, or gel.

[0358] To prepare compositions for pulmonary delivery within the present disclosure, bioactive agents can be combined with various pharmaceutically acceptable additives, as well as bases or carriers for dispersion of the active agent.

[0359] Examples of additives include pH control agents such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and mixtures thereof. Other additives include local anesthetics (e.g., benzyl alcohol), tonicity agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancers (e.g., cyclodextrin and its derivatives), stabilizers (e.g., serum albumin), and reducing agents (e.g., glutathione). When the composition for mucosal delivery is a liquid, the tonicity of the composition, measured relative to the tonicity of 0.9% (w / v) saline taken as a single unit, is typically adjusted to a value that does not induce substantial irreversible tissue damage in the mucosa at the administration site. Generally, the tonicity of the solution is adjusted to 1 / 3 to 3, more typically 1 / 2 to 2, and most often 3 / 4 to 1.7.

[0360] The bioactive agent may be dispersed in a base or vehicle, which may include a hydrophilic compound capable of dispersing the active agent and any desired additives. The base may be selected from a wide range of suitable carriers, including, but not limited to, polycarboxylic acids or their salts, copolymers of carboxylic acid anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, and polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose and hydroxypropylcellulose, natural polymers such as chitosan, collagen, sodium alginate, gelatin, and hyaluronic acid, and non-toxic metal salts thereof. Biodegradable polymers are often selected as the base or carrier, such as polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer, and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters, such as polyglycerin fatty acid esters and sucrose fatty acid esters, can be used as carriers. Hydrophilic polymers and other carriers can be used alone or in combination, and can impart enhanced structural integrity to the carrier by partial crystallization, ionic bonding, crosslinking, etc. Carriers can be supplied in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres, and films for direct application to the nasal mucosa. The use of selected carriers in this context can result in enhanced absorption of the bioactive agent.

[0361] Compositions for mucosal, nasal, or pulmonary delivery may contain hydrophilic low-molecular-weight compounds as bases or excipients. Such hydrophilic low-molecular-weight compounds can provide a passageway for water-soluble active agents, such as physiologically active peptides or proteins, to diffuse through the base to the body surface where they are absorbed. The hydrophilic low-molecular-weight compounds can optionally absorb moisture from the mucous membrane or the administration atmosphere, dissolving the water-soluble active peptide. In some embodiments, the molecular weight of the hydrophilic low-molecular-weight compounds is 10,000 or less, for example, 3,000 or less. Examples of hydrophilic low-molecular-weight compounds include polyol compounds, such as sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentiose, glycerin, polyethylene glycol, and oligosaccharides, disaccharides, and monosaccharides, including mixtures thereof. Further examples of hydrophilic low molecular weight compounds include N-methylpyrrolidone, alcohols (eg, oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.), and mixtures thereof.

[0362] Alternatively, the compositions of the present disclosure may contain pharmaceutically acceptable carriers required for approximate physiological conditions, such as pH adjusting agents and buffering agents, isotonicity adjusting agents, and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof.For solid compositions, conventional non-toxic pharmaceutically acceptable carriers can be used, including, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc.

[0363] In certain embodiments of the present disclosure, the bioactive agent may be administered in a sustained-release formulation, for example, in a composition containing a slow-release polymer. The active agent may be prepared with a carrier that protects against rapid release, for example, a controlled-release vehicle, such as a polymeric microencapsulated delivery system or a bioadhesive gel. Long-term delivery of the active agent in various compositions of the present disclosure may be achieved by including in the composition an agent that delays absorption, such as, for example, aluminum monosterate hydrogel and gelatin.

[0364] In some embodiments, the lipid composition, pharmaceutical composition, or dosage unit contains about 0.01 to about 1000 mg of one or more lipid compounds described herein. In some embodiments, the lipid composition, pharmaceutical composition, or unit dose contains about 0.01, about 0.1, about 0.5, about 1, about 5, about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, 250, about 275, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 mg of one or more lipid compounds described herein.In some embodiments, the lipid composition, pharmaceutical composition, or unit dose is from about 0.01 to about 750 mg, from about 0.01 to about 500 mg, from about 0.01 to about 250 mg, from about 0.01 to about 100 mg, from about 0.01 to about 50 mg, from about 0.01 to about 25 mg, from about 0.01 to about 10 mg, from about 0.01 to about 5 mg, from about 0.01 to about 0.1 mg, from about 0.1 to about 1000 mg, from about 0.1 to about 750 mg, from about 0.1 to about 500 mg, from about 0.1 to about 250 mg g, about 0.1 to about 100 mg, about 0.1 to about 50 mg, about 0.1 to about 25, about 0.1 to about 10 mg, about 0.1 to about 5 mg, about 0.1 to about 1 mg, about 1 to about 1000 mg, about 1 to about 750 mg, about 1 to about 500 mg , about 1 to about 250 mg, about 1 to about 100 mg, about 1 to about 50 mg, about 1 to about 25 mg, about 1 to about 10 mg, about 1 to about 5 mg, about 5 to about 1000 mg, about 5 to about 750 mg, about 5 to about 500 mg, about 5 to about 250 mg , about 5 to about 100 mg, about 5 to about 50 mg, about 5 to about 25 mg, about 5 to about 10 mg, about 10 to about 1000 mg, about 10 to about 750 mg, about 10 to about 500, about 10 to about 250 mg, about 10 to about 100 mg, 10 to about Approximately 50 mg, approximately 10 to approximately 25 mg, approximately 25 to approximately 1000 mg, approximately 25 to approximately 750 mg, approximately 25 to approximately 500 mg, approximately 25 to approximately 250 mg, approximately 25 to approximately 100 mg, approximately 25 to approximately 50 mg, approximately 50 to approximately 1000, mg approximately 5 The lipid compound(s) may comprise 0 to about 750 mg, about 50 to about 500 mg, about 50 to about 250 mg, about 50 to about 100 mg, about 100 to about 1000 mg, about 100 to about 750 mg, about 100 to about 500 mg, about 100 to about 250 mg, about 250 to about 1000 mg, about 250 to about 750 mg, about 250 to about 500 mg, about 500 to about 1000 mg, about 500 to about 750 mg, or about 750 to about 1000 mg of one or more lipid compounds described herein.

[0365] Methods of Use of Lipid Compositions Another aspect of the present disclosure provides a method for delivering a therapeutic agent to a subject (e.g., a patient) in need thereof, comprising administering to the subject (e.g., a patient) a pharmaceutical composition comprising a lipid nanoparticle composition comprising a lipid compound of Formulas (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), (IIIA)-(IIIE), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), and (VC-1)-(VC-6), a pharmaceutically acceptable salt thereof, and / or a stereoisomer of any of the foregoing, and a therapeutic agent.

[0366] Another aspect of the present disclosure relates to a method for extrahepatic delivery of a therapeutic agent to at least one organ other than the liver (e.g., the pancreas, one or both lungs, or the spleen) of a subject in need thereof, with minimal delivery of the therapeutic agent to other parts of the body, such as the liver. The method includes administering to the subject a pharmaceutical composition comprising a lipid compound of Formulas (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), (IIIA)-(IIIE), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), and (VC-1)-(VC-6), a pharmaceutically acceptable salt thereof, and / or a stereoisomer of any of the foregoing, and a lipid nanoparticle composition comprising a therapeutic agent. In some embodiments, the method delivers the therapeutic agent to the pancreas and / or one or both lungs of a subject in need thereof, with minimal delivery to other parts of the body, such as the liver.

[0367] In some embodiments, less than 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the total therapeutic agent administered to a subject is delivered to the subject's liver, hi some embodiments, less than 6%, 7%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total therapeutic agent administered to a subject is delivered to the subject's liver.

[0368] In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the total therapeutic agent administered to a subject is delivered to the subject's pancreas, spleen, and / or one or both lungs. In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the total therapeutic agent administered to a subject is delivered to the subject's pancreas. In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the total therapeutic agent administered to a subject is delivered to the subject's lungs. In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the total therapeutic agent administered to a subject is delivered to the subject's spleen.

[0369] In some embodiments, the total therapeutic agent administered to a subject has a spleen-to-liver ratio of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the total therapeutic agent administered to a subject has a spleen-to-liver ratio of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150. In some embodiments, the total therapeutic agent administered to a subject has a spleen-to-liver ratio of at least 1. In some embodiments, the total therapeutic cargo administered to the subject has a spleen-to-liver ratio of at least 5. In some embodiments, the total therapeutic agent administered to the subject has a spleen-to-liver ratio of at least 10. In some embodiments, the total therapeutic agent administered to the subject has a spleen-to-liver ratio of at least 25. In some embodiments, the total therapeutic agent administered to the subject has a spleen-to-liver ratio of at least 70. In some embodiments, the total therapeutic agent administered to the subject has a spleen-to-liver ratio of at least 75. In some embodiments, the total therapeutic agent administered to the subject has a spleen-to-liver ratio of at least 100. In some embodiments, the total therapeutic agent administered to the subject has a spleen-to-liver ratio of at least 110.

[0370] As used herein, the percentage of the total therapeutic agent administered to a subject and delivered to a site in the subject is measured by protein expression levels or mRNA knockdown levels.

[0371] In some embodiments, the method for delivering a therapeutic agent disclosed above comprises administering to a subject a lipid composition containing the therapeutic agent. In some embodiments, the lipid nanoparticles in the lipid composition are formed from one or more compounds selected from ionizable lipids of formulas (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), (IIIA)-(IIIIE), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), and (VC-1)-(VC-6), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formulas (I), (IA-1), or (IA-2), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formulas (IIA)-(IIC), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formula (IIA-1), pharmaceutically acceptable salts thereof, and any stereoisomers of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formulas (IIIA)-(IIIIE), pharmaceutically acceptable salts thereof, and any stereoisomers of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formulas (IVA-1)-(IVA-3), pharmaceutically acceptable salts thereof, and any stereoisomers of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formulas (IVC-1)-(IVC-3), pharmaceutically acceptable salts thereof, and any stereoisomers of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formulas (VC-1)-(VC-6), pharmaceutically acceptable salts thereof, and any stereoisomers of the foregoing.

[0372] In some embodiments, the lipid compositions disclosed herein can be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells in vitro and / or in vivo. Accordingly, in some embodiments, methods are provided for treating or preventing a disease or disorder in a subject in need thereof, comprising administering a lipid composition to the subject. In some embodiments, the lipid composition encapsulates or is associated with a suitable therapeutic agent, and the lipid composition comprises one or more of the novel ionizable lipids described herein, pharmaceutically acceptable salts thereof, and / or stereoisomers of any of the foregoing.

[0373] In some embodiments, the lipid compositions of the present disclosure are useful for delivering therapeutic agents. In some embodiments, the therapeutic agent is selected from one or more nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antimir), messenger RNA interference complementary RNA (micRNA), DNA, polyvalent RNA, Dicer substrate RNA, complementary DNA (cDNA), etc. Thus, in some embodiments, disclosed herein are methods for inducing expression of a desired protein in vitro and / or in vivo by contacting cells with a lipid composition comprising one or more of the novel ionizable lipids described herein, wherein the lipid nanoparticles encapsulate or are associated with a nucleic acid that is expressed to produce the desired protein (e.g., a messenger RNA or plasmid encoding the desired protein), or a nucleic acid that blocks a process that terminates mRNA expression (e.g., a miRNA inhibitor). In some embodiments, disclosed herein are methods for reducing target gene and protein expression in vitro and / or in vivo by contacting cells with lipid compositions comprising one or more of the novel ionizable lipids described herein, wherein the lipid nanoparticles encapsulate or are associated with a nucleic acid (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)) that reduces target gene expression. In some embodiments, disclosed herein are methods for co-delivery of one or more nucleic acids (e.g., mRNA and plasmid DNA), individually or in combination, which may be useful for producing effects requiring co-localization of different nucleic acids (e.g., mRNA encoding a suitable gene-modifying enzyme and a DNA segment for integration into the host genome).

[0374] In some embodiments, the lipid composition is useful for upregulating endogenous protein expression by delivering miRNA inhibitors that target a specific miRNA or a group of miRNAs that regulate one target mRNA or several mRNAs. In some embodiments, provided herein is a method for upregulating endogenous protein expression, comprising delivering miRNA inhibitors that target one or more miRNAs that regulate one or more mRNAs. In some embodiments, the lipid composition is useful for downregulating (e.g., silencing) the protein level and / or mRNA level of a target gene. In some embodiments, provided herein is a method for downregulating (e.g., silencing) the protein and / or mRNA level of a target gene.

[0375] In some embodiments, the lipid compositions are useful for delivery of mRNA and plasmids for expression of transgenes. In some embodiments, methods are provided herein for delivery of mRNA and plasmids for expression of transgenes.

[0376] In some embodiments, the lipid compositions are useful for inducing a pharmacological effect resulting from the expression of a protein, such as increased production of red blood cells by delivery of a suitable erythropoietin mRNA, or protection against infection by delivery of mRNA encoding a suitable antigen or antibody. In some embodiments, provided herein are methods for inducing a pharmacological effect resulting from the expression of a protein, such as increased production of red blood cells by delivery of a suitable erythropoietin mRNA, or protection against infection by delivery of mRNA encoding a suitable antigen or antibody.

[0377] Non-limiting exemplary embodiments of the ionizable lipids of the present disclosure, lipid compositions comprising same, and their uses for delivering agents (e.g., therapeutic agents such as nucleic acids) and / or modulating gene and / or protein expression are described in further detail below.

[0378] In some embodiments, the present disclosure relates to methods of gene editing comprising contacting a cell with an LNP composition, hi some embodiments, the present disclosure relates to any method of gene editing described herein comprising cleaving DNA.

[0379] In some embodiments, the present disclosure relates to methods of cleaving DNA comprising contacting a cell with an LNP composition.

[0380] In some embodiments, the present disclosure relates to any method of cleaving DNA described herein, wherein the cleaving step comprises introducing a single-stranded DNA nick. In some embodiments, the present disclosure relates to any method of cleaving DNA described herein, wherein the cleaving step comprises introducing a double-stranded DNA break. In some embodiments, the present disclosure relates to any method of cleaving DNA described herein, wherein the LNP composition comprises a Class 2 Cas mRNA and a guide RNA nucleic acid. In some embodiments, the present disclosure relates to any method of cleaving DNA described herein, further comprising introducing at least one template nucleic acid into a cell. In some embodiments, the present disclosure relates to any method of cleaving DNA described herein, comprising contacting a cell with an LNP composition comprising a template nucleic acid.

[0381] In some embodiments, the present disclosure relates to any method of gene editing described herein, comprising administering an LNP composition to an animal, e.g., a human. In some embodiments, the present disclosure relates to any method of gene editing described herein, comprising administering an LNP composition to a cell, such as a eukaryotic cell.

[0382] In some embodiments, the present disclosure relates to any method of gene editing described herein, comprising administering mRNA formulated in a first LNP composition and a second LNP composition comprising one or more of mRNA, gRNA, gRNA nucleic acid, and template nucleic acid. In some embodiments, the present disclosure relates to any method of gene editing described herein, wherein the first and second LNP compositions are administered simultaneously. In some embodiments, the present disclosure relates to any method of gene editing described herein, wherein the first and second LNP compositions are administered sequentially.

[0383] In some embodiments, the present disclosure relates to any method of gene editing described herein, comprising administering mRNA and guide RNA nucleic acids formulated in a single LNP composition. In some embodiments, the present disclosure relates to any method of gene editing described herein, wherein the gene editing results in a gene knockout. In some embodiments, the present disclosure relates to any method of gene editing described herein, wherein the gene editing results in a gene correction.

[0384] In some embodiments, the present disclosure relates to methods for in vivo delivery of interfering RNA to the lungs of a mammalian subject.

[0385] In some embodiments, the methods relate to methods of treating a disease or disorder in a mammalian subject, hi some embodiments, the methods comprise administering a therapeutically effective amount of a lipid composition of the present disclosure to a subject having a disease or disorder associated with expression or overexpression of a gene that can be reduced, decreased, downregulated, or silenced by the lipid composition. [Example]

[0386] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0387] Example 1. Synthesis of Compound 2230 [ka] Step 1: To a solution of 3-(dimethylamino)propanoic acid (0.2 g, 1.30 mmol, 1 equiv) and oxalyl dichloride (826.30 g, 6.51 mmol, 569.86 μL, 5 equiv) in DCM (5 mL) was added two drops of DMF (9.52 mg, 130.20 μmol, 10.02 μL, 0.1 equiv). The mixture was degassed and purged with N three times and stirred under N atmosphere at 20 °C for 10 h. The reaction mixture was concentrated under reduced pressure to give 3-(dimethylamino)propanoyl chloride (0.2 g, crude, HCl) as a yellow oil.

[0388] Step 2: To a solution of [5-(1-octylnonoxy)-5-oxo-pentyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (100.00 mg, 135.48 μmol, 1 equiv.), 3-(dimethylamino)propanoyl chloride (93.24 mg, 541.91 μmol, 4 equiv., HCl) in DCM (3 mL) was added TEA (123.38 mg, 1.22 mmol, 169.71 μL, 9 equiv.) at 0° C. The mixture was stirred at 20° C. for 12 h. The reaction mixture was diluted with 20 mL of HO and extracted with 60 mL of EtOAc (20 mL × 3). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate / NH3·H2O = 1 / 0 / 0.1 to 3 / 1 / 0.1) and preparative HPLC (column: Phenomenex Luna C18 100 × 30 mm × 5 μm, mobile phase: [water (HCl) to ACN]; B%: 45% to 75%, 10 min) to give a residue. The residue was adjusted to pH = 7 with saturated aqueous NaHCO3 and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was then purified by column chromatography (SiO, petroleum ether / ethyl acetate / NH H O = 1 / 0 / 0.1 to 5 / 1 / 0.1) to give 2230, [5-(1-octylnonoxy)-5-oxo-pentyl](2S)-4-[3-(dimethylamino)propanoyloxy]-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (17 mg, 20.30 μmol, 14.99% yield, 100% purity) as a yellow oil. 1H NMR(400MHz,CDCl3),5.21-5.27(m,1H),4.86-4.89(m,1H),4.14-4.16(m, 2H),4.06(t,J=6.4Hz,2H),3.44-3.55(m,1H),3.10-3.26(m,1H),2.45-2.7 3(m,7H),2.32-2.33(m,10H),2.16-2.28(m,1H),2.05-2.06(m,1H),1.60- 1.65(m,6H),1.51-1.52(m,6H),1.27-1.31(m,46H),0.89(t,J=6.8Hz,9H). LCMS:(1 / 2M+H + ):419.2@2.971 minutes.

[0389] Example 2. Synthesis of Compound 2260 [ka] Step 1: A solution of heptadecan-9-ol (10 g, 38.99 mmol, 1 equiv), 5-bromopentanoic acid (7.06 g, 38.99 mmol, 1 equiv), DMAP (952.72 mg, 7.80 mmol, 0.2 equiv), and EDCI (7.47 g, 38.99 mmol, 1 equiv) in DCM (70 mL) was stirred for 12 h at 20° C. The combined organic phase was diluted with 200 mL of EtOAc, washed with 600 mL (200 mL × 3) of water and 400 mL (200 mL × 2) of brine, dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 10 / 1) to give 1-octylnonyl 5-bromopentanoate (25 g, 59.60 mmol, yield 76.42%) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.85-4.91(m,1H),3.42(t,J=6.8Hz,2H),2.34(t,J=7.2Hz,2H),1.90-1 .93(m,2H),1.79-1.81(m,2H),1.52-1.57(m,4H),1.27-1.51(m,24H),0.89(t,J=6.4Hz,6H).

[0390] Step 2: A mixture of (2S)-1-tert-butoxycarbonyl-4-hydroxy-pyrrolidine-2-carboxylic acid (2.30 g, 9.93 mmol, 1 equiv.), 1-octylnonyl 5-bromopentanoate (5 g, 11.92 mmol, 1.2 equiv.), and CsCO (7.12 g, 21.85 mmol, 2.2 equiv.) in DMF (60 mL) was stirred at 20 °C for 12 h under a N atmosphere. The reaction mixture was quenched by adding 10 mL of HO at 0 °C. The mixture was extracted with 30 mL of EtOAc (10 mL × 3), and the combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 3 / 1) to give O1-tert-butyl O2-[5-(1-octylnonoxy)-5-oxo-pentyl](2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (4.5 g, 3.95 mmol, 39.75% yield, 50% purity) as a white solid. 1 H NMR(400MHz,CDCl3),4.86-4.89(m,1H),4.17-4.39(m,4H),3.50-3.69(m,2H),2.33-2.34 (m,2H),1.61-1.72(m,4H),1.42-1.52(m,14H),1.25-1.30(m,25H),0.88(t,J=6.8Hz,6H).

[0391] Step 3: To a solution of O1-tert-butyl O2-[5-(1-octylnonoxy)-5-oxo-pentyl](2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (4.00 g, 7.02 mmol, 1 equiv) in DCM (50 mL) was added TFA (23.10 g, 202.60 mmol, 15.00 mL, 28.86 equiv). The mixture was stirred at 20 °C for 5 h. The reaction mixture was adjusted to pH = 7 with saturated aqueous NaHCO3, extracted with 600 mL (200 mL × 3) of EtOAc, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate=0 / 1 to ethyl acetate / MeOH=3 / 1) to give [5-(1-octylnonoxy)-5-oxo-pentyl](2S)-4-hydroxypyrrolidine-2-carboxylate (2.5 g, 4.52 mmol, 64.45% yield, 85% purity) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.86-4.89(m,1H),4.37-4.45(m,1H),4.15-4.25(m,2H),3.82-4.14(m,1H),2.97-3.15 (m,2H),2.33-2.35(m,2H),1.69-1.70(m,4H),1.50-1.52(m,4H),1.26-1.32(m,26H),0.88(t,J=6.8Hz,6H).

[0392] Step 4: To a solution of [5-(1-octylnonoxy)-5-oxo-pentyl](2S)-4-hydroxypyrrolidine-2-carboxylate (500.00 mg, 1.06 mmol, 1 equiv.), undecyl 6-bromohexanoate (446.26 mg, 1.28 mmol, 1.2 equiv.) in DMF (10 mL) was added K2CO3 (441.37 mg, 3.19 mmol, 3 equiv.). The mixture was stirred at 80 °C for 12 h. The reaction mixture was diluted with 20 mL of HO and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate / NH H O = 10 / 1 / 1 to 1 / 1 / 0.5) to give 2260, [5-(1-octylnonoxy)-5-oxo-pentyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (0.6 g, 715.32 μmol, 67.20% yield, 88% purity) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.86-4.91(m,1H),4.28-4.49(m,1H),4.13-4.16(m,2H),4.06(t,J=6.4Hz,2H),3.07-3.54(m,2H),2.48-2.65 (m,3H),2.28-2.34(m,4H),1.95-2.23(m,2H),1.60-1.64(m,6H),1.50-1.52(m,6H),1.27-1.35(m,44H),0.89(t,J=6.4Hz,9H),(M+H + ): 738.3. LCMS: (M+H + ):738.3@2.843 minutes.

[0393] Example 3. Synthesis of Compound 2231 [ka] Step 1: To a solution of 3-(dimethylamino)propanoic acid (0.2 g, 1.30 mmol, 1 equiv., HCl) and oxalyl dichloride (826.30 mg, 6.51 mmol, 569.86 mL, 5 equiv.) in DCM (5 mL) was added two drops of DMF (9.52 mg, 130.20 μmol, 10.02 μL, 0.1 equiv.) at 20° C. The mixture was stirred at 20° C. for 10 h under a N atmosphere. The reaction mixture was concentrated under reduced pressure to give 3-(dimethylamino)propanoyl chloride (0.2 g, crude, HCl) as a yellow oil.

[0394] Step 2: To a suspension of [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (200 mg, 256.34 μmol, 1 equiv.), DMAP (6.26 mg, 51.27 μmol, 0.2 equiv.), TEA (207.51 mg, 2.05 mmol, 285.44 μL, 8 equiv.), and 4A molecular sieves (100 mg) in DCM (15 mL) was added 3-(dimethylamino)propanoyl chloride (220.52 mg, 1.28 mmol, 5 equiv., HCl) in DCM (10 mL) at 0° C. The mixture was stirred under a N atmosphere. 、The mixture was stirred at 20°C for 8 hours. The reaction mixture was diluted with 20 mL of H2O and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate / NH3·H2O = 1 / 0 / 0.1 to 3 / 1 / 0.1) and preparative HPLC (column: Phenomenex Luna C18 100 × 30 mm × 5 μm; mobile phase: [water (HCl) to ACN]; B%: 55%-85%, 10 min) to give a residue. The residue was adjusted to pH = 7 with saturated aqueous NaHCO3 and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate / NH H O = 1 / 0 / 0.1 to 5 / 1 / 0.1) to give 2231, [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-[3-(dimethylamino)propanoyloxy]-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (40 mg, 45.49 μmol, 13.33% yield, 100% purity) as a yellow oil. 1 H NMR(400MHz,CDCl3),5.21-5.27(m,1H),4.84-4.90(m,1H),4.04-4.13(m,4H),3.08-3.54(m,2H),2.32-2.65(m,7H),2 .29-2.31(m,10H),2.27-2.28(m,2H),1.63-1.65(m,8H),1.60-1.62(m,6H),1.27-1.52(m,48H),0.89(t,J=6.4Hz,9H). LCMS:(1 / 2M+H + ):879.6@2.155 minutes.

[0395] Example 4. Synthesis of Compound 2270 [ka] Step 1: To a mixture of 8-bromooctanoic acid (10 g, 44.82 mmol, 1 equiv.) in DCM (1000 mL), DMAP (1.10 g, 8.96 mmol, 0.2 equiv.), heptadecan-9-ol (11.50 g, 44.82 mmol, 1 equiv.), and EDCI (8.59 g, 44.82 mmol, 1 equiv.) were added, degassed, and purged with N2 three times. The mixture was stirred under N2 atmosphere at 20 °C for 8 h. The reaction was diluted with 200 mL of EtOAc, washed with 600 mL of water (200 mL × 3) and 400 mL of brine (200 mL × 2), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 1 / 0) to give μ 1-octylnonyl-5-bromopentanoate (25 g, 59.60 mmol, yield 76.42%) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.86-4.89(m,1H),3.41(t,J=7.2Hz,2H),2.29(t,J=7.6Hz,2H),1.80-1.90(m,2H) ),1.60-1.63(m,2H),1.44-1.51(m,4H),1.34-1.35(m,2H),1.27-1.33(m,28H),0.89(t,J=6.8Hz,6H).

[0396] Step 2: A mixture of (2S)-1-tert-butoxycarbonyl-4-hydroxy-pyrrolidine-2-carboxylic acid (3.34 g, 14.44 mmol, 1 equiv.), 1-octylnonyl 8-bromooctanoate (8 g, 17.33 mmol, 1.2 equiv.), and CsCO (10.35 g, 31.78 mmol, 2.2 equiv.) in DMF (60 mL) was stirred at 20 °C under a N atmosphere for 8 h. The reaction mixture was quenched by adding 10 mL of HO at 0 °C. The mixture was extracted with 30 mL of EtOAc (10 mL × 3), and the combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 3 / 1) to give O1-tert-butyl O2-[8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (6 g, 9.81 mmol, 67.89% yield) as a white solid.

[0397] Step 3: To a solution of O1-tert-butyl O2-[8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (5.5 g, 8.99 mmol, 1 equiv.) in DCM (50 mL) was added TFA (23.10 g, 202.59 mmol, 15 mL, 22.54 equiv.). The mixture was stirred at 20 °C for 5 h. The reaction mixture was adjusted to pH = 7 with saturated aqueous NaHCO3, extracted with 600 mL (200 mL × 3) of EtOAc, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 0 / 1 to ethyl acetate / MeOH = 3 / 1) to give [5-(1-octylnonoxy)-5-oxo-pentyl](2S)-4-hydroxypyrrolidine-2-carboxylate (2.5 g, 4.52 mmol, 64.45% yield, 85% purity) as a yellow oil. 1H NMR(400MHz,CDCl3),4.85-4.89(m,1H),4.45-4.47(m,1H),4.02-4.18(m,3H),2.99-3.19(m,2H),2.29-2.31(m,4H),2 .07-2.27(m,1H),2.05-2.06(m,1H),1.61-1.66(m,4H),1.50-1.52(m,4H),1.26-1.35(m,30H),0.88(t,J=6.8Hz,6H).

[0398] Step 4: To a solution of [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxypyrrolidine-2-carboxylate (2 g, 3.91 mmol, 1 equiv.), undecyl 6-bromohexanoate (1.64 g, 4.69 mmol, 1.2 equiv.) in DMF (20 mL) was added K2CO3 (1.62 g, 11.72 mmol, 3 equiv.). The mixture was stirred at 80 °C for 8 h. The reaction mixture was diluted with 20 mL of HO and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate / NH H O = 10 / 1 / 1 to 1 / 1 / 0.5) to give 2270 [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (3 g, 3.85 mmol, 98.39% yield, 100% purity) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.84-4.90(m,1H),4.49-4.52(m,1H),4.04-4.39(m,5H),3.05-3.66(m,2H),2.48-2.69 (m,2H),1.94-2.32(m,6H),1.60-1.66(m,8H),1.50-1.52(m,6H),1.27-1.34(m,48H),0.89(t,J=6.8Hz,9H). LCMS:(M+H + ):780.5@2.889 minutes.

[0399] Example 5. General reaction scheme for the synthesis of exemplary ionizable lipid compounds A general reaction scheme for the synthesis of exemplary ionizable lipid compounds containing a heterocyclic core structure (eg, an N-containing core) is shown in Scheme 1. [ka] Scheme 1

[0400] A general reaction scheme for the synthesis of exemplary ionizable lipid compounds containing a cycloalkyl core structure is shown in Scheme 2. [ka] Scheme 2

[0401] In Scheme 1 or 2, non-limiting examples of alternative starting heterocyclic or cycloalkyl core structures for preparing exemplary ionizable lipid compounds are shown in Scheme 3. [ka] Scheme 3

[0402] Example 6. Preparation of lipid nanoparticle compositions with or without cargo Exemplary Lipid Nanoparticle Compositions Exemplary lipid nanoparticle compositions were prepared to obtain a molar ratio of ionizable lipid:structural lipid:sterol:PEG lipid of 50:10:38.5:1.5, respectively. For example, exemplary lipid nanoparticle compositions of this example are shown in the table below. The exemplary ionizable lipids used in each exemplary lipid nanoparticle composition were compounds 2230, 2231, 2260, and 2270 (LNP 2230, LNP 2231, LNP 2260, LNP 2270). [Table 3]

[0403] To prepare these compounds, lipids according to the chart above were solubilized in ethanol, mixed in the molar ratios above, and diluted in ethanol (organic phase) to give a total lipid concentration of 5.5 mM.

[0404] Comparison of lipid nanoparticle compositions A lipid nanoparticle composition containing C12-200 (LNP C12-200) was prepared as a control, with a molar ratio of C12-200:DOPE:cholesterol (14:0):DMPE-PEG2k of 35:16:46.5:2.5. C12-200 is a commercially available ionizable lipid with the chemical name 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol). The lipids were solubilized in ethanol. These lipids were mixed in the above molar ratio and diluted in ethanol (organic phase) to obtain a total lipid concentration of 5.5 mM.

[0405] Another lipid nanoparticle composition containing MC3 (LNP MC3) was prepared as a control, resulting in a molar ratio of 50:38.5:10:1.5: MC3:DSPC:cholesterol:14:0 DMPE-PEG2k. MC3 is a commercially available ionizable lipid with the chemical name (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate. The lipids were solubilized in ethanol. These lipids were mixed in the above molar ratio and diluted in ethanol (organic phase) to obtain a total lipid concentration of 5.5 mM.

[0406] Lipid nanoparticle compositions encapsulating mRNA.

[0407] The mRNA solution (aqueous phase, fluc:EPO mRNA) was prepared in RNAse-free water and 100 mM citrate buffer, pH 3, to give a final concentration of 50 mM citrate buffer and an mRNA concentration of 0.167 mg / mL (1:1 fluc:EPO). Formulations maintained an ionized lipid to mRNA ratio of 15:1 for the LNP C12-200 control and 6:1 for the exemplary lipid nanoparticle compositions (LNP 2230, LNP 2231, LNP 2260, LNP 2270) and the LNP MC3 control.

[0408] For each LNP composition, the lipid mixture and mRNA solution were mixed in a NanoAssemblr Ignite (Precision Nanosystems) at a volume ratio of 1:3, respectively, at a total flow rate of 9 mL / min. The resulting composition was then loaded into a Slide-A-Lyzer G2 dialysis cassette (10k MWCO) and dialyzed against 200x the sample volume of 1x PBS at room temperature for 2 hours with gentle agitation. The PBS was refreshed, and the composition was further dialyzed for at least 14 hours at 4°C with gentle agitation. The dialyzed composition was then collected and concentrated by centrifugation at 2000 x g using an Amicon Ultra centrifugal filter (100k MWCO). The concentrated particles were characterized for size, polydispersity, and particle concentration using a Zetasizer Ultra (Malvern Panalytical) and for mRNA encapsulation efficiency using a Quant-iT RiboGreen RNA Assay Kit (ThermoFisher Scientific).

[0409] For pKa measurements, the TNS assay was performed as described by Sabnis et al., Molecular Therapy, 26(6):1509-19, which is incorporated herein by reference in its entirety. Briefly, 20 buffer solutions (10 mM sodium phosphate, 10 mM sodium borate, 10 mM sodium citrate, and 150 mM sodium chloride in distilled water) with unique pH values ​​ranging from 3.0 to 12.0 were prepared using 1 M sodium hydroxide and 1 M hydrochloric acid. 3.25 μL of LNP composition (0.04 mg / mL mRNA in PBS) was incubated with 2 μL of TNS reagent (0.3 mM in DMSO) and 90 μL of buffer solution (described above) at each pH value in a 96-well black-walled plate. Each pH condition was performed in triplicate wells. TNS fluorescence was measured using a Biotek Cytation Plate reader at excitation / emission wavelengths of 321 / 445 nm. The fluorescence values ​​were then plotted and fitted using a four-parameter sigmoidal curve. From the fit, the pH value resulting in half-maximal fluorescence was calculated and reported as the apparent LNP pKa value.

[0410] Particle characterization data for each exemplary lipid nanoparticle composition (LNP 2230, LNP 2231, LNP 2260, LNP 2270) is provided in the table below. [Table 4]

[0411] Example 7. In vivo bioluminescence imaging Exemplary lipid nanoparticle compositions (LNP 2230, LNP 2231, LNP 2260, LNP 2270) and comparative lipid nanoparticle compositions (LNP C12-200 and LNP MC3) prepared according to Example 6 and loaded with mRNA (EPO) were used in this example.

[0412] Bioluminescence screening Eight- to nine-week-old female Balb / c mice were used in this bioluminescence-based ionized lipid screening approach. Mice were obtained from Jackson Laboratories (JAX Stock: 000651) and allowed to acclimate for one week before treatment. After placing the animals under a heat lamp for several minutes, they were introduced into a restraining chamber. The tail was wiped with an alcohol pad (Fisher Scientific), and for each of the LNP compositions described above, 100 μL of lipid nanoparticle composition containing 10 μg of total mRNA (5 μg Fluc + 5 μg EPO) was intravenously injected using a 29G insulin syringe (Covidien). Four to six hours after administration, the animals were injected with 200 μL of 15 mg / mL D-luciferin (GoldBio) and placed in the nose cone of an IVIS Lumina LT imaging system (PerkinElmer). LivingImage software was used for imaging. Whole-body bioluminescence was captured by automated exposure, after which the animals were removed from the IVIS and placed in a CO2 chamber for euthanasia. After being placed in dorsal recumbency, each animal underwent cardiac puncture and blood was collected using a 25G insulin syringe (BD). Once all blood samples were collected, the tubes were spun at 2000G for 10 minutes using a tabletop centrifuge, and the plasma was aliquoted into individual Eppendorf tubes (Fisher Scientific) and stored at -80°C for subsequent EPO quantification. Plasma EPO levels were determined using an EPO MSD kit (Meso Scale Diagnostics).

[0413] hEPO MSD measurement Reagents used to measure hEPO levels included: MSD Wash Buffer (#R61AA-1) MSD EPO Kit (#K151VXK-2) oMSD GOLD 96 Small Spot Streptavidin Plate o Diluent 100 o Diluent 3 o Diluent 43 o Calibrator 9 o Capture Ab o Detection Ab oMSD GOLD reading buffer B

[0414] General Procedure: Plates were coated. 200 μL of biotinylated capture antibody was added to 3.3 mL of Diluent 100 and mixed by vortexing. 25 μL of the above solution was added to each well of the provided MSD GOLD Small Spot Streptavidin Plate. The plate was sealed with an adhesive plate seal and incubated with shaking at room temperature for 1 hour or at 2-8°C overnight. The plate was washed three times with at least 150 μL / well of 1X MSD Wash Buffer.

[0415] Preparation of Calibrator Standards. Calibrator vials were allowed to reach room temperature. Each vial of calibrator was reconstituted by adding 250 μL of Diluent 43 to the glass vial to obtain a 5x concentrated calibrator stock. The reconstituted calibrator was inverted at least three times, equilibrated at room temperature for 15-30 minutes, and then briefly vortexed. Calibrator Standard 1 was prepared by adding 50 μL of reconstituted calibrator to 200 μL of Diluent 43 and vortexing. Calibrator Standard 2 was prepared by adding 75 μL of Calibrator Standard 1 to 225 μL of Diluent 43 and vortexing. The four-fold serial dilution was repeated five more times to generate a total of seven calibrator standards, with mixing by vortexing between each serial dilution. Diluent 43 was used as Calibrator Standard 8 (zero calibrator).

[0416] Addition of samples and calibrators. Add 25 μL of Diluent 43 to each well. Add 25 μL of prepared calibrator standard or sample to each well. Seal the plate with adhesive plate seals and incubate at room temperature with shaking for 1 hour.

[0417] Preparation and addition of detection antibody solution. The detection antibody solution was provided as a 100x stock solution. The working solution was 1x. 60 μL of the provided 100x detection antibody was added to 5940 μL of Diluent 3. The plate was washed three times with at least 150 μL / well of 1x MSD Wash Buffer. 50 μL of the detection antibody solution prepared above was added to each well. The plate was sealed with an adhesive plate seal and incubated at room temperature with shaking for 1 hour.

[0418] Sample reading: Plates were washed three times with at least 150 μL / well of 1× MSD wash buffer. 150 μL of MSD GOLD read buffer B was added to each well. Plates were analyzed on the MSD instrument to read EPO levels.

[0419] The average radiance levels determined by in vivo bioluminescence imaging for each exemplary lipid nanoparticle composition (LNP 2230, LNP 2231, LNP 2260, LNP 2270) are shown in the table below. [Table 5]

[0420] The spleen:liver ratio of mean radiance was determined for the exemplary lipid nanoparticle compositions (LNP 2230, LNP2231) compared to the comparative lipid nanoparticle compositions (LNP C12-200, LNP MC3), and the results are shown in Figure 1. As shown in the figure, the exemplary lipid nanoparticle compositions (LNP 2230, LNP 2231) exhibited significantly higher spleen-to-liver ratios than the comparative lipid nanoparticle compositions (LNP C12-200, LNP MC3) (>>1 vs <<0.1), indicating that instead of the typical delivery primarily via the liver exhibited by the comparative lipid nanoparticle compositions, the exemplary lipid nanoparticle compositions exhibited surprisingly high delivery to the spleen in addition to liver delivery.

[0421] Thus, lipid nanoparticles using the novel ionized lipids described herein demonstrate selective delivery of therapeutic cargo outside the liver and are expected to have low hepatotoxicity due to low hepatic lipid levels.

[0422] Example 8. Synthesis of exemplary ionizable lipid compounds 8.1. Synthesis of Compound 2290 [ka] Step 1: To a solution of 8-bromooctanoic acid (4.35 g, 19.50 mmol, 1 equiv.) and heptadecan-9-ol (5 g, 19.50 mmol, 1 equiv.) in DCM (100 mL) was added EDCI (4.48 g, 23.39 mmol, 1.2 equiv.) and DMAP (1.19 g, 9.75 mmol, 0.5 equiv.). The mixture was stirred at 15 °C for 8 h. The reaction mixture was quenched by adding 200 mL of HO at 15 °C and then extracted with 600 mL (200 mL × 3) of EtOAc. The combined organic layers were washed with 400 mL (200 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 20 / 1) to give 1-octylnonyl 8-bromooctanoate (35 g, 75.83 mmol, yield 97.24%) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.84-4.90(m,1H),3.41(t,J=6.8Hz,2H),2.29(t,J=7.6Hz,2H),1.82-1 .88(m,2H),1.62-1.65(m,2H),1.42-1.52(m,6H),1.25-1.36(m,28H),0.89(t,J=7.2Hz,6H).

[0423] Step 2: A mixture of 1-octylnonyl 8-bromooctanoate (1 g, 2.17 mmol, 1.2 equiv.), (2S)-1-tert-butoxycarbonyl-4-hydroxy-pyrrolidine-2-carboxylic acid (417.51 ​​mg, 1.81 mmol, 1 equiv.), and CsCO (1.29 g, 3.97 mmol, 2.2 equiv.) in DMF (10 mL) was degassed and purged with N three times, and then the mixture was stirred under N atmosphere at 15 °C for 8 h. The reaction mixture was quenched by adding 50 mL of HO at 15 °C and then extracted with 150 ml of EtOAc (50 mL × 3). The combined organic layers were washed with 100 mL (50 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 20 / 1 to 3 / 1) to give O1-tert-butyl O2-[8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (4.55 g, 7.44 mmol, 82.37% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.84-4.90(m,1H),4.18-4.52(m,3H),4.06-4.10(m,1H),3.42-3.72 (m,2H),2.21-2.39(m,3H),2.07-2.11(m,1H),1.25-1.67(m,48H),0.88(t,J=6.8Hz,6H).

[0424] Step 3: To a solution of O1-tert-butyl O2-[8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (4.5 g, 7.35 mmol, 1 equiv.) in DCM (30 mL) was added TFA (23.10 g, 202.59 mmol, 15 mL, 27.55 equiv.). The mixture was stirred at 15 °C for 3 h. The reaction mixture was quenched by adding 60 mL of aqueous NaHCO3 at 15 °C, and then extracted with 150 mL of EtOAc (50 mL × 3). The combined organic layers were washed with 100 mL of brine (50 mL×2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxypyrrolidine-2-carboxylate (3.76 g, 7.35 mmol, 100.00% yield) as a colorless oil.

[0425] Step 4: To a solution of [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxypyrrolidine-2-carboxylate (2 g, 3.91 mmol, 1 equiv.) and undecyl 6-bromohexanoate (1.64 g, 4.69 mmol, 1.2 equiv.) in DMF (40 mL) was added KCO (1.62 g, 11.72 mmol, 3 equiv.) and KI (324.37 mg, 1.95 mmol, 0.5 equiv.). The mixture was stirred at 50 °C for 8 h. The reaction mixture was quenched by adding 50 mL of HO at 15 °C and extracted with 150 mL of EtOAc (50 mL × 3). The combined organic layers were washed with 100 mL of brine (50 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 20 / 1 to 1 / 1) to give [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (1.6 mg, 2.05 μmol, 52.48% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.86-4.90(m,1H),4.24-4.53(m,1H),4.04-4.15(m,4H),2.99-3.69(m,2H),1 .84-2.84(m,8H),1.59-1.68(m,8H),1.45-1.54(m,6H),1.15-1.44(m,50H),0.89(t,J=7.8Hz,9H). LCMS:(M+H + ):780.4@13.579 minutes.

[0426] Step 5: To a solution of 3-pyrrolidin-1-ylpropanoic acid (100 mg, 698.41 μmol, 1 equiv.) in DCM (5 mL) was added (COCl) (443.23 mg, 3.49 mmol, 305.68 μL, 5 equiv.) and DMF (5.10 mg, 69.84 μmol, 5.37 μL, 0.1 equiv.). The mixture was stirred at 15° C. for 2 h. The reaction mixture was concentrated under reduced pressure to give 3-pyrrolidin-1-ylpropanoyl chloride (138 mg, 696.65 μmol, 99.75% yield, HCl) as a yellow solid.

[0427] Step 6: To a suspension of [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (200 mg, 256.34 μmol, 1 equiv.), TEA (77.82 mg, 769.03 μmol, 107.04 μL, 3 equiv.), and DMAP (15.66 mg, 128.17 μmol, 0.5 equiv.) in DCM (3 mL) was added [3-pyrrolidin-1-ylpropanoyl chloride (126.95 mg, 640.85 μmol, 2.5 equiv., HCl) in DCM (1 mL) dropwise at 15° C. The mixture was stirred at 15° C. for 2 h under a N atmosphere. The reaction mixture was quenched by adding 10 mL of saturated NaHCO3 at 15 °C, and then extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 20 mL (10 mL × 2) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was analyzed by preparative TLC (SiO 2、 Purification with EtOAc:MeOH=10:1) gave [8-(1-octylnonoxy)-8-oxo-octyl](2S)-1-(6-oxo-6-undecoxy-hexyl)-4-(3-pyrrolidin-1-ylpropanoyloxy)pyrrolidine-2-carboxylate (100 mg, 108.24 μmol, 49.00% yield, 98% purity) as a colorless oil. 1 H NMR(400MHz,CDCl3),5.19-5.31(m,1H),4.84-4.89(m,1H),4.04-4.15(m,4H),3.43-3.55(m,1H),3.09-3.26(m,1H),2.49-2.81(m,1 0H),2.24-2.36(m,5H),1.95-2.22(m,1H),1.80(s,3H),1.59-1.68(m,8H),1.44-1.54(m,6H),1.12-1.42(m,50H),0.85-0.93(m,9H). LCMS: (M+H + ):905.4@1.950 / 2.035 minutes.

[0428] 8.2. Synthesis of Compound 2291 [ka] Step 1: To a suspension of [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (1 g, 1.28 mmol, 1 equiv.), TEA (648.47 mg, 6.41 mmol, 891.99 µL, 5 equiv.), and DMAP (78.29 mg, 640.85 µmol, 0.5 equiv.) in DCM (9 mL) was added dropwise a solution of prop-2-enoyl chloride (464.02 mg, 5.13 mmol, 418.04 µL, 4 equiv.) in DCM (3 mL). The mixture was stirred at 15 °C under a N atmosphere for 3 h. The reaction mixture was quenched by adding 10 mL of HO at 15 °C and then extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 20 mL (10 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 20 / 1 to 8 / 1) to give [8-(1-octylnonoxy)-8-oxo-octyl](2S)-1-(6-oxo-6-undecoxy-hexyl)-4-prop-2-enoyloxy-pyrrolidine-2-carboxylate (300 mg, 359.60 μmol, 28.06% yield) as a colorless oil.

[0429] Step 2: A mixture of [8-(1-octylnonoxy)-8-oxo-octyl](2S)-1-(6-oxo-6-undecoxy-hexyl)-4-prop-2-enoyloxy-pyrrolidine-2-carboxylate (300 mg, 359.60 μmol, 1 equiv.), 2-(methylamino)ethanol (27.01 mg, 359.60 μmol, 28.89 μL, 1 equiv.) in toluene (3 mL) was degassed and purged with N three times, then the mixture was stirred under N atmosphere at 90° C. for 8 h. The reaction mixture was concentrated under reduced pressure to provide a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 100 × 30 mm × 5 μm; mobile phase: [water (HCl) to ACN]; B%: 45% to 75%, 10 min) to give a solution. The solution was adjusted to approximately pH 7 with saturated NaHCO3 and extracted with EtOAc (20 mL × 3). The organic layer was washed with brine (20 mL × 2), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-[3-[2-hydroxyethyl(methyl)amino]propanoyloxy]-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (30 mg, 32.59 μmol, 9.06% yield, 98.8% purity) as a colorless oil. 1 H NMR(400MHz,CDCl3),5.15-5.31(m,1H),4.81-4.93(m,1H),4.01-4.18(m,4H),3.39-3.72(m,3H),3.04-3.3 1(m,1H),1.97-2.88(m,18H),1.59-1.66(m,8H),1.47-1.55(m,6H),1.23-1.38(m,48H),0.84-0.95(m,9H). LCMS:(M+H + ):909.7@9.772 minutes.

[0430] 8.3. Synthesis of Compound 2292 [ka] Step 1: To a solution of 3-(dimethylamino)propanoic acid (100 mg, 651.01 mmol, 1 equiv., HCl) in DCM (5 mL) was added (COCl) (354.59 mg, 2.79 mmol, 244.55 μL, 4 equiv.) and DMF (5.10 mg, 69.84 μmol, 5.37 μL, 0.1 equiv.). The mixture was stirred at 15 °C for 2 h. The mixture was concentrated under reduced pressure to give 3-(dimethylamino)propanoyl chloride (112 mg, 650.96 μmol, 99.99% purity, HCl) as a yellow solid.

[0431] Step 2: To a solution of [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (200 mg, 256.34 μmol, 1 equiv.) in DCM (10 mL) was added TEA (129.69 mg, 1.28 mmol, 178.40 μL, 5 equiv.) and 3-(dimethylamino)propanoyl chloride (112 mg, 650.96 μmol, 2.54 equiv., HCl). ℃ The mixture was stirred at 15°C for 3 hours. The reaction mixture was quenched by adding 10 mL of NaHCO3 at 15°C and then extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 20 mL (10 mL × 2) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO2, EtOAc:MeOH = 10:1) to give [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-[4-(dimethylamino)butanoyloxy]-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (33 mg, 36.42 μmol, 14.7% yield, 98.6% purity) as a colorless oil. 1H NMR(400MHz,CDCl3),5.11-5.34(m,1H),4.84-4.90(m,1H),4.03-4.14(m,4H),3.43-3.54(m,1H),3.09-3.25(m,1H), 2.03-2.77(m,19H),1.77-1.82(m,2H),1.60-1.65(m,8H),1.49-1.52(m,6H),1.27-1.34(m,48H),0.87-0.90(m,9H). LCMS:(M / 2+1):893.4@10.022 minutes.

[0432] 8.4. Synthesis of Compound 2293 [ka] Step 1: A solution of (2S)-1-tert-butoxycarbonyl-4-hydroxy-pyrrolidine-2-carboxylic acid (10 g, 43.24 mmol, 1 equiv) in MeOH (50 mL) and HO (20 mL) was adjusted to pH = 7.0 with dicesium carbonate (8.45 g, 25.95 mmol, 0.6 equiv). The mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in DMF (100 mL), and BnBr (7.40 g, 43.24 mmol, 5.14 mL, 1 equiv) was added at 25 °C. The mixture was degassed and purged with N three times, then stirred under N atmosphere at 25 °C for 8 h. The reaction mixture was diluted with 50 mL of HO and extracted with 300 mL (100 mL × 3) of EtOAc. The combined organic layers were washed with 100 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 8 / 1 to 2 / 1) to give O2-benzyl O1-tert-butyl (2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (13 g, 40.45 mmol, 93.54% yield) as a colorless oil. 1H NMR(400MHz,CDCl3),7.31-7.40(m,5H),4.99-5.16(m,3H),4.23-4.29(m,2H),3.35-3. 55(m,1H),3.10-3.30(m,1H),2.10-2.40(m,1H),1.80-1.90(m,1H),1.20-1.40(m,9H).

[0433] Step 2: To a solution of O2-benzyl O1-tert-butyl(2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (13 g, 40.45 mmol, 1 equiv) in EtOAc (80 mL) was added HCl / EtOAc (4 M, 80 mL, 7.91 equiv) dropwise at 20 °C. The mixture was stirred at 25 °C under a N2 atmosphere for 4 h. The reaction mixture was filtered, and the residue was collected and concentrated under reduced pressure to give benzyl (2S)-4-hydroxypyrrolidine-2-carboxylate (9 g, 34.92 mmol, 86.33% yield, HCl) as a white solid.

[0434] Step 3: To a solution of (2S)-4-hydroxypyrrolidine-2-carboxylate (1 g, 3.88 mmol, 1 equiv., HCl) in DMF (70 mL) was added DIEA (1.00 g, 7.76 mmol, 1.35 mL, 2 equiv.) at 25 °C and stirred for 0.5 h under a N atmosphere. To the mixture, undecyl 6-bromohexanoate (1.36 g, 3.88 mmol, 1 equiv.) and KI (128.83 mg, 776.06 μmol, 0.2 equiv.) were added and stirred for 8 h at 50 °C under a N atmosphere. The reaction mixture was diluted with 50 mL of HO and extracted with 300 mL (150 mL × 2) of EtOAc. The combined organic layers were washed with 200 mL (100 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 20 / 1 to 5 / 1) to give benzyl (2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (2.8 mg, 5.72 mmol, 49.12% yield) as a colorless oil. 1H NMR(400MHz,CDCl3),7.25-7.55(m,5H),5.14-5.25(m,2H),4.25-4.48(m,1H),4.05(t,J=6.8Hz,2H),3.15-3.65(m,2H),2.55-2.75(m ,1H),2.40-2.55(m,1H),2.30-2.40(m,1H),2.20-2.30(m,2H),2.05-2.15(m,1H),1.90-2. 00(m,1H),1.55-1.75(m,4H),1.40-1.50(m,2H),1.20-1.35(m,18H),0.89(t,J=6.4Hz,3H).

[0435] Step 4: A mixture of methyl 8-chloro-8-oxo-octanoate (3.22 g, 15.60 mmol, 2.21 mL, 1 equiv.), heptadecan-9-ol (4 g, 15.60 mmol, 1 equiv.), and pyridine (1.23 g, 15.60 mmol, 1.26 mL, 1 equiv.) in THF (20 mL) was stirred at 70 °C under a N atmosphere for 6 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 20 / 1) to give O1-methyl O8-(1-octylnonyl)octanedioate (3.6 g, 8.44 mmol, 54.10% yield) as a colorless oil.

[0436] Step 5: To a solution of O1-methyl O8-(1-octylnonyl)octanedioate (3.6 g, 8.44 mmol, 1 equiv.) in THF (15 mL) was added LiOH.HO (424.88 mg, 10.12 mmol, 1.2 equiv.) in HO (1 mL) dropwise at 25 °C. The mixture was stirred at 25 °C for 4 h under a N2 atmosphere. The reaction mixture was diluted with 500 mL of HO and extracted with 60 mL of EtOAc (30 mL × 2). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=10 / 1 to 5 / 1) to give 8-(1-octylnonoxy)-8-oxo-octanoic acid (2.1 g, 5.09 mmol, 60.32% yield) as a colorless oil.

[0437] Step 6: To a solution of 8-(1-octylnonoxy)-8-oxo-octanoic acid (2.1 g, 5.09 mmol, 1 equiv) in DCM (25 mL) was added (COCl) (3.23 g, 25.45 mmol, 2.23 mL, 5 equiv) and DMF (37.20 mg, 508.91 μmol, 39.15 μL, 0.1 equiv) dropwise at 0 °C. The mixture was stirred at 25 °C under a N atmosphere for 2 h. The reaction mixture was concentrated under reduced pressure to give the crude product 1-octylnonyl 8-chloro-8-oxo-octanoate (2.3 g, crude) as a colorless oil, which was used in the next step without further purification.

[0438] Step 7: A mixture of (2S)-4-hydroxy-1-(6-oxo-6-undecoxyhexyl)pyrrolidine-2-carboxylate (0.4 g, 816.85 μmol, 1 equiv.), 1-octylnonyl 8-chloro-8-oxo-octanoate (704.27 mg, 1.63 mmol, 2 equiv.), and pyridine (64.61 mg, 816.85 μmol, 65.93 μL, 1 equiv.) in THF (15 mL) was stirred at 60° C. under a N atmosphere for 8 h. The reaction mixture was filtered, and the filtrate was diluted with 10 mL of HO and then extracted with 60 mL (30 mL × 2) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 10 / 1) to give O1-[(5S)-5-benzyloxycarbonyl-1-(6-oxo-6-undecoxy-hexyl)pyrrolidin-3-yl]O8-(1-octylnonyl)octanedioate (330 mg, 373.17 mmol, 45.68% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3),7.31-7.39(m,5H),5.14-5.25(m,3H),4.85-4.89(m,1H),4.06(t,J=6.8Hz,2H),3.10-3.55(m,2H), 2.20-2.75(m,11H),2.05-2.20(m,1H),1.58-1.75(m,6H),1.40-1.55(m,6H),1.20-1.35(m,48H),0.89(t,J=6.4Hz,9H).

[0439] Step 8: To a solution of Pd / C (500 mg, 10% purity) in EtOAc (400 mL) was added O1-[(5S)-5-benzyloxycarbonyl-1-(6-oxo-6-undecoxy-hexyl)pyrrolidin-3-yl]O8-(1-octylnonyl)octanedioate (500 mg, 565.41 μmol, 1 equiv.). The mixture was stirred under H2 at 15 Psi at 25 °C for 5 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give (2S)-4-[8-(1-octylnonoxy)-8-oxo-octanoyl]oxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylic acid (350 mg, crude) as a colorless oil.

[0440] Step 9: To a solution of (2S)-4-[8-(1-octylnonoxy)-8-oxo-octanoyl]oxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylic acid (200 mg, 251.83 μmol, 1 equiv.) and CsCO (164.10 mg, 503.66 μmol, 2 equiv.) in DMF (5 mL), 2-bromo-N,N-dimethyl-ethanamine (45.94 mg, 302.19 μmol, 1.2 equiv.) was added. The mixture was stirred at 25 °C for 8 h. The mixture was added to HO (10 mL) and extracted with EtOAc (10 mL × 3). The organic layer was washed with brine (10 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) and preparative TLC (SiO, ethyl acetate:MeOH = 1:0 with 1% NH.H.sub.2O) to give O1-[(5S)-5-[2-(dimethylamino)ethoxycarbonyl]-1-(6-oxo-6-undecoxy-hexyl)pyrrolidin-3-yl] O8-(1-octylnonyl)octanedioate (125 mg, 143.01 μmol, 56.79% yield, 99% purity) as a colorless oil. 1H NMR(400MHz,CDCl3),5.14-5.25(m,1H),4.85-4.89(m,1H),4.23-4.28(m,2H),4.05(t,J=6.8Hz,2H),3.10-3.60(m,2H), 2.25-2.80(m,18H),2.00-2.10(m,1H),1.58-1.65(m,8H),1.40-1.55(m,6H),1.20-1.35(m,46H),0.89(t,J=6.4Hz,9H). LCMS: (M+H + ):865.7@10.196 / 10.709 minutes.

[0441] 8.5. Synthesis of Compound 2294 [ka] Step 1: To a solution of heptadecan-9-ol (10 g, 38.99 mmol, 1 equiv.) and 7-bromoheptanoic acid (8.82 g, 42.17 mmol, 1.08 equiv.) in DCM (100 mL) was added DMAP (2.38 g, 19.50 mmol, 0.5 equiv.) and EDCI (8.97 g, 46.79 mmol, 1.2 equiv.). The mixture was stirred at 20 °C for 8 h. The mixture was added to HO (200 mL) and extracted with EtOAc (50 mL × 3). The organic layer was washed with brine (50 mL × 2), dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 5 / 1) to give 1-octylnonyl-7-bromoheptanoate (12 g, 26.81 mmol, yield 68.77%) as a colorless oil.

[0442] Step 2: To a solution of (2S)-1-tert-butoxycarbonyl-4-hydroxy-pyrrolidine-2-carboxylic acid (950 mg, 4.11 mmol, 1 equiv.) and 1-octylnonyl 7-bromoheptanoate (2.02 g, 4.52 mmol, 1.1 equiv.) in DMF (50 mL) was added CsCO (2.94 g, 9.04 mmol, 2.2 equiv.). The mixture was stirred at 20 °C for 8 h. The mixture was added to HO (50 mL) and extracted with EtOAc (20 mL × 3). The organic layer was washed with brine (20 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 5 / 1) to give O1-tert-butyl O2-[7-(1-octylnonoxy)-7-oxo-heptyl](2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (1.5 g, 2.51 mmol, 61.07% yield) as a colorless oil.

[0443] Step 3: A solution of O1-tert-butyl O2-[9-(1-octylnonoxy)-9-oxo-nonyl](2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (1.5 g, 2.40 mmol, 1 equiv.) in DCM (30 mL) and TFA (6.93 g, 60.78 mmol, 4.50 mL, 25.36 equiv.) was added to 20 mL of HCl. ℃ The mixture was stirred at rt for 2 h. The mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in EtOAc (20 mL), and the organic layer was washed with saturated NaHCO (50 mL × 4), brine (20 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure to give [9-(1-octylnonoxy)-9-oxo-nonyl](2S)-4-hydroxypyrrolidine-2-carboxylate (1 g, 1.90 mmol, 79.36% yield) as a colorless oil.

[0444] Step 4: To a solution of [7-(1-octylnonoxy)-7-oxo-heptyl](2S)-4-hydroxypyrrolidine-2-carboxylate (0.5 g, 1.00 mmol, 1 equiv.), KCO (416.49 mg, 3.01 mmol, 3 equiv.), and KI (83.38 mg, 502.26 μmol, 0.5 equiv.) in DMF (20 mL) was added undecyl 6-bromohexanoate (386.02 mg, 1.10 mmol, 1.1 equiv.). The mixture was stirred at 50 °C for 8 h. The mixture was added to HO (20 mL) and extracted with EtOAc (20 mL × 3). The organic layer was washed with brine (20 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 5 / 1) and preparative HPLC (column: Phenomenex Luna C18 100 × 30 mm × 5 μm; mobile phase: [water (HCl) to ACN]; B%: 55% to 85%, 10 min) to give a solution. Saturated NaHCO3 was added to the solution until the pH reached approximately 7, and the solution was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine (20 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give [7-(1-octylnonoxy)-7-oxo-heptyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (1 g, 1.31 mmol, 64.96% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.84-4.91(m,1H),4.25-4.55(m,1H),4.04-4.20(m,4H),3.05-3.75(m,2H),1 .91-2.85(m,9H),1.60-1.70(m,8H),1.45-1.55(m,6H),1.20-1.40(m,46H),0.89(t,J=6.4Hz,9H). LCMS:(M+H + ):766.4@13.405 minutes.

[0445] Step 5: To a solution of 3-(dimethylamino)propanoic acid (300 mg, 1.95 mmol, 1 equiv., HCl) in DCM (5 mL) was added (COCl) (991.60 mg, 7.81 mmol, 683.86 μL, 4 equiv.) and DMF (14.27 mg, 195.30 μmol, 15.03 μL, 0.1 equiv.) and stirred at 20° C. for 2 h. The mixture was concentrated under reduced pressure to give 3-(dimethylamino)propanoyl chloride (336 mg, 1.95 mmol, 99.99% yield, HCl) as a yellow solid.

[0446] Step 6: To a solution of [7-(1-octylnonoxy)-7-oxo-heptyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (200 mg, 261.03 μmol, 1 equiv.), DMAP (15.95 mg, 130.52 μmol, 0.5 equiv.), and TEA (132.07 mg, 1.31 mmol, 181.66 μL, 5 equiv.) in DCM (10 mL) was added 3-(dimethylamino)propanoyl chloride (112.00 mg, 650.96 μmol, 2.49 equiv., HCl) under N2. ℃The mixture was then stirred at 20°C for 1 hour. The mixture was added to saturated NaHCO3 (20 mL) and extracted with EtOAc (10 mL x 3). The organic layer was washed with brine (10 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) and then preparative HPLC (column: Phenomenex Luna C18 100 x 30 mm x 5 μm; mobile phase: [water (HCl) to ACN]; B%: 45% to 75%, 10 min) to obtain a solution. Saturated NaHCO3 was added to the solution until the pH reached approximately 7, and the solution was extracted with EtOAc (20 mL x 3). The organic layer was washed with brine (20 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure to give [7-(1-octylnonoxy)-7-oxo-heptyl](2S)-4-[3-(dimethylamino)propanoyloxy]-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (70 mg, 80.90 μmol, 30.99% yield, 100% purity) as a yellow oil. 1 H NMR(400MHz,CDCl3),5.20-5.28(m,1H),4.83-4.90(m,1H),4.03-4.15(m,4H),3.43-3.55(m,1H),3.09-3.2 7(m,1H),2.00-2.80(m,17H),1.55-1.70(m,8H),1.45-1.55(m,6H),1.20-1.40(m,48H),0.86-0.90(m,9H). LCMS:(M+H + ):865.4@9.871 / 9.920 minutes.

[0447] 8.6. Synthesis of Compound 2295 [ka] Step 1: To a solution of heptadecan-9-ol (10 g, 38.99 mmol, 1 equiv.) and 9-bromononanoic acid (10 g, 42.17 mmol, 1.08 equiv.) in DCM (100 mL) was added DMAP (2.38 g, 19.50 mmol, 0.5 equiv.) and EDCI (8.97 g, 46.79 mmol, 1.2 equiv.). The mixture was stirred at 20 °C for 8 h. The mixture was added to HO (200 mL) and extracted with EtOAc (100 mL × 3). The organic layer was washed with brine (100 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 5 / 1) to give 1-octylnonyl 9-bromononanoate (15 g, 31.54 mmol, yield 80.89%) as a colorless oil.

[0448] Step 2: To a solution of (2S)-1-tert-butoxycarbonyl-4-hydroxy-pyrrolidine-2-carboxylic acid (5 g, 21.62 mmol, 1 equiv.) and 1-octylnonyl 9-bromononanoate (12.34 g, 25.95 mmol, 1.2 equiv.) in DMF (100 mL) was added CsCO (15.50 g, 47.57 mmol, 2.2 equiv.). The mixture was stirred at 20 °C for 8 h. The mixture was added to HO (200 mL) and extracted with EtOAc (100 mL × 3). The organic layer was washed with brine (100 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 5 / 1) to give O1-tert-butyl O2-[9-(1-octylnonoxy)-9-oxo-nonyl](2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (10 g, 15.98 mmol, 73.89% yield) as a colorless oil. 1H NMR(400MHz,CDCl3),4.85-4.89(m,1H),4.05-4.55(m,4H),3.40-3.80(m,2H),2.25-2.40(m,3H),2 .05-2.15(m,1H),1.60-1.75(m,4H),1.40-1.60(m,14H),1.20-1.35(m,32H),0.88(t,J=6.4Hz,6H).

[0449] Step 3: A solution of O1-tert-butyl O2-[9-(1-octylnonoxy)-9-oxo-nonyl](2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (10 g, 15.98 mmol, 1 equiv.) in DCM (60 mL) and TFA (57.75 g, 506.48 mmol, 37.50 mL, 31.70 equiv.) was added to 20 mL of HCl. ℃ The mixture was stirred at rt for 2 hours. The mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in EtOAc (100 mL), washed with saturated NaHCO (200 mL × 2) and brine (200 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure to give [9-(1-octylnonoxy)-9-oxo-nonyl](2S)-4-hydroxypyrrolidine-2-carboxylate (7 g, crude) as a yellow oil.

[0450] Step 4: To a solution of [9-(1-octylnonoxy)-9-oxo-nonyl](2S)-4-hydroxypyrrolidine-2-carboxylate (2 g, 3.80 mmol, 1 equiv.), K2CO3 (1.58 g, 11.41 mmol, 3 equiv.), and KI (315.71 mg, 1.90 mmol, 0.5 equiv.) in DMF (100 mL) was added undecyl 6-bromohexanoate (1.59 g, 4.56 mmol, 1.2 equiv.). The mixture was stirred at 50 °C for 8 h. The mixture was added to HO (20 mL) and extracted with EtOAc (20 mL × 3). The organic layer was washed with brine (20 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 5 / 1) and then by preparative HPLC (column: Phenomenex Luna C18 100 × 30 mm × 5 μm; mobile phase: [water (HCl) to ACN]; B%: 55% to 80%, 10 min) to give [9-(1-octylnonoxy)-9-oxo-nonyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (2 g, 2.52 mmol, 66.20% yield) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.85-4.90(m,1H),4.20-4.55(m,1H),4.00-4.15(m,4H),3.05-3.60(m,2H),1 .90-2.80(m,9H),1.55-1.75(m,8H),1.45-1.55(m,6H),1.20-1.40(m,50H),0.88(t,J=6.4Hz,9H).

[0451] Step 5: To a solution of 3-(dimethylamino)propanoic acid (100 mg, 651.01 mmol, 1 equiv., HCl) in DCM (5 mL) was added (COCl) (330.53 mg, 2.60 mmol, 227.95 μL, 4 equiv.) and DMF (4.76 mg, 65.10 μmol, 5.01 μL, 0.1 equiv.). The mixture was stirred at 20 °C for 2 h. The mixture was concentrated under reduced pressure to give 3-(dimethylamino)propanoyl chloride (112 mg, crude, HCl) as a yellow solid. Then, 3-(dimethylamino)propanoyl chloride (112 mg, 650.96 μmol, 2.59 equiv., HCl) was added to a solution of [9-(1-octylnonoxy)-9-oxo-nonyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (200 mg, 251.81 μmol, 1 equiv.), DMAP (15.38 mg, 125.91 μmol, 0.5 equiv.), and TEA (127.41 mg, 1.26 mmol, 175.25 μL, 5 equiv.) in DCM (5 mL) under N at 0° C., and the mixture was stirred at 20° C. for 1 h. The mixture was added to saturated NaHCO (20 mL) and extracted with EtOAc (10 mL × 3). The organic layer was washed with brine (10 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) and further purified by preparative TLC (SiO2, ethyl acetate / MeOH = 5:1 with 3% NH3.HO) to give [9-(1-octylnonoxy)-9-oxo-nonyl](2S)-4-[3-(dimethylamino)propanoyloxy]-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (100 mg, 108.58 μmol, 43.12% yield, 97% purity) as a yellow oil. 1H NMR(400MHz,CDCl3),5.20-5.29(m,1H),4.85-4.89(m,1H),4.03-4.18(m,4H),3.44-3.55(m,1H),3.09-3.2 6(m,1H),2.05-2.80(m,19H),1.60-1.65(m,8H),1.45-1.55(m,6H),1.18-1.40(m,50H),0.86-0.91(m,9H). LCMS:(M+H + ):893.5@10.397 / 10.417 minutes.

[0452] 8.7. Synthesis of Compound 2296 [ka] Step 1: To a solution of 5-bromopentan-1-ol (10 g, 59.86 mmol, 1 equiv.) and dodecanoic acid (12.59 g, 62.86 mmol, 1.05 equiv.) in DCM (100 mL) was added EDCI (22.95 g, 119.73 mmol, 2 equiv.) and DMAP (3.66 g, 29.93 mmol, 0.5 equiv.) at 0 °C. The mixture was stirred at 20 °C for 8 h. The reaction mixture was quenched by adding 200 mL of HO at 0 °C and then extracted with 300 mL (100 mL × 3) of EtOAc. The combined organic layers were washed with 300 mL of saturated brine (100 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 5 / 1) to give 5-bromopentyl dodecanoate (15 g, 42.94 mmol, yield 71.72%) as a white solid.

[0453] Step 2: To a solution of [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxypyrrolidine-2-carboxylate (800 mg, 1.56 mmol, 1 equiv.) in DMF (10 mL) was added K2CO3 (648.13 mg, 4.69 mmol, 3 equiv.) and KI (129.75 mg, 781.59 μmol, 0.5 equiv.). 5-Bromopentyl dodecanoate (600.70 mg, 1.72 mmol, 1.1 equiv.) was then added to the mixture. The mixture was stirred at 50 °C for 8 h. The reaction mixture was quenched by adding 20 mL of HO at 0 °C and then extracted with 60 mL of EtOAc (20 mL × 3). The combined organic layers were washed with 60 mL (20 mL × 3) of saturated brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) to give [8-(1-octylnonoxy)-8-oxo-octyl](2S)-1-(5-dodecanoyloxypentyl)-4-hydroxy-pyrrolidine-2-carboxylate (820 mg, 1.05 mmol, 67.23% yield) as a yellow oil.

[0454] Step 3: To a solution of 3-(dimethylamino)propanoic acid (400 mg, 2.60 mmol, 1 equiv., HCl) in DCM (10 mL) was added DMF (9.52 mg, 130.20 μmol, 10.02 μL, 0.05 equiv.) and (COCl) (396.63 mg, 3.12 mmol, 273.54 μL, 1.2 equiv.) at 0° C. The mixture was stirred at 0° C. for 2 h. The mixture was concentrated under reduced pressure to give 3-(dimethylamino)propanoyl chloride (450 mg, crude, HCl) as a yellow solid.

[0455] Step 4: To a solution of [8-(1-octylnonoxy)-8-oxo-octyl](2S)-1-(5-dodecanoyloxypentyl)-4-hydroxy-pyrrolidine-2-carboxylate (400 mg, 512.68 μmol, 1 equiv.) in DCM (10 mL), TEA (518.78 mg, 5.13 mmol, 713.59 μL, 10 equiv.) and 3-(dimethylamino)propanoyl chloride (352.83 mg, 2.05 mmol, 4 equiv., HCl) were added at 0° C. The mixture was stirred at 20° C. for 3 h. The reaction mixture was quenched by adding 10 mL of HO at 0° C. and then extracted with 30 mL of EtOAc (10 mL × 3). The combined organic layers were washed with 30 mL (10 mL × 3) of saturated brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 100 × 30 mm × 5 μm; mobile phase: [water (HCl) to ACN]; B%: 45% to 75%, 10 min) to obtain a solution. The solution was adjusted to pH 8 with saturated NaHCO3 and extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-[3-(dimethylamino)propanoyloxy]-1-(5-dodecanoyloxypentyl)pyrrolidine-2-carboxylate (108 mg, 51.58 μmol, 10.06% yield, 42% purity) as a colorless oil. 1 H NMR(400MHz,CDCl3),5.21-5.28(m,1H),4.84-4.88(m,1H),4.10-4.13(m,2H),4.06(t,J=6.8Hz,2H),3.12-3.55(m,2H), 1.97-2.35(m,7H),2.40-2.57(m,12H),1.60-1.65(m,6H),1.50-1.52(m,6H),1.26-1.38(m,50H),0.89(t,J=6.8Hz,9H). LCMS: (M+H + ):879.4@10.062 minutes.

[0456] 8.8. Synthesis of Compound 2297 [ka] Step 1: To a solution of 4-benzyloxybutanoic acid (2 g, 10.30 mmol, 1.82 mL, 1 equiv) in DCM (20 mL) was added DMF (15.05 mg, 205.95 μmol, 15.85 μL, 0.02 equiv) and (COCl) (1.57 g, 12.36 mmol, 1.08 mL, 1.2 equiv) at 0 °C. The mixture was stirred at 0 °C for 2 h. The mixture was concentrated under reduced pressure to give 4-benzyloxybutanoyl chloride (2.2 g, crude) as a white solid.

[0457] Step 2: To a solution of [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (1.5 g, 1.92 mmol, 1 equiv.) in DCM (20 mL) was added TEA (1.95 g, 19.23 mmol, 2.68 mL, 10 equiv.) and 4-benzyloxybutanoyl chloride (2.04 g, 9.61 mmol, 5 equiv.) at 0° C. The mixture was stirred at 20° C. for 3 h. The reaction mixture was quenched by adding 20 mL of HO at 0° C. and then extracted with 60 mL of EtOAc (20 mL × 3). The combined organic layers were washed with 60 mL (20 mL × 3) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate=20 / 1 to 1 / 1) to give [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-(4-benzyloxybutanoyloxy)-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (1.3 g, 1.36 mmol, 70.70% yield) as a yellow oil.

[0458] Step 3: To a solution of [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-(4-benzyloxybutanoyloxy)-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (600 mg, 627.34 μmol, 1 equiv.) in EtOAc (10 mL) was added Pd / C (0.3 g, 10% purity) and Pd(OH) / C (0.3 g, 427.25 μmol, 20% purity, 6.81 e-1 equiv.). The mixture was stirred under a H atmosphere (15 Psi) at 20° C. for 8 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 100 × 30 mm × 5 μm; mobile phase: [water (HCl) to ACN]; B%: 50% to 80%, 10 min). The mixture was then adjusted to pH = 8 with saturated NaHCO3 and extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-(4-hydroxybutanoyloxy)-1-(6-oxo-6-undecoxy-hexyl)pyrrolidine-2-carboxylate (191 mg, 220.48 μmol, 35.15% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3),5.15-5.28(m,1H),4.85-4.88(m,1H),4.03-4.12(m,4H),3.27-3.73(m,4H),2.26-2.72(m ,11H),1.86-1.91(m,2H),1.60-1.70(m,8H),1.45-1.55(m,6H),1.26-1.34(m,48H),0.89(t,J=5.2Hz,9H).(M+H + ):866.8. LCMS: (M+H + ):866.8@13.884 minutes.

[0459] 8.9. Synthesis of Compound 2298 [ka] Step 1: To a solution of 7-bromoheptan-1-ol (3.60 g, 18.46 mmol, 1.05 equiv.) and 2-octyldecanoic acid (5 g, 17.58 mmol, 1 equiv.) in DCM (100 mL) was added DMAP (1.07 g, 8.78 mmol, 0.5 equiv.) and EDCI (4.04 g, 21.10 mmol, 1.2 equiv.). The mixture was stirred at 20 °C for 8 h. The mixture was added to HO (200 mL) and extracted with EtOAc (200 mL × 3). The organic layer was washed with brine (200 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 5 / 1) to give 7-bromoheptyl 2-octyldecanoate (7 g, 15.17 mmol, crude) as a colorless oil.

[0460] Step 2: To a solution of (2S)-1-tert-butoxycarbonyl-4-hydroxy-pyrrolidine-2-carboxylic acid (3 g, 12.96 mmol, 1 equiv.) and 7-bromoheptyl 2-octyldecanoate (6.99 g, 15.18 mmol, 1.17 equiv.) in DMF (100 mL) was added CsCO (9.30 g, 28.53 mmol, 2.2 equiv.). The mixture was stirred at 20 °C for 8 h. The mixture was added to HO (20 mL) and extracted with EtOAc (20 mL × 3). The organic layer was washed with brine (20 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 5 / 1) to give O1-tert-butyl O2-[7-(2-octyldecanoyloxy)heptyl](2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (5 g, 8.17 mmol, 62.99% yield) as a colorless oil.

[0461] Step 3: A solution of O1-tert-butyl O2-[7-(2-octyldecanoyloxy)heptyl](2S)-4-hydroxypyrrolidine-1,2-dicarboxylate (5 g, 8.18 mmol, 1 equiv.) in DCM (30 mL) and TFA (23.10 g, 101.30 mmol, 15.02 mL, 24.79 equiv.) was added to 20 mL of HCl. ℃ The mixture was stirred at rt for 2 hours. The mixture was concentrated under reduced pressure to give a residue, which was dissolved in EtOAc (20 mL). The organic layer was washed with saturated NaHCO (50 mL × 4) and brine (20 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure to give 7-(2-octyldecanoyloxy)heptyl (2S)-4-hydroxypyrrolidine-2-carboxylate (4 g, crude) as a colorless oil.

[0462] Step 4: To a solution of 7-(2-octyldecanoyloxy)heptyl (2S)-4-hydroxypyrrolidine-2-carboxylate (2 g, 3.91 mmol, 1 equiv.), K2CO3 (1.62 g, 11.72 mmol, 3 equiv.), and KI (324.36 mg, 1.95 mmol, 0.5 equiv.) in DMF (20 mL) was added 5-bromopentyl dodecanoate (1.50 g, 4.30 mmol, 1.1 equiv.). The mixture was stirred at 50 °C for 8 h. The mixture was added to HO (20 mL) and extracted with EtOAc (20 mL × 3). The organic layer was washed with brine (20 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 5 / 1) and preparative HPLC (column: Phenomenex Luna C18 100 × 30 mm × 5 μm; mobile phase: [water (HCl) to ACN]; B%: 50% to 80%, 10 min) to give a solution. Saturated NaHCO3 was added until the solution reached pH = ~7, and the mixture was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine (20 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give 7-(2-octyldecanoyloxy)heptyl (2S)-1-(5-dodecanoyloxypentyl)-4-hydroxy-pyrrolidine-2-carboxylate (2 g, 2.56 mmol, 65.59% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.25-4.55(m,1H),3.95-4.25(m,6H),3.05-3.75(m,2H),1.80-2.85 (m,8H),1.60-1.70(m,8H),1.40-1.55(m,6H),1.23-1.40(m,48H),0.89(t,J=6.4Hz,9H). LCMS: (M+H + ):780.4@13.242&13.267 minutes.

[0463] Step 5: To a solution of 3-(dimethylamino)propanoic acid (300 mg, 1.95 mmol, 1 equiv., HCl) in DCM (5 mL) was added (COCl) (991.60 mg, 7.81 mmol, 683.86 μL, 4 equiv.) and DMF (14.27 mg, 195.30 μmol, 15.03 μL, 0.1 equiv.). The mixture was stirred at 20 °C for 2 h. The mixture was concentrated under reduced pressure to give 3-(dimethylamino)propanoyl chloride (336 mg, 1.95 mmol, 99.99% yield, HCl) as a yellow solid.

[0464] Step 6: To a solution of 7-(2-octyldecanoyloxy)heptyl (2S)-1-(5-dodecanoyloxypentyl)-4-hydroxy-pyrrolidine-2-carboxylate (350 mg, 448.60 μmol, 1 equiv.), DMAP (27.40 mg, 224.30 μmol, 0.5 equiv.), and TEA (226.97 mg, 2.24 mmol, 312.20 μL, 5 equiv.) in DCM (10 mL) was added 3-(dimethylamino)propanoyl chloride (294.00 mg, 1.71 mmol, 3.81 equiv., HCl) under N at 0 °C, and the mixture was stirred at 20 °C for 1 h. The mixture was added to saturated NaHCO (20 mL) and extracted with EtOAc (10 mL × 3). The organic layer was washed with brine (10 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) and then by preparative HPLC (column: Phenomenex Luna C18 100 × 30 mm × 5 μm; mobile phase: [water (HCl) to ACN]; B%: 50% to 80%, 10 min) to obtain a solution. Saturated NaHCO3 was added until the solution reached pH = ∼7, and the solution was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine (20 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure to give 7-(2-octyldecanoyloxy)heptyl(2S)-4-[3-(dimethylamino)propanoyloxy]-1-(5-dodecanoyloxypentyl)pyrrolidine-2-carboxylate (132 mg, 148.61 μmol, 45.40% yield, 99% purity) as a yellow oil. 1 H NMR(400MHz,CDCl3),5.20-5.30(m,1H),4.03-4.17(m,6H),3.25-3.55(m,1H),3.09-3.30(m,1H),2 .00-2.80(m,18H),1.57-1.70(m,8H),1.45-1.55(m,6H),1.15-1.40(m,48H),0.89(t,J=6.8Hz,9H). LCMS:(M+H + ):879.7@9.868 minutes.

[0465] 8.10. Synthesis of Compound 2304 [ka] Step 1: To a solution of undecan-1-ol (5 g, 29.02 mmol, 1 equiv.) and DMAP (3.55 g, 29.02 mmol, 1 equiv.) in DMF (10 mL) was added oxepane-2,7-dione (4.46 g, 34.82 mmol, 1.2 equiv.). The mixture was stirred at 20 °C for 8 h. The mixture was added to HO (20 mL) and extracted with EtOAc (20 mL × 3). The organic layer was washed with brine (20 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 5 / 1) to give 6-oxo-6-undecoxy-hexanoic acid (4 g, 13.31 mmol, 45.88% yield) as a white solid. 1 H NMR(400MHz, CDCl3), 4.07(t,J=6.8Hz,2H),2.33-2.41(m,4H),1.60-1.71(m,6H),1.19 - 1.41(m,17H),0.89(m,J=7.2Hz,3H).

[0466] Step 2: To a solution of 6-oxo-6-undecoxy-hexanoic acid (2 g, 6.66 mmol, 1 equiv.) in DCM (10 mL) was added (COCl) (2.53 g, 19.97 mmol, 1.75 mL, 3 equiv.) and DMF (4.87 mg, 66.57 μmol, 5.12 μL, 0.01 equiv.). The mixture was stirred at 20 °C for 2 h. The mixture was concentrated under reduced pressure to give undecyl 6-chloro-6-oxo-hexanoate (2.12 g, crude) as a white solid.

[0467] Step 3: To a solution of [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxypyrrolidine-2-carboxylate (3 g, 5.86 mmol, 1 equiv.), TEA (1.78 g, 17.59 mmol, 2.45 mL, 3 equiv.), and DMAP (71.61 mg, 586.20 μmol, 0.1 equiv.) in DCM (5 mL) was added undecyl 6-chloro-6-oxo-hexanoate (1.91 g, 5.99 mmol, 1.02 equiv.). The mixture was stirred at 20 °C for 5 h. The mixture was added to HO (20 mL) and extracted with EtOAc (20 mL × 3). The organic layer was washed with brine (20 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 5 / 1) to give [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexanoyl)pyrrolidine-2-carboxylate (2 g, 2.52 mmol, 42.96% yield) as a white solid. 1 H NMR(400MHz,CDCl3),4.80-4.90(m,1H),4.40-4.65(m,2H),4.05-4.25(m,4H),3.49-3.88(m,2H),2.20- 2.40(m,8H),1.60-1.75(m,10H),1.40-1.53(m,4H),1.23-1.38(m,46H),0.89(t,J=6.8Hz,9H).

[0468] Step 4:

[0469] To a solution of 3-(dimethylamino)propanoic acid (400 mg, 2.60 mmol, 1 equiv., HCl) in DCM (5 mL) was added (COCl) (1.32 g, 10.42 mmol, 911.81 μL, 4 equiv.) and DMF (19.03 mg, 260.40 μmol, 20.03 μL, 0.1 equiv.). The mixture was stirred at 20 °C for 2 h. The mixture was concentrated under reduced pressure to give 3-(dimethylamino)propanoyl chloride (448 mg, crude, HCl) as a yellow solid.

[0470] Step 5:

[0471] To a solution of [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-hydroxy-1-(6-oxo-6-undecoxy-hexanoyl)pyrrolidine-2-carboxylate (500 mg, 629.57 μmol, 1 equiv.), TEA (318.53 mg, 3.15 mmol, 438.14 μL, 5 equiv.), and DMAP (38.46 mg, 314.79 μmol, 0.5 equiv.) in DCM (10 mL) was added 3-(dimethylamino)propanoyl chloride (433.28 mg, 2.52 mmol, 4 equiv., HCl) under N at 0° C., and the mixture was stirred at 20° C. for 1 h. The mixture was added to saturated NaHCO (20 mL) and extracted with EtOAc (10 mL × 3). The organic layer was washed with brine (10 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) to obtain the product. The product was dissolved in petroleum ether (2 mL) and washed with ACN (2 mL × 2). The petroleum ether phase was concentrated under reduced pressure to obtain [8-(1-octylnonoxy)-8-oxo-octyl](2S)-4-[3-(dimethylamino)propanoyloxy]-1-(6-oxo-6-undecoxy-hexanoyl)pyrrolidine-2-carboxylate (200 mg, 223.88 μmol, 35.56% yield, 100% purity) as a yellow oil. 1H NMR(400MHz,CDCl3),5.33-5.37(m,1H),4.80-4.90(m,1H),4.40-4.75(m ,1H),4.11-4.15(m,2H),4.00-4.10(m,2H),3.85-3.88(m,1H),3.61-3.7 0(m,1H),2.52-2.65(m,2H),2.40-2.50(m,3H),2.15-2.38(m,13H),1.66 -1.70(m,8H),1.45-1.53(m,6H),1.23-1.38(m,46H),0.86-0.91(m,9H). LCMS:(M+H + ):893.7@13.385 / 13.687 minutes.

[0472] 8.11. Synthesis of Compound 2305 [ka] Step 1: A mixture of (2S,4R)-1-tert-butoxycarbonyl-4-hydroxy-pyrrolidine-2-carboxylic acid (835.02 mg, 3.61 mmol, 1 equiv.), 1-octylnonyl 8-bromooctanoate (2 g, 4.33 mmol, 1.2 equiv.), and CsCO (2.59 g, 7.94 mmol, 2.2 equiv.) in DMF (30 mL) was degassed and purged with N three times, and then the mixture was stirred under N atmosphere at 15 °C for 8 h. The reaction mixture was quenched by adding 100 mL of HO at 15 °C and then extracted with 300 mL (100 mL × 3) of EtOAc. The combined organic layers were washed with 200 mL (100 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 20 / 1 to 3 / 1) to give O1-tert-butyl O2-[8-(1-octylnonoxy)-8-oxo-octyl](2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (11 g, 17.71 mmol, 98.08% yield, 98.5% purity) as a colorless oil. 1H NMR(400MHz,CDCl3),4.85-4.88(m,1H),4.36-4.51(m,2H),3.64-3.68(m,1H),3.42-3.56 (m,1H),2.06-2.33(m,4H),1.60-1.64(m,5H),1.20-1.55(m,45H),0.88(t,J=6.4Hz,6H). 【...

Claims

1. Formula: 【Chemistry 1】 (IIA-1), or 【Chemistry 2】 (III-1), A compound having, a pharmaceutically acceptable salt thereof, or any of the stereoisomers described above, During the ceremony, A does not exist, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-,-N(R 7 ) C(O)N(R 7 )-, -S-, -S-S-, or a divalent heterocycle, X and Z are independent of each other, nonexistent, -O-, -CO-, -N(R) 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 ) - or -S-, Each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl, Each M is independently -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, -C(O-R13)-O-, -C(O)O(CH2)r-, -C(O)N(R7)(CH2)r-, or -C(O-R13)-O-(CH2)r- Each R7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl. R13 is a branched or unbranched C3-C10 alkyl group, and r is 1, 2, 3, 4, or 5. R 30 , R 40 , R 50 , R 60 , R 80 , R 90 , R 110 , and R 120 Each of these atoms is independently interrupted by H, optionally by a heteroatom, or substituted by OH, SH, or a halogen. 1 -C 16 Branched or unbranched alkyl or C 1 -C 16 Branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl, l and m are integers from 1 to 10, t1 is an integer from 0 to 10, and W is a compound, a pharmaceutically acceptable salt thereof, or any of the stereoisomers thereof, wherein W is a hydroxyl, a substituted or unsubstituted hydroxyalkyl, a substituted or unsubstituted amino, a substituted or unsubstituted aminocarbonyl, or a substituted or unsubstituted heterosilyl or heteroaryl.

2. A does not exist, -O-, -N(R7)-, -N(R7)C(O)-, 【Transformation 3】 -OC(O)- or -C(O)O-, where R6 is independently H, alkyl, hydroxyl, hydroxyalkyl, amino, aminoalkyl, thiol, thiolalkyl, or N+(R7)3-alkylene-Q-, where R7 is H or C1-C3 alkyl. X does not exist, -O-, or -C(O)-, Z is -O-, -C(O)O-, or -OC(O)-, Each of R30, R40, R50, and R60 is H or a C1-C4 branched or unbranched alkyl group. l is an integer between 3 and 7. m is an integer from 1 to 5, t1 is 0, 1, 2, 3, or 4, and / or W is a hydroxyl, hydroxyalkyl, or one of the following parts: 【Chemistry 4】 During the ceremony, Each Q is independently nonexistent, -O-, -C(O)-, -C(S)-, -C(O)O-, -C(R7)2-, -C(O)N(R7)-, -C(S)N(R7)-, or -N(R7)- Each R6 is independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, or N+(R7)3-alkylene-Q-. Each R8 is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thioalkyl, or two R8s may form a ring with a nitrogen atom. Each q is independently 0, 1, 2, 3, 4, or 5, and Each p is independently 0, 1, 2, 3, 4, or 5. The compound according to claim 1.

3. M is -OC(O)- or -C(O)O-, Each of R 30, R 40, R 50, and R 60 is H, 【Transformation 5】 They are independent, 【Transformation 6】 Selected from the group consisting of, where t is 0, 1, 2, 3, 4, or 5. and / or, W is OH, 【Transformation 7】 And, During the ceremony, q is 0, Each R 8 H and C are independent of each other. 1 -C 3 Alkyl, or hydroxyalkyl, or two R 8 It forms a five-membered ring in which the nitrogen atom is optionally substituted with one or more alkyl groups. Each R 6 These are independently H, hydroxyl, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, and C. 1 -C 3 Alkyl, or -Q-alkylene-N + (R 7 ) 3 And, Each Q is independent, nonexistent, -O-, -C(O)-, -N(R) 7 )-,-C(R 7 ) 2 -, -C(O)O-, -C(O)N(R 7 )-, or-C(S)N(R 7 ) - and Each R 7 H and C are independent of each other. 1 -C 3 The compound according to claim 1, wherein it is alkyl, hydroxy, or hydroxyalkyl.

4. W is OH, 【Transformation 8】 The compound according to claim 1. 【Request Item 5】 【Chemistry 9】 but, OH, 【Chemistry 10】 And, In the formula, each R c H or C 1 -C 3 The compound according to claim 1, wherein each t1 is independently 1, 2, 3, or 4. 【Request Item 6】 【Chemistry 11】 but, OH, 【Chemistry 12】 The compound according to claim 1.

7. X does not exist, -O-, or -C(O)-, Z is -O-, -C(O)O-, or -OC(O)-, M is -OC(O)- or -C(O)O-, 【Chemistry 13】 teeth, OH, 【Chemistry 14】 And, Each R c H or C 1 -C 3 It is alkyl, Each t1 is independently 1, 2, 3, or 4. R 30 , R 40 , R 50 , and R 60 Each of these is H or C 1 -C 4 It is a branched or unbranched alkyl group. R 70 H is R 80 and R 90 Each is independently H or C 1 -C 12 It is a branched or unbranched alkyl group. R 100 H is R 110 and R 120 Each is independently H or C 1 -C 12 It is a branched or unbranched alkyl group, however, R 80 and R 90 At least one of them is R, not H. 110 and R 120 At least one of them is not H, l is 3 to 7, and The compound according to claim 1, wherein m is 1 to 5.

8. formula: 【Chemistry 15】 It has, During the ceremony, Each m1 is an integer between 3 and 6, independently. Each l1 is an independent integer between 4 and 8. m2 and l2 are each independent integers between 0 and 3. R 80 and R 90 Each of them is independent of the non-substituted C 5 -C 8 Alkyl, or R 80 is H or unsubstituted C 1 -C 4 It is alkyl, and R 90 is unsubstituted C 5 -C 11 It is alkyl, and R 110 and R 120 Each of them is independent of the non-substituted C 5 -C 8 Alkyl, or R 110 is H or unsubstituted C 1 -C 4 It is alkyl, and R 120 is unsubstituted C 5 -C 11 The compound according to claim 1, wherein it is alkyl.

9. formula: 【Chemistry 16】 【change】 It has, During the ceremony, 【Chemistry 17】 teeth, OH, [Chemistry 18] The compound according to claim 8.

10. R 80 is H or unsubstituted C 1 -C 2 alkyl, and R 90 is unsubstituted C 6 -C 10 alkyl, and R 110 and R 120 Each of them is independent of the non-substituted C 5 -C 8 It is alkyl; or, The compound according to claim 8, wherein R80, R90, R110, and R120 are each independently unsubstituted C5-C8 alkyl groups.

11. formula: 【Chemistry 19】 It has, During the ceremony, Each m1 is an integer between 3 and 6, independently. Each l1 is an independent integer between 4 and 8. m2 and l2 are each independent integers between 0 and 3. R 80 and R 90 each independently is unsubstituted C 5 -C 8 alkyl, or R 80 is H or unsubstituted C 1 -C 4 alkyl, and R 90 is unsubstituted C 5 -C 11 alkyl, and R 110 and R 120 Each of them is independent of the non-substituted C 5 -C 8 Alkyl, or R 110 is H or unsubstituted C 1 -C 4 It is alkyl, and R 120 is unsubstituted C 5 -C 11 The compound according to claim 1, wherein it is alkyl.

12. formula: 【Chemistry 20】 It has, During the ceremony, 【Chemistry 21】 teeth, 【Chemistry 22】 The compound according to claim 11.

13. R 80 is H or unsubstituted C 1 -C 2 It is alkyl, R 90 is unsubstituted C 6 -C 10 It is alkyl, and R 110 and R 120 Each of them is independent of the non-substituted C 5 -C 8 It is alkyl; or, The compound according to claim 11, wherein R80, R90, R110, and R120 are each independently unsubstituted C5-C8 alkyl groups.

14. The compound according to claim 1, having one of the following structures. Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12

15. A lipid composition comprising a compound according to any one of claims 1 to 14, wherein the lipid composition is LNP.

16. The lipid composition according to claim 15, further comprising a second lipid, wherein the lipid composition may optionally contain the compound and the second lipid in a ratio of about 1:

1.

17. The lipid composition according to claim 15, further comprising sterols, PEG lipids, phospholipids, and / or neutral lipids.

18. A pharmaceutical composition comprising the lipid composition according to claim 15, a therapeutic agent, and a pharmaceutically acceptable excipient.

19. The pharmaceutical composition according to claim 18, wherein the therapeutic agent is a nucleic acid molecule, a protein, or a small molecule drug.

20. The pharmaceutical composition according to claim 19, wherein the nucleic acid molecule is RNA or DNA; optionally, the nucleic acid molecule is RNA containing mRNA.

21. Use of the pharmaceutical composition according to claim 18 for the manufacture of a pharmaceutical for delivering a therapeutic agent to a target organ or target organ.

22. The therapeutic agent is formulated to be delivered to a target organ selected from the group consisting of the pancreas, spleen, and lungs, The use according to claim 21, wherein 50%, 30%, or less than 10% of the therapeutic agent is formulated to be delivered to the liver; and / or 50%, 70%, or more than 90% of the therapeutic agent is formulated to be delivered to the pancreas, spleen, and / or lungs of the subject.

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