Lipid Compositions and Methods for Nucleic Acid Delivery

Abstract

The present invention relates to ionizable lipids and lipid nanoparticle compositions thereof that are useful for the delivery of therapeutic agents, such as nucleic acids.

Description

[Technical field]

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 333,153, filed April 21, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Effective delivery of bioactive agents such as messenger RNA (mRNA) is an ongoing challenge. Although mRNA has significant therapeutic potential, delivery efficiency into target cells remains challenging. In particular, delivery of nucleic acids such as RNA into cells is made difficult by their relative instability and poor cell permeability. Currently available delivery methods and nanoparticle compositions are unable to deliver more than 1% of the loaded mRNA. Thus, there is a need to develop new lipids and their compositions to facilitate the delivery of bioactive agents such as nucleic acids into cells. Summary of the Invention

[0003] In various aspects and embodiments, the present invention provides lipid nanoparticle compositions comprising ionizable lipid compounds having multiple nitrogen atoms in the backbone with lipophilic substituents as described in detail herein. In some embodiments, the lipophilic substituents are selected to allow interaction with encapsulated nucleic acids, for example, via cyclic and / or substantially planar portions. Such ionizable lipid compounds provide advantages for nucleic acid delivery to cells, including but not limited to RNA delivery (e.g., mRNA delivery).

[0004] In various embodiments, the lipid nanoparticle composition has formula (I): [ka] wherein each R1 is independently H or a substituent; L1 is -OC(=O)- or -C(=O)O-; L2 is -OC(=O)- or -C(=O)O-; L3 is [ka] is a group selected from each of n, t, and p is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; X and Y are each independently selected from lipophilic moieties having at least 6 carbon atoms.

[0005] In various embodiments, L3 is [ka] and t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; when t is 0, the two N atoms are directly connected to the bicycle.

[0006] In various embodiments, each 1 is independently H, or (C1-C6) alkyl, substituted (C 1~ C6) alkyl, (C1-C6) alkenyl, substituted (C1-C6) alkenyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclo.

[0007] In some embodiments, each R is independently selected from (halo)(C1-C6)alkyl, (alkoxy)(C1-C6)alkyl, (hydroxy)(C1-C6)alkyl, -(CH2) n -S-(C1-C6) alkyl, -(CH2) n -O-(C1-C6) alkyl, -C(O)(C1-C6) alkyl, (C3-C 12 ) cycloalkyl, and (C3-C 12 ) cycloalkenyl, any of which is independently optionally substituted as allowed by valence.

[0008] In some embodiments, each R is independently -(CH)n CHZR', -CHZR', -CZ(R')2, and -(CH2) n Z, wherein Z is selected from -OC(O)(C1-C6)alkyl, -C(O)O(C1-C6)alkyl, -OC(O)(C1-C6)alkenyl, -C(O)O(C1-C6)alkenyl, (C3-C 12 ) cycloalkyl, and (C3-C 12 )cycloalkenyl, each of which is independently optionally substituted as allowed by valence. R' at each occurrence is independently halo, hydroxyl, cyano, nitro, oxo, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C2-C6)alkenyl, (C1-C6)alkynyl, (C3-C 12 )Cycloalkyl, (C3-C 12 ) is a substituent such as a substituent selected from cycloalkenyl, heterocyclo, aryl, and heteroaryl.

[0009] In certain embodiments, each R1 is independently C1-C3 alkyl, optionally methyl, ethyl, or isopropyl.

[0010] In certain embodiments, each R is independently: [ka] is selected from the group consisting of m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A1 or A2 is H, C1-C3 alkyl, optionally methyl, ethyl, or isopropyl.

[0011] In yet other embodiments, each R is independently -(CH) n Z, where Z is aryl or heterocyclo. For example, each R is independently -(CH) nZ, where each Z is selected from phenyl, morpholinyl, pyrrolidinyl, imidazolidinyl, imidazolyl, pyrazolidinyl, pyrazolyl, oxazolidinyl, oxazolyl, pyridinyl, piperidinyl, diazinanyl, and diazinyl, and is optionally substituted (e.g., by one or more substituents).

[0012] In various embodiments of formula I, X and Y are lipophilic moieties and may be independently selected from linear or branched alkyl, linear or branched alkenyl, sterol, polyphenol, flavonoid, and tocopherol. For example, in some embodiments, one or both of X and Y are cholesteryl moieties. In some embodiments, one or both of X and Y are esters of tocopherol, which are optionally alpha, beta, gamma, or delta tocopherol. In embodiments, one or both of X and Y are branched alkyl or alkenyl having 8 to 30 carbon atoms. In some embodiments, one or both of X and Y are: [ka]

[0013] In various embodiments, X and / or Y are flavonoids, optionally selected from quercetin, rutin, maclaxanthone, genistein, scopoletin, daidzein, taxifolin, naringenin, abyssinone, eriodictyol, fisetin, theaflavin, peonidin, diosmetin, tricin, biochanin, hesperidin, epicatechin, myricetin, kaempferol, luteolin, and apigenin.

[0014] In various embodiments of the compound of formula I, t is an integer from 2 to 10. In some embodiments, t is 3, 4, 5, 6, 7, or 8.

[0015] In various embodiments of the compound of Formula I, n and p, which may be the same or different, are each an integer ranging from 3 to 10. In some embodiments, n and p, which may be the same or different, are selected from 6, 7, and 8. In some embodiments, n and p are the same.

[0016] In various embodiments, the lipid nanoparticle composition comprises an ionizable lipid of formula (II), (III), or (IV), where n, t, p, L1, L2, and R1 are defined as in formula I. [ka]

[0017] In various embodiments, the composition comprises an ionizable lipid as shown in Table 1. Such compounds can be made according to the methods and schemes described in Example 1. Thus, the ionizable lipid of formula 1 can be selected from the following: (propane-1,3-diylbis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (propane-1,3-diylbis(ethylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (octane-1,8-diylbis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (ethane-1,2-diylbis(benzylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), ((((1R,3S)-cyclohexane-1,3-diyl)bis(methylene))bis(azanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (((1s,4s)-cyclohexane-1,4-diyl)bis(azanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (cyclohexane-1,2-diylbis(azanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (((1R,2S)-cyclohexane-1,2-diyl)bis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (propane-1,3-diylbis(isopropylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (ethane-1,2-diylbis(tert-butylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), and (bicyclo[1.1.1]pentane-1,3-diylbis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate).

[0018] In some embodiments, lipid nanoparticles, e.g., lipid nanoparticles for encapsulating nucleic acids such as mRNA, may comprise cationic or ionizable lipids of formula I, neutral lipids, structured lipids, and PEGylated lipids, or may be formulated according to other nanoparticle formulations known in the art. In various embodiments, the maximum dimension of the lipid nanoparticles is about 200 nm or less. In exemplary embodiments, the average diameter of the lipid nanoparticles is in the range of about 50 nm to about 125 nm (e.g., in the range of about 60 to 110 nm).

[0019] In various embodiments, the lipid nanoparticles in the composition encapsulate one or more therapeutic, prophylactic, or diagnostic agents. For example, the lipid nanoparticles may include one or more therapeutic proteins. For example, the lipid nanoparticles may encapsulate one or more polynucleotides, which may be DNA (single-stranded or double-stranded) or RNA, or a mixture of RNA and DNA nucleotides. In some embodiments, the RNA is one or more selected from small RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA, double-stranded RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), double-stranded mRNA, circular RNA (oRNA), guide RNA, crRNA, tracer RNA, sgRNA, self-amplifying mRNA (SAM), and lentiviral RNA (lgRNA). In some embodiments, the polynucleotide is DNA or RNA selected from aptamers, RNA or DNA containing mobile genetic elements (including, for example, transposons and retrotransposons), and RNA or DNA containing sequences derived from viruses. In some embodiments, the polynucleotide is an antisense oligonucleotide (e.g., about 8 to about 25 nucleotides), which may be constructed of DNA, RNA, or a mixture of DNA and RNA nucleotides. In various embodiments, the lipid nanoparticle encapsulates a DNA vector, which in some embodiments is a plasmid or linear DNA construct encoding one or more genes under the control of a promoter suitable for delivery. In some embodiments, the lipid nanoparticle encapsulates one or more nucleic acid analogs, such as, for example, peptide nucleic acid (PNA) or locked nucleic acid (LNA). In some embodiments, the lipid nanoparticle encapsulates one or more non-coding RNAs, including, for example, long non-coding RNAs (lncRNAs).In some embodiments, the lipid nanoparticles encapsulate one or more polynucleotides containing backbone modifications (e.g., including phosphothioate linkages), polynucleotides containing one or more base- or sugar-modified nucleosides, polynucleotides chemically conjugated or complexed with proteins or small molecules. In some embodiments, the lipid nanoparticles encapsulate one or more co-formulations with one or more polynucleotides, one or more proteins, and / or one or more small molecules. In some embodiments, the RNA is an mRNA that encodes a component of an infectious agent (e.g., an antigen), e.g., a component of a virus, which is encapsulated with the LNP to provide an mRNA vaccine composition. In some embodiments, the LNPs encapsulate at least two, or at least three, or at least four open reading frames (as one or more separate RNA molecules), thereby simultaneously combining several immunogens for vaccination or proteins for therapy.

[0020] In other aspects, the present disclosure provides a method for delivering a therapeutic or prophylactic agent, such as a nucleic acid. The method includes administering a lipid nanoparticle composition of the present disclosure to a subject in need thereof. Exemplary subjects in need of treatment include subjects in need of protection from infectious diseases by vaccination, subjects in need of treatment for genetic diseases, or subjects in need of treatment for cancer. In various embodiments, the composition is administered by parenteral administration, for systemic administration or locally to a target tissue. In various embodiments, the composition is administered by routes such as intramuscular, intradermal, subcutaneous, intravenous, or intrathecal administration. In other embodiments, the composition described herein (including, for example, mRNA vaccines) is administered intranasally or by inhalation or administration to a mucosal surface.

[0021] In some embodiments, the nanoparticle compositions of the present disclosure may be useful for treating diseases, disorders, or conditions. In particular, such compositions may be useful for treating diseases, disorders, or conditions characterized by missing or abnormal protein or polypeptide activity. For example, a nanoparticle composition comprising an mRNA encoding a missing or abnormal polypeptide may be administered or delivered to a subject. Diseases, disorders, and / or conditions characterized by dysfunctional or abnormal protein or polypeptide activity to which the compositions may be administered include, but are not limited to, rare diseases, infectious diseases (both vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0022] Various aspects and embodiments of the disclosure are described more fully in connection with the figures and detailed description that follow.

[0023] The following summary, and detailed description of the invention, can be better understood when read in conjunction with the appended drawings. [Brief description of the drawings]

[0024] [Figure 1] 1 illustrates lipid compounds according to certain embodiments of the present disclosure. [Diagram 2] A-C show the efficacy of lipid nanoparticles containing the ionizable lipid GILP-124 in HEK293 cells. A shows the structure of the ionizable lipid GILP-124. B shows the results of in vitro evaluation (delivery of luciferase mRNA) of lipid nanoparticles containing GILP-124 against lipid nanoparticles containing the commercially available ionizable lipid heptadecane-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) (chemical structure shown in FIG. 7) as a control. C shows the normalized in vitro evaluation. [Diagram 3]1A-C illustrate the efficacy of lipid nanoparticles containing the ionizable lipid GILP-124 in HeLa cells. A shows the structure of the ionizable lipid GILP-124. B shows the in vitro evaluation (delivery of luciferase mRNA) of lipid nanoparticles containing the ionizable lipid GILP-124 versus the commercially available ionizable lipid heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) as a control. C shows the normalized in vitro evaluation. [Figure 4] 1A-C illustrate the efficacy of lipid nanoparticles containing the ionizable lipid GILP-126 in HEK293 cells. A shows the structure of the ionizable lipid GILP-126. B shows the in vitro evaluation (delivery of luciferase mRNA) of lipid nanoparticles containing the ionizable lipid GILP-126 versus the control ionizable lipid heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate. C shows the normalized in vitro evaluation. [Diagram 5] 1A-C illustrate the efficacy of lipid nanoparticles containing the ionizable lipid GILP-126 in HeLa cells. A shows the structure of the ionizable lipid GILP-126. B shows the in vitro evaluation (delivery of luciferase mRNA) of lipid nanoparticles containing the ionizable lipid GILP-126 versus the commercially available ionizable lipid heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) as a control. C shows the normalized in vitro evaluation. [Figure 6] The endosomal escape ability of LNPs formulated with ionizable lipids is illustrated using a hemolysis assay. [Figure 7] The chemical structure of heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate, used as a comparison (control), is shown. [Figure 8]A shows the chemical structure of GILP-133. B shows the luciferase expression levels in HEK293 cells of LNPs containing GILP-133 compared to the control (B). [Figure 9] Luciferase expression levels in HeLa cells of LNPs containing GILP-133 compared to controls are shown. [Figure 10] Luciferase expression levels at the injection site (A) and throughout the body (B) after intramuscular injection of LNPs into mice are shown. [Figure 11-1] Luciferase expression levels in major organs including muscle (A) and liver (B) 24 hours after administration to mice are shown. [Figure 11-2] Luciferase expression levels in major organs including dLN (C) and ndLN (D) 24 hours after administration to mice are shown. [Figure 11-3] Luciferase expression levels in major organs including spleen (E) 24 hours after administration to mice are shown. [Figure 12-1] IgG levels after injection of LNPs containing mRNA encoding the SARS-CoV-2 beta spike protein: shown on day 7 (A) and day 21 (B). [Figure 12-2] IgG levels after injection of LNPs containing mRNA encoding the SARS-CoV-2 beta spike protein: day 42 (C). [Figure 13] The number of antigen-specific IFN-γ-producing T cells in the peripheral blood of mice following administration of LNPs containing mRNA encoding the SARS-CoV-2 beta spike protein: day 7 (A) and day 42 (B). [Figure 14] 1 illustrates the delivery efficacy of lipid nanoparticles containing GILP-133 (GIL133) in HEK cells compared to MGNR24, MGNR23, MGNR22, and MGNR18. [Figure 15] 1 illustrates the delivery efficacy of lipid nanoparticles containing GILP-133 (GLB133) in HEK293 cells compared to BCY-001. [Figure 16] 1 shows the cytotoxicity of GILP-133. [Figure 17] The chemical structure of MGNR23 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The following is a detailed description of the present invention. Those skilled in the art may modify and vary the embodiments described herein without departing from the scope and spirit of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety.

[0026] 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 invention belongs.

[0027] Chemical structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds in which one or more hydrogen atoms are replaced by deuterium or tritium, or compounds in which one or more carbon atoms are replaced by 13 C- or 14 Compounds where C-enriched carbons are substituted are within the scope of the present invention.

[0028] The compounds of the present invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms.All stereoisomeric forms of the compound(s) and mixtures thereof, including racemic mixtures, are contemplated as part of this disclosure.Furthermore, this disclosure contemplates all geometric and positional isomers.For example, if a compound contains a double bond, both cis and trans forms (referred to as Z and E, respectively) and mixtures thereof are contemplated.

[0029] Mixtures of stereoisomers, e.g., diastereomeric mixtures, can be separated into their individual stereochemical components on the basis of their physical chemical differences by known methods, such as, for example, chromatography and / or fractional crystallization. Enantiomers can also be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with a suitable optically active compound (e.g., alcohol), separating the resulting diastereomers, and then converting the individual diastereomers into the corresponding pure enantiomers (e.g., by hydrolysis).

[0030] In various aspects and embodiments, the present invention provides lipid nanoparticle compositions comprising ionizable lipid compounds having multiple nitrogen atoms in the backbone with lipophilic substituents as described herein below. In some embodiments, the lipophilic substituents are selected to allow interaction with encapsulated nucleic acids, for example, via cyclic and / or substantially planar portions. Such ionizable lipid compounds provide advantages in nucleic acid delivery to cells, including, but not limited to, RNA delivery (e.g., mRNA delivery). As described herein, lipid nanoparticles comprising ionizable lipids as described herein have advantages in nucleic acid delivery efficiency, including advantages in endosomal escape.

[0031] In various embodiments, the lipid nanoparticle composition has formula (I): [ka] wherein each R1 is independently H or a substituent; L1 is -OC(=O)- or -C(=O)O-; L2 is -OC(=O)- or -C(=O)O-; L3 is [ka] is a group selected from the group consisting of each of n, t, and p is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; X and Y are each independently selected from lipophilic moieties having at least 6 carbon atoms.

[0032] In various embodiments, L3 is [ka] wherein t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0033] In various embodiments, L3 is [ka] and t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and when t is 0, the two N atoms in formula (I) are directly connected to the bicyclic ring.

[0034] In various embodiments, one of the two N atoms may be positively charged, or both N atoms are positively charged, as shown below. These compounds are within the scope of the present invention. [ka]

[0035] In various embodiments, each 1 is independently H, or (C1-C6) alkyl, substituted (C 1~ C6) alkyl, (C1-C6) alkenyl, substituted (C1-C6) alkenyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclo.

[0036] In some embodiments, each R is independently selected from (halo)(C1-C6)alkyl, (alkoxy)(C1-C6)alkyl, (hydroxy)(C1-C6)alkyl, -(CH2) n -S-(C1-C6) alkyl, -(CH2) n-O-(C1-C6) alkyl, -C(O)(C1-C6) alkyl, (C3-C 12 ) cycloalkyl, and (C3-C 12 ) cycloalkenyl, any of which is independently optionally substituted as allowed by valence.

[0037] In some embodiments, each 1 is independently -(CH) n CHZR', -CHZR', -CZ(R')2, and -(CH2) n Z, wherein Z is selected from -OC(O)(C1-C6)alkyl, -C(O)O(C1-C6)alkyl, -OC(O)(C1-C6)alkenyl, -C(O)O(C1-C6)alkenyl, (C3-C 12 ) cycloalkyl, and (C3-C 12 )cycloalkenyl, each of which is independently optionally substituted as allowed by valence. R' is independently selected from halo, hydroxyl, cyano, nitro, oxo, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C2-C6)alkenyl, (C1-C6)alkynyl, (C3-C 12 )Cycloalkyl, (C3-C 12 ) is a substituent such as a substituent selected from cycloalkenyl, heterocyclo, aryl, and heteroaryl.

[0038] In certain embodiments, each R1 is independently C1-C3 alkyl, optionally methyl, ethyl, or isopropyl.

[0039] In certain embodiments, each R is independently: [ka] is selected from the group consisting of m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A1 or A2 is H, C1-C3 alkyl, optionally methyl, ethyl, or isopropyl.

[0040] In still other embodiments, each R is independently -(CH) n Z, where Z is aryl or heterocyclo. For example, each R is independently -(CH) n Z, each Z being selected from phenyl, morpholinyl, pyrrolidinyl, imidazolidinyl, imidazolyl, pyrazolidinyl, pyrazolyl, oxazolidinyl, oxazolyl, pyridinyl, piperidinyl, diazinanyl, and diazinyl.

[0041] In an exemplary embodiment, each R is independently: [ka] wherein each m is independently 0, 1, 2, 3, 4, 5, or 6; each k is independently 0, 1, 2, 3, or 4; R2 is CH2OH or CO2(C1-C6)alkyl; R3 is OH, (C1-C6) alkyl, or aryl; R4 is OH, O(C1-C6)alkyl, SH, or S(C1-C6)alkyl; Each R5 is independently H, (C1-C6) alkyl, -(CH2) k OH, or O(C1-C6)alkyl; Each R6 is independently H, (C1-C6) alkyl, -(CH2) k OH, or O(C1-C6)alkyl; R7 is H or (C1-C6) alkyl; R8 is H or (C1-C6) alkyl; Z1 is NH, O, CH2, or NR5; Z2 is O, S, NR6, N, or NH.

[0042] In various embodiments of formula I, X and Y are independently selected from linear or branched alkyl, linear or branched alkenyl, sterol, polyphenol, flavonoid, and tocopherol. For example, in some embodiments, one or both of X and Y are cholesteryl esters. In some embodiments, one or both of X and Y are esters of tocopherol, which are optionally alpha, beta, gamma, or delta tocopherol. In embodiments, one or both of X and Y are branched alkyl or alkenyl having 8 to 30 carbon atoms, and optionally 10 to 20 carbon atoms, or 12 to 20 carbon atoms. In some embodiments, one or both of X and Y are: [ka]

[0043] In various embodiments, X and / or Y are flavonoids, optionally selected from quercetin, rutin, maclaxanthone, genistein, scopoletin, daidzein, taxifolin, naringenin, abyssinone, eriodictyol, fisetin, theaflavin, peonidin, diosmetin, tricin, biochanin, hesperidin, epicatechin, myricetin, kaempferol, luteolin, and apigenin.

[0044] In certain embodiments, X is a cholesteryl ester and Y is [ka] It is.

[0045] In certain embodiments, X is tocopherol, optionally alpha-tocopherol, and Y is [ka] It is.

[0046] In various embodiments of the compound of formula I, t is an integer from 2 to 10, e.g., 2, 3, 4, 5, 6, 7, or 8. In some embodiments, t is 3.

[0047] In various embodiments of the compound of Formula I, n and p, which may be the same or different, are each an integer ranging from 3 to 10. In some embodiments, n and p, which may be the same or different, are selected from 6, 7, and 8. In some embodiments, n and p are the same.

[0048] In various embodiments, the lipid nanoparticle composition comprises an ionizable lipid of formula (II), (III), or (IV), where n, t, p, L1, L2, and R1 are defined as in formula I. [ka]

[0049] In various embodiments, the composition comprises an ionizable lipid as shown in Table 1. Such compounds can be made according to the methods and schemes described in Example 1. Thus, the ionizable lipid of formula I can be selected from the following: (propane-1,3-diylbis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (propane-1,3-diylbis(ethylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (octane-1,8-diylbis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (ethane-1,2-diylbis(benzylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate) (((1R,2R)-cyclohexane-1,2-diyl)bis(methylazanediyl))bis(pentane-5,1-diyl)bis(2-hexyldecanoate), (propane-1,3-diylbis(isopropylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (ethane-1,2-diylbis(tert-butylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (bicyclo[1.1.1]pentane-1,3-diylbis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), and ((((1R,3S)-Cyclohexane-1,3-diyl)bis(methylene))bis(azanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate).

[0050] In some embodiments, for example, lipid nanoparticles for encapsulating mRNA may include cationic or ionizable lipids of formula I, neutral lipids, structured lipids, and PEGylated lipids. Lipid particle formulations used in embodiments of the present disclosure include those described in US8,058,069, US9,738,593, US9,867,888, US10,221,127, US10,166,298, US10,266,485, and US10,442,756, which are incorporated herein by reference in their entirety. Other lipid nanoparticle formulations known in the art may also be used, including those that include PLGA or PLA polymers, or polybeta aminoester polymers.

[0051] In some embodiments, the lipid nanoparticle (or LNP) comprises a structured lipid. Exemplary structured lipids can be selected from one or more of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and tocopherol (e.g., alpha-tocopherol). In some embodiments, the structured lipid is cholesterol.

[0052] In some embodiments, the LNP comprises one or more phospholipids. Exemplary phospholipids are selected from the group consisting of cardiolipin, sterol-modified lipids (lipids modified with a cholesterol moiety attached to the sn-2 carbon of the glycerol backbone), mixed acylglycerophospholipids, and symmetric acylglycerophospholipids. Head groups of acylglycerophospholipids include, for example, phosphatidic acid, lysophosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphoinositides, and phosphatidylserine.Exemplary phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

[0053] In various embodiments, the lipid nanoparticle composition further comprises one or more PEG lipids. The PEG lipid is a lipid modified with polyethylene glycol. Exemplary PEG lipids are selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The PEG lipid can be selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-cholesterol, PEG-tocopherol, or PEG-DSPE lipid.

[0054] In some embodiments, the composition comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG).

[0055] In various embodiments, the lipid nanoparticle composition comprises a structured lipid, a PEG lipid, and a phospholipid, each optionally according to the preceding paragraph. In an exemplary embodiment, the LNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG).

[0056] In various embodiments, the molar ratio of the compound of formula I to the phospholipid in the composition ranges from about 1:1 to about 10:1, or from about 2:1 to about 9:1, or from about 3:1 to about 8:1, or from about 4:1 to about 7:1, or from about 4:1 to about 6:1, and optionally is about 5:1.

[0057] In various embodiments, the molar ratio of phospholipid to structural lipid in the composition ranges from about 1:1 to about 1:10, or from about 1:2 to about 1:9, or from about 1:3 to about 1:8, or from about 1:3 to about 1:7, or from about 1:3 to about 1:5, and optionally is about 1:4.

[0058] In various embodiments, the molar ratio of structured lipid to PEG lipid ranges from about 50:1 to about 1:0.025, or from about 40:1 to about 5:1, or from about 40:1 to about 10:1, or from about 30:1 to about 15:1, or from about 30:1 to about 20:1, and optionally is about 50:1.5.

[0059] In various embodiments, the molar ratio of the compound of formula I, the phospholipid, the structured lipid, and the PEG lipid is about 50: about 10: about 38.5: about 1.5, respectively.

[0060] The lipid nanoparticle composition may include, in addition to the ionizable lipid according to Formula I, one or more additional cationic and / or ionizable lipids (i.e., lipids that may have a positive charge or a partial positive charge at physiological pH). The cationic lipid and / or ionizable lipid may be selected from the following non-limiting group: 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylanino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 15,18,21,24-tetraaza-octatriacontane (KL26), 15,18,21,24-tetraaza-octatriacontane (KL27), 15,18,21,24-tetraaza-octatriacontane (KL28), 15,18,21,24-tetraaza-octatriacontane (KL29), 15,18,21,24-tetraaza-octatriacontane (KL30), 15,18,21,24-tetraaza-octatriacontane (KL31), 15,18,21,24-tetraaza-octatriacontane (KL32), 15,18,21,24-tetraaza-octatriacontane (KL33), 15,18,21,24-tetraaza-octatriacontane (KL34), 15,18,21,24-tetraaza-octatriacontane (KL35), 15 ), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-MC3-DMA). in-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2S)-2-({8-[(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), and (2S)-2-({8-[(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)). In addition to these, the cationic lipid may also be a lipid containing a cyclic amine group.

[0061] In various embodiments, the maximum dimension of the lipid nanoparticles is about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about 200 nm or less (e.g., about 200 nm or less, about 175 nm or less, about 150 nm or less, about 125 nm, about 100 nm, about 75 nm, about 50 nm or less). Particle size or diameter can be quantified by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. In exemplary embodiments, the average diameter of the lipid nanoparticles is in the range of about 50 nm to about 125 nm (e.g., in the range of about 60 to 110 nm).

[0062] In various embodiments, the lipid nanoparticles in the composition encapsulate one or more therapeutic or diagnostic agents.

[0063] For example, the lipid nanoparticles can encapsulate one or more polynucleotides, which can be DNA (single-stranded or double-stranded) or RNA, or a mixture of RNA and DNA nucleotides. In some embodiments, the RNA is one or more selected from small RNA, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA, double-stranded RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), double-stranded mRNA, circular RNA (oRNA), and self-amplifying mRNA (SAM). In some embodiments, the polynucleotide is an antisense oligonucleotide (e.g., about 8 to about 25 nucleotides), which can be constructed of DNA, RNA, or a mixture of DNA and RNA nucleotides. The antisense oligonucleotides may contain a variety of sugar modifications known in the art (e.g., LNA, cET, 2'-MOE, 2'-OMe, 2'-F), as well as a variety of backbone modifications known in the art (e.g., phosphorothioate, morpholino, PNA).

[0064] In various embodiments, the lipid nanoparticles encapsulate mRNA. The mRNA useful in the present disclosure typically comprises a first region of linked nucleosides (e.g., coding region) that encodes a polypeptide of interest, a first adjacent region (e.g., 5'-UTR) located at the 5' end of the first region, and a second adjacent region (e.g., 3'-UTR) located at the 3' end of the first region. The mRNA may comprise a 5' cap region and a 3' stabilization region (e.g., polyA tail). In some embodiments, the nucleic acid or polynucleotide comprises a Kozak sequence (e.g., in the 5'-UTR). In some cases, the mRNA may comprise one or more intron nucleotide sequences that can be excised from the polynucleotide.

[0065] Nucleic acids and polynucleotides may contain the naturally occurring "standard" nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), and T (thymidine). Nucleic acids and polynucleotides may further contain one or more non-standard nucleotides, such as 1-methylpseudouridine (mlΨ) and pseudouridine (Ψ), in place of some or all of the uridines in the RNA. Modified nucleosides are described in U.S. Pat. No. 8,691,966 and WO2013022990A1, which are incorporated by reference in their entireties.

[0066] According to the present disclosure, the mRNA comprises modified uridines. In some embodiments, the modified uridines are selected from pseudouridine (Ψ), N1-methyl-pseudouridine, and 5-methoxy-uridine. For example, at least about 10%, or at least about 25%, or at least about 50%, or at least about 75%, or all of the uridines can be modified uridines, such as pseudouridine, N1-methyl-pseudouridine, and / or 5-methoxy-uridine. In some embodiments, substantially all of the uridines in the mRNA are replaced with pseudouridine and / or N1-methyl-pseudouridine.

[0067] In some embodiments, the nucleic acid (e.g., RNA or mRNA) is selected from the group consisting of 2-thiouridine, 5-azauridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxypseudouridine, 5-ethoxyuridine, 5-ethoxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-aminopseudour ... Lysine, 5-formylpseudouridine, 5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5 -hydroxy-5-azacytidine, 5-methyl-pseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine,2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N 4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5-aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy cy-5-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methylpseudoisocytidine,2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5- Azacitidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4- Hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8 -azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine,and 6-thio-7-deaza-8-azaguanosine.

[0068] In various embodiments, the lipid nanoparticles encapsulate DNA vectors, which in some embodiments are plasmids or linear DNA constructs encoding one or more genes under the control of a suitable promoter for delivery.

[0069] In some cases, the polynucleotide is more than 10 nucleotides long (e.g., an oligonucleotide). In various embodiments, the polynucleotide is at least 25, or at least about 50 nucleotides long, or at least about 100 nucleotides long (e.g., a small RNA or siRNA). In various embodiments, the length is at least 100 nucleotides. In some embodiments, the length of the polynucleotide (e.g., an mRNA or coding DNA) is at least about 200 nucleotides, or at least about 300 nucleotides, or at least about 500 nucleotides long, or at least about 700 nucleotides long, or at least about 1000 nucleotides, or at least about 1200 nucleotides long, or at least about 1500 nucleotides long, or at least about 2000 nucleotides long, or at least about 3000 nucleotides, or at least about 4000 nucleotides, or at least about 5000 nucleotides, or at least about 6000 nucleotides, or at least about 7000 nucleotides, or at least about 8000 nucleotides, or at least about 9000 nucleotides, or at least about 10000 nucleotides.

[0070] In some embodiments, the RNA is an mRNA that encodes a component of an infectious agent, such as a component of a virus, and is encapsulated within the LNP to provide an mRNA vaccine composition. In some embodiments, the LNP encapsulates at least two or at least three, or at least four open reading frames, thereby simultaneously combining several immunogens for vaccination or proteins for therapy.

[0071] In some embodiments, the RNA is an RNA described in WO2022 / 016077, US2022 / 0370599, or WO2021 / 113774.

[0072] In some embodiments, the mRNA encodes one or more proteins of a virus, or one or more polypeptides derived from a viral protein, e.g., a DNA or RNA virus. Examples include those of the Paramyxoviridae and / or Pneumovirus or Morbillivirus genera. Examples of viruses include human metapneumovirus (hMPV), parainfluenza virus (hPIV), (types 1, 2, and 3), respiratory syncytial virus (RSV), and measles virus (MeV). In some embodiments, the RNA virus is a coronavirus (CoV) (family Coronaviridae, subfamily Coronavirinae). In some embodiments, the coronavirus is a betacoronavirus, such as SARS-CoV or MERS-CoV. In some embodiments, the RNA virus is SARS-CoV-2, or a naturally occurring variant thereof. In other embodiments, the virus is a herpes virus, e.g., herpes simplex virus or varicella zoster virus. In other embodiments, the virus is RSV, hepatitis virus, or adenovirus. In yet other embodiments, the virus is an Ebola virus.

[0073] In some embodiments, the mRNA encodes one or more viral structural proteins, or one or more polypeptides derived from viral proteins, e.g., proteins contained in the viral envelope, such as the spike protein (S) of coronaviruses. Alternatively, or in addition, the mRNA encodes other CoV structural proteins, e.g., the M (membrane) glycoprotein, the E (envelope) protein, and / or the N (nucleocapsid) protein. Alternatively, the mRNA encoding the spike protein or other structural proteins may be encapsulated into particles that include or are modified with one or more CoV structural proteins or portions thereof.

[0074] In some embodiments, the mRNA encodes one or more influenza proteins, such as neuraminidase (NA), hemagglutinin (HA), matrix protein 2 (M2), and / or nucleoprotein (NP). In some embodiments, the mRNA encodes at least one neuraminidase and at least one hemagglutinin.

[0075] In some embodiments, the mRNA encodes one or more varicella antigens, such as glycoprotein E, glycoprotein B, glycoprotein H, glycoprotein L, or glycoprotein I.

[0076] In some embodiments, the mRNA encodes one or more cancer-associated epitopes or neoantigens.

[0077] In some embodiments, the mRNA is targeted for expression in a tissue or organ selected from, for example, liver (e.g., hepatocytes), skin (e.g., keratinocytes), skeletal muscle, endothelial cells, epithelial cells of various organs including lungs, or hematopoietic or immune cells (T cells, B cells, or macrophages). For example, the mRNA is designed to encode a polypeptide of interest selected from a vaccine target, an enzyme (including a metabolic enzyme or an endonuclease such as a Cas endonuclease), an antibody or an antigen-binding fragment thereof, or an antibody mimetic (single-chain antibody such as a nanobody or a single-chain variable fragment), a secreted protein or peptide (including a cytokine, growth factor, or a soluble receptor thereof), a plasma membrane protein, a cytoplasmic or cytoskeletal protein, an intracellular membrane-associated protein, a nuclear protein, a protein associated with a human disease (including a protein with a loss-of-function or gain-of-function mutation associated with a human disease). In some embodiments, the therapeutic protein comprises one or more cancer-associated epitopes (e.g., one or more mutations associated with cancer, including neo-antibodies) that can be used in a cancer vaccine. In an exemplary embodiment in which the mRNA encodes an antibody, the open reading frames encoding the heavy and light chains can be expressed from different mRNA molecules.

[0078] In various embodiments, the nucleic acid encodes a therapeutic protein, e.g., for the treatment of a disease or disorder. Exemplary diseases characterized by dysfunctional or abnormal protein activity include cystic fibrosis, sickle cell anemia, epidermolysis bullosa, amyotrophic lateral sclerosis, and glucose-6-phosphate dehydratase deficiency. In various embodiments, the nucleic acid (e.g., mRNA) encodes a protein that overcomes the activity of an abnormal protein present in the subject's cells. A specific example of a dysfunctional protein is a missense mutation variant of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produces a dysfunctional protein variant of the CFTR protein that causes cystic fibrosis. Other diseases characterized by a lack or substantial reduction in protein activity (where proper, normal, or physiological protein function does not occur) include cystic fibrosis, Niemann-Pick disease type C, beta thalassemia major, Duchenne muscular dystrophy, Hurler syndrome, Player syndrome, and hemophilia A. In these conditions, proteins important for cellular function may be absent or essentially non-functional. The present invention provides nanoparticle compositions for treating such conditions or diseases. The nanoparticles encapsulate nucleic acids, such as mRNA, that encode proteins that replace the protein activity missing from the subject.

[0079] In some embodiments where the composition is a vaccine, the composition may include one or more adjuvants. Such adjuvants include, for example, emulsion adjuvants such as MIPLA, R848, QS-21, aluminum salt-based adjuvants, MF59 (squalene, polysorbate 80, sorbitan trioleate, trisodium citrate dehydrate) and AS03 (polysorbate 80, squalene, DL-α-tocopherol); TLR agonist-based adjuvants such as CpG ODN and AS04 (3'-O-deacylated monophosphoryl lipid A (MPL) + aluminum salt); AS01B (MPL + QS-21); and any analogues of the above. Other adjuvants for use with the present invention may include glucopyranosyl lipid adjuvants (GLA), CpG oligodeoxynucleotides (e.g., class A or B), poly(I:C), aluminum hydroxide, Pam3CSK4, and analogs of any of the above. Other adjuvants for use with the present invention may include lipid-based adjuvants such as GLA-SE and GLA-AF; emulsions such as Montanide ISA 51 and Montanide ISA 720; saponins such as matrix M and ASO2; nucleotides such as cyclic dinucleotides (CDNs), CpG, ODNs, dsRNA, IL-12, and Pika adjuvants; cytokines such as IL-2, IL-12, IL-15, and granulocyte macrophage colony stimulating factor (GM-CSF); calcium phosphate, bacterial flagellin, virosomes, and analogs of any of the above.

[0080] In some embodiments, for encapsulation of nucleic acids, the ratio of amines of the ionizable lipid of formula I to phosphates of the polynucleotide (N:P ratio) is about 2:1 to about 50:1, or about 2:1 to about 40:1, or about 2:1 to about 20:1, or about 2:1 to about 15:1, or about 2:1 to about 12:1, or about 35:1 to about 45:1, or about 2:1 to about 10:1, or about 3:1 to about 10:1, or about 4:1 to about 10:1, or about 5:1 to about 7:1, or about 35:1 to about 45:1. In some embodiments, the N:P ratio is about 6:1. In some embodiments, the N:P ratio is about 12:1.

[0081] In some embodiments, the lipid nanoparticles of the present disclosure encapsulate RNA, which in various embodiments is present in the composition at a concentration of about 0.01 to about 2.0 mg / mL, or about 0.01 to about 1.0 mg / mL, or about 0.05 to about 0.5 mg / mL, or about 0.1 mg / mL.

[0082] In other embodiments, in addition to a nucleic acid, the therapeutic agent may be another biologically active substance or "active agent." The therapeutic agent and / or prophylactic agent may be a substance that, when delivered to a cell or organ, produces a desired change in the cell, organ, or other body tissue or system. In some embodiments, the therapeutic agent and / or prophylactic agent is a small molecule drug useful for treating a particular disease, disorder, or condition. Examples of drugs useful in the nanoparticle compositions include antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antineoplastic agents (e.g., actinomycin D, vincristine, vinblastine, cystine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic receptor blockers (e.g., propranolol, timolol, and labetolol), antihypertensive agents, (e.g., clonidine and hydrazine), antidepressants (e.g., imipramine, amitriptyline, and doxepime), anticonvulsants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, narcotics, and contrast media.

[0083] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that induces an immune response. A cytotoxin or cytotoxic agent includes any agent that is detrimental to a target cell.

[0084] The nanoparticle compositions described herein are stable for storage and / or transportation when refrigerated or frozen (e.g., when stored at temperatures below 4° C., e.g., at temperatures of about −150° C. to about 0° C., or about −80° C. to about −20° C.). In some embodiments, the pharmaceutical compositions are stable when refrigerated for storage and / or transportation, e.g., at about 0° C., or about −10° C., or about −20° C., or about −30° C., or about −40° C., or −50° C., or −60° C., or −70° C., or about −80° C.

[0085] In certain embodiments, the composition is stable at refrigerated temperatures. For example, in some embodiments, the lipid nanoparticles are stable at 2° C. for at least 3 months, or are stable at 2° C. for at least 6 months. In some embodiments, the composition is stable at 4° C. for at least 3 months, or are stable at 4° C. for at least 6 months. In some embodiments, the composition is stable at 8° C. for at least 3 months, or are stable at 8° C. for at least 6 months. Thus, the composition can be stored and / or distributed at temperatures ranging from 2-8° C., providing a substantial advantage over currently approved mRNA vaccines.

[0086] Degradation or instability can be determined by an increase or decrease in the average size of the particles in the formulation (eg, an average size that is at least about 10% or at least about 20% larger or smaller than a control).

[0087] RNA degradation can be determined by the presence of smaller RNA species and the disappearance of the desired RNA size, for example, as determined by high performance liquid chromatography (HPLC).

[0088] In various embodiments, the population of LNPs encapsulating RNA is relatively homogenous as determined by the polydispersity index (PDI), which indicates the particle size distribution of lipid nanoparticles. A small PDI (e.g., less than 0.3) indicates a narrow particle size distribution. The LNPs may have a PDI of about 0 to about 0.25. In some embodiments, the PDI is about 0.10 to about 0.20.

[0089] In various embodiments, the compositions have a relatively low charge, positive or negative, since more highly charged species may have undesirable interactions with cells or tissues in the body upon administration, hi some embodiments, the zeta potential of the composition can be from about -20 mV to about +20 mV, or from about -10 mV to about +10 mV.

[0090] The efficiency of encapsulation of a therapeutic and / or prophylactic agent refers to the amount of therapeutic and / or prophylactic agent that is encapsulated or otherwise associated with a nanoparticle composition after preparation relative to the initial amount provided. It is desirable for the encapsulation efficiency to be high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing the nanoparticle composition before and after dissolving the nanoparticle composition with one or more organic solvents or surfactants. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic and / or prophylactic agent can be at least about 50%, or at least about 70%, or at least about 80%, or at least about 90%.

[0091] Nanoparticle compositions can be designed for one or more specific applications or targets. The components of the nanoparticle composition can be selected based on a particular application or target, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more of the components. Similarly, a particular formulation of a nanoparticle composition can be selected for a particular application or target, for example, according to the efficacy and toxicity of a particular combination of components. In various embodiments, the composition includes excipients, which can include one or more antioxidants, non-ionic surfactants, one or more stabilizers, and pH buffers.

[0092] Exemplary antioxidants include methionine, propyl gallate, ascorbic acid, citric acid, monothioglycerol, phosphoric acid, potassium metabisulfite, alpha-tocopherol, sodium sulfite, cysteine, sodium metabisulfite, t-cysteine-HCL, vitamin E TPGS, HP-β-CD, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), L-methionine, L-cysteine, citric acid / citrate salts, vitamin A, vitamin C (ascorbic acid), vitamin E, or other antioxidants approved for injectable use in humans. Additional exemplary antioxidants not yet approved for injectable use may also be used, such as polyphenols or vitamin P. In various embodiments, the concentration of the antioxidant is from 0.01% to about 1.50% w / v, or from about 0.02% to about 0.2%, or from about 0.05% to about 0.5%, or from about 0.10% to about 0.25% w / v.

[0093] In various embodiments, the composition includes a non-ionic surfactant, such as a polysorbate or poloxamer. In some embodiments, the polysorbate is polysorbate 20, polysorbate 40, polysorbate 60, and / or polysorbate 80. In some embodiments, the non-ionic surfactant is polysorbate 20. In various embodiments, the concentration of polysorbate-20 is about 0.001% to about 0.1% w / v, or about 0.005% to about 0.05% w / v, or about 0.01% w / v. In some embodiments, the poloxamer is poloxamer 188, poloxamer 124, poloxamer 182, poloxamer 331, poloxamer 335, poloxamer 407, or other poloxamers.

[0094] In various embodiments, the stabilizer is selected from one or more of glycine, sorbitol, and gelatin. In some embodiments, the stabilizer is glycine, and the glycine may be present in the composition at a concentration of about 0.25% to about 15% w / v, or about 0.25% to about 10% w / v, or about 0.25% to about 5% w / v, or about 0.5% to about 2.5% w / v. In some embodiments, the concentration of glycine is about 1.5% w / v. In these or other embodiments, the stabilizer is sorbitol, and optionally, is present in the composition at about 1% to about 20%, e.g., about 10% w / v. In these or other embodiments, the stabilizer(s) includes gelatin, and the gelatin is optionally present in the composition at about 1% to about 20% w / v, or about 5% to about 15% w / v, e.g., about 10% w / v.

[0095] In various embodiments, the pH is buffered to a pH of about 6.0 to about 8.0. In certain embodiments, the pH is buffered to about 6.0, about 7.4, or about 8.0. In various embodiments, the composition is pH buffered at about pH 7.4. In various embodiments, the pH buffer is a phosphate buffer. In still other embodiments, the pH buffer is a Tris-EDTA (TE) buffer. In some embodiments, the pH buffer is a histidine buffer. In some embodiments, the histidine buffer is L-histidine. In some embodiments, the buffer is a TE buffer consisting of Tris-HCL and disodium EDTA. In some embodiments, the buffer is Tris-acetate, which may consist of Tris base and sodium acetate. In some embodiments, the buffer is a sodium citrate buffer, which may consist of sodium citrate dihydrate and citric acid. In some embodiments, the buffer is PBS, which may consist of potassium chloride, potassium phosphate monobasic, sodium chloride, and sodium phosphate dibasic dihydrate.

[0096] In some embodiments, the composition further comprises a metal chelator. For example, the chelator may be selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethyleneglycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), iminodisuccinic acid, polyaspartic acid, ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine diacetate (MGDA), L-glutamic acid N,N-diacetate (GLDA), or salts thereof. In some embodiments, the metal ion chelator is EDTA or a salt thereof, which is optionally disodium EDTA. In some embodiments, the concentration of EDTA or disodium EDTA is about 0.01 mM to about 1 mM, or about 0.05 mM to about 0.5 mM, or about 0.1 mM.

[0097] In various embodiments, the composition further comprises an excipient that reduces exposure of the RNA to water, hi some embodiments, the excipient that reduces exposure of the mRNA to water is a sugar, such as sucrose.

[0098] In some embodiments, the composition further comprises an excipient that reduces RNA degradation by free radical oxidation. In some embodiments, the excipient that reduces RNA degradation by free radical oxidation is one or more of ethanol and histidine. In some embodiments, ethanol is included as an excipient at 200 mM or less, or about 150 mM or less, or less than about 100 mM, or about 50 mM or less to avoid affecting LNP size. In some embodiments, the excipient(s) that reduces RNA degradation comprises or consists of histidine. Histidine may be present in the composition at a concentration of about 0.01% w / v to about 1% w / v, or about 0.05% w / v to about 0.5% w / v, or about 0.1% w / v.

[0099] In another aspect, the present disclosure provides a method for delivering a therapeutic agent. The method comprises administering a lipid nanoparticle composition of the present disclosure to a subject in need thereof. Exemplary subjects and conditions or disorders in need of treatment (including protection from infectious diseases by vaccination) have been described above.

[0100] In some aspects, the disclosure provides methods for preventing or reducing the likelihood of a viral infection, e.g., a SARS-CoV-2 infection, in a patient or population. In these embodiments, the methods include administering an mRNA vaccine of the disclosure expressing one or more viral proteins, such as the SARS-CoV-2 spike protein and / or other SARS-CoV-2 structural proteins described herein. In some embodiments, the mRNA vaccine is administered as a single dose. In some embodiments, the mRNA vaccine is administered as multiple (e.g., two or three) doses with a booster one, two, or three weeks after the initial dose. Periodic boosters can be administered as needed. According to various aspects, the disclosure provides for simplified global distribution over currently available mRNA vaccines due to the lack of sub-zero conditions required for storage and distribution and / or improved vaccine stability.

[0101] In some embodiments of this aspect, the disclosure provides methods for expressing a therapeutic protein in a patient comprising administering an mRNA composition described herein. For example, diseases, disorders, and / or conditions for treatment or prevention include autoimmune disorders (e.g., diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis), inflammatory disorders (e.g., arthritis, pelvic inflammatory disease), infectious diseases (e.g., viral infections, bacterial infections, fungal infections, and sepsis), neurological disorders (e.g., Alzheimer's disease, Huntington's disease, autism, Duchenne muscular dystrophy), cardiovascular disorders (e.g., angiogenic disorders such as atherosclerosis, hypercholesterolemia, thrombosis, coagulation disorders, macular degeneration), and metabolic disorders. and liver disorders (e.g., ornithine transcarbamylase deficiency); proliferative disorders (e.g., cancer, benign neoplasms); respiratory disorders (e.g., chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis); digestive disorders (e.g., inflammatory bowel disease, ulcers); musculoskeletal disorders (e.g., fibromyalgia, arthritis); endocrine, metabolic, and nutritional disorders (e.g., diabetes, osteoporosis); urinary disorders (e.g., renal disease); psychiatric disorders (e.g., depression, schizophrenia); skin disorders (e.g., wounds, eczema); and blood and lymphatic disorders (e.g., anemia, hemophilia).

[0102] In some embodiments, the therapeutic agent (such as RNA) of the pharmaceutical composition according to the present disclosure can be administered at a dose of about 1 μg to 500 μg, or about 5 μg to 450 μg, or about 10 μg to 400 μg, or about 15 μg to 400 μg, or about 20 μg to 350 μg, or about 25 μg to 325 μg, or about 30 μg to 300 μg, or about 35 μg to 275 μg, or about 40 μg to 250 μg, or about 45 μg to 225 μg, or about 50 μg to 200 μg, or about 60 μg to 180 μg, or about 70 μg to 150 μg, or about 80 μg to 125 μg, or about 90 μg to 100 μg. In some embodiments, the therapeutic agent is an mRNA vaccine.

[0103] In various embodiments, the subject is a mammal or a bird. In some embodiments, the subject is a human. Other exemplary subjects include pigs, dogs, cats, cows, horses, sheep, and chickens.

[0104] In various embodiments, the composition is administered parenterally for systemic administration or locally to the target tissue. In various embodiments, the composition is administered by routes such as intramuscular, intradermal, subcutaneous, intravenous, or intrathecal administration. In other embodiments, the composition (e.g., mRNA vaccine) described herein is administered intranasally or by inhalation.

[0105] In various embodiments, the nanoparticle compositions of the present disclosure may target or accumulate in a particular type or class of cells or tissues, such as the liver, kidney, spleen, lung, heart, muscle, or CNS. Specific delivery to a particular class of cells, organs, or systems or groups thereof means, for example, that upon administration of the nanoparticle composition, a higher percentage of nanoparticles are delivered to the destination (e.g., tissue) of interest compared to other destinations. In some embodiments, specific delivery may result in a 2-fold, 5-fold, 10-fold, 15-fold, or greater than 20-fold increase in the amount of therapeutic and / or prophylactic agent per gram of tissue in the targeted destination (e.g., a tissue of interest such as the liver) compared to another destination (e.g., the spleen). In some embodiments, the target tissue is a tumor.

[0106] In some embodiments, the nanoparticle compositions of the present disclosure may be useful for treating diseases, disorders, or conditions. In particular, such compositions may be useful for treating diseases, disorders, or conditions characterized by missing or abnormal protein or polypeptide activity. For example, a nanoparticle composition comprising an mRNA encoding a missing or abnormal polypeptide may be administered or delivered to a cell. Subsequent translation of the mRNA produces the polypeptide, thereby reducing or eliminating problems caused by the lack or abnormal activity of the polypeptide. Diseases, disorders, and / or conditions characterized by dysfunctional or abnormal protein or polypeptide activity for which the compositions may be administered include, but are not limited to, rare diseases, infectious diseases (both vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases. Several diseases, disorders, and / or conditions may be characterized by a lack of protein activity (or a substantial reduction such that proper protein function does not occur). Such proteins may be absent or they may be essentially non-functional. A specific example of a dysfunctional protein is a missense mutation variant in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which generates a dysfunctional protein variant of the CFTR protein that causes cystic fibrosis.

[0107] definition As used in this specification and the appended claims, the articles "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless the context clearly indicates otherwise. By way of example, "an element" means one element or more than one element.

[0108] The present disclosure contemplates the inclusion of one or more substituents of the ionizable lipid of formula I. The group or atom that replaces hydrogen atom is also called "substituent". In various embodiments, a particular molecule or group can have one or more substituents depending on the number of hydrogen atoms that can be replaced.

[0109] The term "H" represents a single hydrogen atom and is not a substituent.

[0110] The term "alkyl", when used alone or within other terms such as "haloalkyl" or "alkylamino", embraces straight-chain or branched hydrocarbon radicals. Exemplary alkyls have from 1 to about 30 carbon atoms. Examples of alkyls include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, hexyl, and the like. The term "alkenyl" or "alkylene" embraces bridged divalent alkyl radicals such as methylenyl or ethylenyl.

[0111] The term "alkenyl" embraces straight-chain or branched hydrocarbon radicals having at least one carbon-carbon double bond. Exemplary alkenyl groups have from 2 to about 30 carbon atoms. Examples of alkenyl radicals include ethenyl, propenyl, allyl, propenyl, butenyl, and 4-methylbutenyl. The term "alkenyl" embraces radicals having "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations.

[0112] The term "alkynyl" refers to a straight or branched chain radical having at least one carbon-carbon triple bond. Exemplary alkynyl groups have from 2 to about 30 carbon atoms. Examples of such radicals include propargyl, butynyl, and the like.

[0113] The alkyl, alkylenyl, alkenyl, and alkynyl radicals may be optionally substituted with one or more functional groups, such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, and heterocyclo.

[0114] The term "halo" means halogens such as fluorine, chlorine, bromine, or iodine atoms.

[0115] The term "haloalkyl" includes radicals in which any one or more of the alkyl carbon atoms are substituted with halo as defined above. Specifically included are monohaloalkyl, dihaloalkyl, and polyhaloalkyl radicals, including perhaloalkyl. For example, monohaloalkyl radicals may have either iodo, bromo, chloro, or fluoro atoms within the radical. Dihalo and polyhaloalkyl radicals may have two or more of the same halo atoms, or a combination of different halo radicals. Examples of haloalkyl radicals include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl.

[0116] The term "hydroxyalkyl" embraces linear or branched alkyl radicals, e.g., having from 1 to about 30 carbon atoms, any one of which may be substituted with one or more hydroxyl radicals. Examples of such radicals include hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and hydroxyhexyl.

[0117] The term "alkoxy" embraces straight-chain or branched oxy-containing radicals, each having an alkyl portion of, for example, 1 to about 30 carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy, and tert-butoxy. Alkoxy radicals may be further substituted with one or more halo atoms, for example, fluoro, chloro, or bromo, to provide "haloalkoxy" radicals. Examples of such radicals include fluoromethoxy, chloromethoxy, trifluoromethoxy, trifluoroethoxy, fluoroethoxy, and fluoropropoxy.

[0118] The term "aryl", alone or in combination, means a carbocyclic aromatic system containing one or more rings, which may be joined together in a fused fashion. The term "aryl" encompasses aromatic radicals such as phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl. An "aryl" group may have one or more substituents, such as lower alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy, and lower alkylamino.

[0119] The term "heterocyclyl" (or "heterocyclo") embraces saturated, partially saturated, and unsaturated heteroatom-containing ring radicals, where the heteroatoms may be selected from nitrogen, sulfur, and oxygen. It does not include rings containing -OO-, -OS-, or -SS- moieties. "Heterocyclyl" groups may have 1 to 4 substituents, such as hydroxyl, Boc, halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, lower alkoxy, amino, and lower alkylamino.

[0120] Examples of saturated heterocyclic radicals include saturated 3- to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms [e.g., pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl, piperazinyl], saturated 3- to 6-membered heteromonocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g., morpholinyl], and saturated 3- to 6-membered heteromonocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g., thiazolidinyl]. Examples of partially saturated heterocyclyl radicals include dihydrothienyl, dihydropyranyl, dihydrofuryl, and dihydrothiazolyl.

[0121] Examples of unsaturated heterocyclic radicals, also referred to as "heteroaryl" radicals, include unsaturated 5-6 membered heteromonocyclyl groups containing 1-4 nitrogen atoms, e.g., pyrrolyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrimidyl, pyranidyl, pyridazinyl, triazolyl [e.g., 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl]; unsaturated 5-6 membered heteromonocyclic groups containing an oxygen atom, e.g., pyranyl, 2-furyl, 3-furyl, etc.; unsaturated 5-6 membered heteromonocyclic groups containing a sulfur atom, e.g., pyranyl, 2-furyl, 3-furyl, etc. Examples of monocyclic groups include 2-thienyl, 3-thienyl, etc., unsaturated 5- to 6-membered heteromonocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as oxazolyl, isoxazolyl, oxadiazolyl [e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl], and unsaturated 5- to 6-membered heteromonocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, such as thiazolyl, thiadiazolyl [e.g., 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl].

[0122] The term heterocyclyl, (or heterocyclo) also encompasses radicals in which a heterocyclic radical is fused / condensed with an aryl radical: unsaturated fused heterocyclic groups containing 1-5 nitrogen atoms, e.g., indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl [e.g., tetrazolo[1,5-b]pyridazinyl]; ...2 oxygen atoms and 1-3 unsaturated fused heterocyclic groups containing nitrogen atoms [e.g., benzoxazolyl, benzoxadiazolyl], unsaturated fused heterocyclic groups containing 1-2 sulfur atoms and 1-3 nitrogen atoms [e.g., benzothiazolyl, benzothiadiazolyl], and saturated, partially unsaturated, and unsaturated fused heterocyclic groups containing 1-2 oxygen or sulfur atoms [e.g., benzofuryl, benzothienyl, 2,3-dihydro-benzo[1,4]dioxinyl, and dihydrobenzofuryl]. Examples of heteroaryl radicals include quinolyl, isoquinolyl, imidazolyl, pyridyl, thienyl, thiazolyl, oxazolyl, furyl, and pyrazinyl. Other heteroaryl radicals are 5- or 6-membered heteroaryl containing one or two heteroatoms selected from sulfur, nitrogen, and oxygen, and are selected from thienyl, furyl, pyrrolyl, indazolyl, pyrazolyl, oxazolyl, triazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, pyridyl, piperidinyl, and pyrazinyl.

[0123] Particular examples of unsubstituted heteroaryls include pyranyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, benzofuryl, and benzothienyl.

[0124] Specific examples of partially saturated and saturated heterocyclyls include pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl, pyrazolidinyl, piperazinyl, morpholinyl, tetrahydropyranyl, thiazolidinyl, dihydrothienyl, 2,3-dihydro-benzo[1,4]dioxanyl, indolinyl, isoindonyl, dihydrobenzothienyl, dihydrobenzofuryl, isochromanyl, chromanyl, 1,2-dihydroquinolyl, 1,2,3,4-tetrahydro-iso quinolyl, 1,2,3,4-tetrahydro-quinolyl, 2,3,4,4a,9,9a-hexahydro-1H-3-aza-fluorenyl, 5,6,7-trihydro-1,2,4-triazolo[3,4-a]isoquinolyl, 3,4-dihydro-2H-benzo[1,4]oxazinyl, benzo[1,4]dioxanyl, 2,3-dihydro-1H-1λ′-benzo[d]isothiazol-6-yl, dihydropyranyl, dihydrofuryl, and dihydrothiazolyl.

[0125] Thus, the term "heterocyclo" includes the following ring systems: [ka] [ka] [ka] etc.

[0126] The terms "carboxy" or "carboxyl," whether used alone or with other terms, such as "carboxyalkyl," refer to --CO.sub.2H.

[0127] The term "carbonyl", whether used alone or with other terms, such as "aminocarbonyl", refers to --(C.dbd.O)--.

[0128] The term "cycloalkyl" embraces saturated carbocyclic radicals. Examples of such radicals include cyclopentyl, cyclopropyl, and cyclohexyl.

[0129] The term "cycloalkenyl" includes carbocyclic groups having one or more carbon-carbon double bonds, including "cycloalkyldienyl" compounds.

[0130] The term "cholesteryl moiety" refers to the following structure: [ka]

[0131] Wavy line [ka] indicates a connection point.

[0132] For purposes of describing and claiming the present invention, the open-ended term "comprising" is used herein as a synonym for terms such as including, containing, or having, although the invention, or embodiments thereof, may alternatively be described using alternative terms, such as "consisting of" or "consisting essentially of."

[0133] The symbol "-" represents a covalent bond and can also be used on radical groups to indicate the point of attachment to another group. In chemical structures, this symbol is commonly used to represent a methyl group in a molecule.

[0134] The term "excipient" means any pharma- ceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API), that is normally included in the formulation and / or administration to a patient.

[0135] The term "therapeutically effective amount" means an amount of a compound that ameliorates, attenuates, or eliminates one or more symptoms of a particular disease or condition, or prevents or delays the onset of one or more symptoms of a particular disease or condition.

[0136] The term "pharmaceutical acceptable" means that the compound or composition described herein, or the referred substance, such as a salt thereof, or a formulation containing a compound described herein or a particular excipient, is suitable for administration to a patient.

[0137] Terms such as "treating," "treat," or "treatment" include preventative (eg, prophylactic) and palliative treatment.

[0138] As used herein, the term "about" means ±10% of the associated numerical value.

[0139] All patents, patent applications, and other publications cited herein are hereby incorporated by reference.

[0140] Other aspects and embodiments of the present invention will become apparent from the following examples. EXAMPLES

[0141] The present teachings generally described herein may be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the present disclosure.

[0142] Messenger RNA (mRNA) has significant therapeutic potential but continues to face limitations due to the efficiency of delivery vehicles. Effective formulations require safe transport of mRNA, which is inherently unstable due to its polyanionic nature, into the cytosol of target cells. In these examples, ionizable cationic lipids were designed and synthesized with two or more nitrogen atoms in the backbone. According to embodiments of the present disclosure, such lipids may allow for a reduction in the amount of lipid required for LNP formulation compared to traditional lipid structures. These ionizable lipids were formulated with luciferase mRNA and three additional lipid components: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG) in a molar ratio of 50:10:38.5:1.5 with an N / P ratio of 12 by microfluidic mixing. N / P is the ratio between the amines in the ionizable lipid and the amines of the anionic phosphate of the mRNA. Apparent pKa represents lipid nanoparticle (LNP) surface ionization and is indirectly measured by TNS assay. Apparent pKa of LNP correlates with mRNA delivery efficiency. Formulated LNPs were evaluated for particle size, size distribution range, and mRNA encapsulation efficiency. The following example demonstrates a new class of ionizable lipids (an embodiment of which is shown in Figure 1) that show efficient mRNA delivery to cells via LNPs.

[0143] Example 1: General synthetic scheme The following general synthetic scheme for an embodiment of the present invention is shown below for the synthesis of (propane-1,3-diylbis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate). [ka]

[0144] 6-Hydroxyhexyl 2-hexyldecanoate (3): [ka]

[0145] To a solution of 2-hexyldecanoic acid (1 equiv.) in anhydrous dichloromethane was added N,N'-diisopropylcarbodiimide (2 equiv.) on an ice bath (0°C) under argon atmosphere. The reaction mixture was stirred at 0°C for 30 min, then 1,6-hexane-diol (1.05 equiv.) and DMAP (0.5 equiv.) were added slowly to the mixture. The reaction mixture was warmed to room temperature and stirred at room temperature overnight (16 h). The reaction mixture was washed with 30 mL of saturated NaHCO3 solution and extracted with 2 x 30 mL of dichloromethane. The combined organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Purification by Teledyne ISCO NextGen300+ silica flash chromatography (0-100% EtOAc in hexanes over 25 min) afforded the desired product.

[0146] 6-Oxohexyl 2-hexyldecanoate (4) [ka]

[0147] To a solution of 6-hydroxyhexyl 2-hexyldecanoate (1 equiv.) in anhydrous dichloromethane was added pyridinium chlorochromate (1.5 equiv.) slowly over 10 min under an argon atmosphere. The reaction mixture was stirred at room temperature for 2 h. The mixture was filtered through a Celite pad and the collected fractions were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. Purification by Teledyne ISCO NextGen300+ silica flash chromatography (0-100% EtOAc in hexanes over 25 min) gave the desired product.

[0148] (Propane-1,3-diylbis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate) (6): [ka]

[0149] To a solution of 6-oxyl 2-hexyldecanoate (3.0 equiv.) in anhydrous dichloromethane was added N,N-dimethyl-1,3 propanediamine (1.0 equiv.), sodium triacetoxyborohydride (3.0 equiv.), and a few drops of acetic acid. The reaction mixture was stirred at room temperature under argon for 16 h. The reaction mixture was washed with 30 mL of saturated aqueous NaHCO3 and extracted with 2 x 30 mL of dichloromethane. The combined organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Purification by Teledyne ISCO NextGen300+ silica flash chromatography (0-20% MeOH in CH2Cl2, 25 min) afforded the desired product.

[0150] Lipids synthesized according to this general scheme are shown in Table 1.

[0151] Example 2: LNP formulation Unless otherwise stated, only RNAase- and DNAase-free materials were used. All lipid stock solutions were warmed to 37°C. Stock solutions were visually inspected to ensure the absence of crystals. Sonication (times varied) and / or a heat gun (max 20 sec) were used as necessary to dissolve crystals. Luc-mRNA was thawed at 4°C (no vortexing or sonication). Lipid mix and ethanol were added to a vial (organic phase). The mRNA and acetate buffer (pH 4) were combined to obtain the aqueous phase. After addition of mRNA to the acidic buffer, the mRNA stock solution was returned to the -80°C freezer. The aqueous phase was loaded into the appropriate syringe, avoiding air bubbles. Similarly, the organic phase was loaded into another syringe, avoiding air bubbles.

[0152] The cartridge and 15 mL Falcon tube were inserted into a NANOASSEMBLR Ignite (Precision NanoSystems). Formulations were made with the following settings: flow ratio (FRR) 3:1, total flow rate (TFR) 12 mL / min, starting waste 0.25 mL, and final waste 0.25 mL.

[0153] After pressing the start setting, the formulation was collected in a Falcon tube and diluted with approximately 10-fold volume of PBS.

[0154] The formulations were transferred to a 10K Amicon filter (15 mL) and centrifuged at 2000 rcf at 4° C. for 90 minutes or until the volume was reduced to approximately 400 μL. The formulations were harvested and characterized for size and PDI using DLS. The formulations were further analyzed for RNA encapsulation using the Quant-iT™ RiboGreen® RNA Assay Kit.

[0155] The formulations were kept at 4°C until used in luciferase expression assays to evaluate the potency and toxicity of LNPs using LDH assays in HeLa and HEK293 cells. LNPs were used in TNS assays to determine the experimental (apparent) pKa of ionizable lipids. Hemolysis assays were used to determine the indirect endosomal escape (EE) ability of LNPs. LNPs were evaluated in vivo for luciferase expression in mice 6 and 24 hours after dose by im injection of 5 mg per mouse. Mice were sacrificed at the endpoint and then organs were harvested for ex vivo imaging. TIFF2025514748000028.tif41170

[0156] Lipid nanoparticles containing GILP-124 (described above and shown in Figures 2A and 3A) were tested for the efficiency of mRNA delivery using HEK293 cells and HeLa cells. In these studies, encapsulation of luciferase mRNA was used. Figures 2B and 2C show the results of HEK293 cells for lipid nanoparticles containing the control ionizable lipid eptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate shown in Figure 7. Figure 2C shows the normalized values ​​for the in vitro evaluation (i.e., the control value was set as 1 and the relevant ratios for the test samples were calculated based on it). As shown, nanoparticles containing GILP-124 provided high efficiency of mRNA delivery to HEK293 cells, showing high delivery levels even at 25 μg / mL. GLP-124 showed a significant increase in efficiency over the control. See Figures 2B and 2C. Similar results were obtained in HeLa cells, although the results were more dose-dependent for GILP-124 LNPs, see Figures 3B and 3C.

[0157] Lipid nanoparticles containing GILP-126 (described above and shown in Figures 4A and 5A) were similarly tested. Nanoparticles containing GILP-126 also performed significantly better than the positive control LNPs in both HEK293 and HeLa cells. See Figures 4B and 4C, and Figures 5B and 5C.

[0158] Lipid nanoparticles containing GILP-133 (described above and shown in Figures 8A and 9A) were similarly tested and found to perform significantly better than the positive control LNPs in both HEK293 and HeLa cells, see Figures 8B and 9B.

[0159] Figure 6 shows the endosomal escape potential of LNPs formulated with ionizable lipids using a hemolysis assay. Surprisingly, lipid nanoparticles formulated with either GILP-124 or GILP-126 exhibited negligible hemolysis at neutral pH (pH 7.4) (suggesting low toxicity) and strong hemolysis at acidic pH (pH 5.5), suggesting strong endocytic escape potential. LNPs containing GILP-124 or GILP-126 appear to have substantially stronger endocytic escape potential compared to control LNPs.

[0160] Example 3. Animal luciferase expression studies Two formulations containing GILP-133 (shown in Table 3) were selected to assess luciferase expression in vivo. TIFF2025514748000029.tif46170

[0161] Each sample was prepared as described above in Example 1 and stored in the presence of 10% glycerol, 10% sucrose, 80% PBS aqueous solution at -80°C. After freeze-thawing, acceptable particle size, PDI and EE were maintained, as shown in Table 4. TIFF2025514748000030.tif54170

[0162] These samples were injected into mice by intramuscular injection at the leg site at a dose of 5 μg of mRNA per mouse. The composition containing the control LNP of FIG. 7 was used as a positive control. Six and 24 hours after injection, the mice were subjected to the IVIS imaging system to acquire whole-body images. At the 6-hour time point, sample numbers 2 and 3 showed lower luciferase expression at the injection site than sample number 1, indicating a low correlation between in vitro and in vivo. Sample 4 (1.5%) with a higher ratio of DMG-PEG showed much higher luciferase expression compared to sample 2 (0.5%) with a lower ratio of DMG-PEG. The same trend was observed in the whole-body imaging results. The results are shown in FIGs. 10A and 10B.

[0163] At 24 hours, mice were sacrificed and major organs including heart, liver, spleen, lung, kidney, muscle, dLN and ndLN were collected and imaged. Except for sample no. 3, all other GIL-133 LNP formulations showed similar luciferase expression in muscle. Sample no. 2 and 3 showed no luciferase expression in liver, indicating that they may have potential for delivery beyond liver. In dLN, sample no. 1 and 4 showed similar luciferase expression levels as the positive control, indicating that they may have potential for inducing a strong immune response. In ndLN and spleen, the positive control showed higher levels of luciferase expression than all GIL-133 formulations. All results are shown in 11A, 11B, 11C, 11D, and 11E.

[0164] Example 4. Animal immune response assay Vaccine formulations containing GILP124, GILP126, GILP133, and GILP124 / 124Q (a mixture of GILP124:GILP124Q at approximately 4:1, denoted as GLB Quat / Tet 124 / 124Q in Figures 12 and 13) and the appropriate right nitrogen quaternary (positively charged) were selected for study in mouse immune response assays. mRNA encoding the SARS-CoV-2 spike protein, beta variant, was encapsulated in the LNP formulation. C57BL / 6 mice were immunized with 5 μg of vaccine in a prime and boost regimen 21 days apart (primary day 0, boost day 21). Mice were euthanized on day 42. Blood was collected for antibody and ELISpot analysis of antigen-specific IFN-γ producing T cells. Mice were divided into 5 groups with 5 mice per group (n=5).

[0165] Anti-beta SARS-CoV-2 S1 spike IgG measurement by ELISA.

[0166] ELISA was performed from blood collected on days 7, 21, and 42 to assess seroconversion and IgG levels against the SARS-CoV-2 beta spike protein. On day 7, not all animals seroconverted in groups receiving mRNA formulated in 124 / 124Q and GILP126, but when formulated in GILP124 and GILP133, all animals were observed to seroconvert after a single dose of mRNA, as shown in Figure 12A. At week 3, 21 days after the first dose, all animals in all groups except one mouse in 124 / 124Q had seroconverted between 3.1 and 4.5 (Log 10 ) range of anti-spike IgG levels, and the lower limit of quantification indicated by the dotted black line was Log 10 (See FIG. 12B.) At day 42, 21 days after the boost of formulated mRNA, the levels of anti-spike IgG were seroconverted in all animals in all groups, showing high levels of anti-spike IgG. (See FIG. 12C.) The results show that the tested formulations allow delivery of mRNA that induces a strong antibody response against the SARS-CoV-2 beta spike protein. The endpoint titer was calculated as the dilution that produced an optical density greater than 4 times the background (secondary antibody alone). In these assays, all measurements above the LLOQ were considered positive for anti-spike IgG.

[0167] A commercially available ELISpot kit (BD™ ELISPOT Mouse IFN-γ ELISPOT Set) was used to evaluate peripheral blood of mice 7 days after the first dose of formulated 048 mRNA according to the vendor's protocol. Blood cells were lysed and plated overnight after stimulation with a peptide pool of SARS-CoV-2 beta spike protein. An assay internal positive control was performed using PMA / ION as a stimulus. It was observed that a single dose of formulated mRNA was sufficient to prime antigen-specific IFN-γ producing T cells, as can be seen from the number of spots in Figure 13A. The same assay was performed on day 42, 21 days after a booster dose of mRNA in the different formulations, and the immune response was enhanced several-fold, as shown by the number of spots in Figure 13B. These data suggest that all such formulations successfully deliver encapsulated antigens that induce priming and booster antigen-specific T cell immune responses as measured by IFN-γ ELISpot assay.

[0168] Example 5 - Alternative structures of GILP-133. Alternative structures of GILP-133 were evaluated to see if changing the number of carbons in the linker or the ring structure at the center of the molecule would have any effect. MGNR24, shown in Table 1, differs from GILP-133 in that it incorporates a six carbon linker region rather than a seven carbon linker region. MGNR23, shown in Figure 17, differed in that it incorporated a lineolitic tail. Also, BCY-01, shown in Table 1, differs in that it incorporated a bicyclic ring in L3.

[0169] In a luciferase expression assay in HEK cells as described in Example 2, GILP-133 outperformed a variety of formulations including MGNR24, MGNR23, and BCY-01. See Figures 14 and 15.

[0170] Example 6 - LDH Toxicity Assay GILP-133 was tested in an assay to measure the levels of lactate dehydrogenase (LDH), also known as lactic acid dehydrogenase. The LDH assay protocol is based on an enzyme-coupled reaction. LDH released from cells oxidizes lactate to produce NADH, which reacts with WST to produce a yellow color. The intensity of the resulting color directly correlates with the number of cells lysed. This provides an indication of cytotoxicity by looking at the % of viable cells remaining. In this study, Hek293FT cells were used. 10uL of 1% Triton was added to the positive control wells and incubated for 5 minutes. 50uL of cell culture medium was transferred to a black 96-well plate. 50uL of LDH assay solution was added and incubated for 10 minutes at room temperature. 50ul of stop solution was then added and the fluorescence intensity was read using a microplate reader. As shown in Figure 16, GILP-133 from two separate batches showed comparable % cell viability compared to the control cationic lipid shown in Figure 7. This suggests that GILP-133 is not more toxic at the concentrations tested than the control lipid already approved for human use. TIFF2025514748000031.tif243170TIFF2025514748000032.tif235170TIFF2025514748000033.tif239170 TIFF2025514748000034.tif240170TIFF2025514748000035.tif241170TIFF2025514748000036.tif235170

Claims

1. Formula (I): 【Chemistry 1】 wherein each R １ is independently H or a substituent; L １ is -OC(=O)- or -C(=O)O-, L ２ is -OC(=O)- or -C(=O)O-, L ３ but, 【Chemistry 2】 is selected from the group consisting of each of n, t, and p is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; The lipid nanoparticle composition, wherein X and Y are each independently selected from lipophilic moieties having at least 6 carbon atoms.

2. Each R １ are independently H, (C １ ~C ６ ) alkyl, substituted (C １ ~C ６ ) alkyl, (C １ -C ６ ) alkenyl, substituted (C １ ~C ６ 2. The lipid nanoparticle composition of claim 1, wherein said aryl is selected from the group consisting of alkenyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclo.

3. Each R １ However, independently, (halo) (C １ ~C ６ ) alkyl, (alkoxy) (C １ ~C ６ ) alkyl, (hydroxy) (C １ ~C ６ ) alkyl, -(CH ２ ) ｎ -S-(C １ ~C ６ ) alkyl, -(CH ２ ) ｎ -O-(C １ ~C ６ ) alkyl, —C(O)(C １ ~C ６ ) alkyl, (C ３ ~C １２ ) cycloalkyl, and (C ３ ~C １２ 3. The lipid nanoparticle composition of claim 1 or 2, wherein the aryl, aryl, aryl and / or aryl groups are selected from the group consisting of aryl, aryl, aryl and / or aryl groups, each of which is independently optionally substituted as permitted by valence.

4. Each R １ are independently -(CH ２ ) ｎ CHZR', -CHZR', -CZ(R') ２ , and -(CH ２ ) ｎ Z, wherein Z is —OC(O)(C １ ~C ６ ) alkyl, —C(O)O(C １ ~C ６ ) alkyl, —OC(O)(C １ ~C ６ ) alkenyl, —C(O)O(C １ ~C ６ ) alkenyl, (C ３ ~C １２ ) cycloalkyl, and (C ３ ~C １２ 3. The lipid nanoparticle composition of claim 1 or 2, wherein R' is selected from the group consisting of cycloalkenyl, cycloalkyl ...

5. R' at each occurrence is independently halo, hydroxyl, cyano, nitro, oxo, (C １ ~C ６ ) alkyl, (C １ ~C ６ ) alkoxy, (C １ ~C ６ ) haloalkyl, (C ２ ~C ６ ) alkenyl, (C １ ~C ６ ) alkynyl, (C ３ ~C １２ ) cycloalkyl, (C ３ ~C １２ 5. The lipid nanoparticle composition of claim 4, wherein said aryl is cycloalkenyl, heterocyclo, aryl, or heteroaryl.

6. Each R １ But independently, C １ ~C ３ The lipid nanoparticle composition of claim 1, wherein the alkyl is methyl, ethyl, or isopropyl.

7. Each R １ are independently -(CH ２ ) ｎ The lipid nanoparticle composition of claim 4, wherein Z is aryl or heterocyclo.

8. Each R １ are independently -(CH ２ ) ｎ 8. The lipid nanoparticle composition of claim 7, wherein Z is selected from phenyl, morpholinyl, pyrrolidinyl, imidazolidinyl, imidazolyl, pyrazolidinyl, pyrazolyl, oxazolidinyl, oxazolyl, pyridinyl, piperidinyl, diazinanyl, and diazinyl.

9. Each R １ But independently, 【Chemistry 3】 wherein each m is independently 0, 1, 2, 3, 4, 5, or 6; each k is independently 0, 1, 2, 3, or 4; R ２ But, CH ２ OH, or CO ２ (C １ ~C ６ ) alkyl; R ３ But, OH, (C １ ~C ６ ) alkyl, or aryl; R ４ But OH, O(C １ ~C ６ ) alkyl, SH, or S(C １ ~C ６ ) alkyl; Each R ５ are independently H, (C １ ~C ６ ) alkyl, -(CH ２ ) ｋ OH, or O(C １ ~C ６ ) alkyl; Each R ６ are independently H, (C １ ~C ６ ) alkyl, -(CH ２ ) ｋ OH, or O(C １ ~C ６ ) alkyl; R ７ is H, or (C １ ~C ６ ) alkyl; R ８ is H, or (C １ ~C ６ ) alkyl; Z １ NH, O, CH ２ , or N.R. ５ and Z ２ But O, S, NR ６ , N, or NH.

10. The lipid nanoparticle composition according to any one of claims 1 to 9, wherein X and Y are independently selected from linear or branched alkyl, linear or branched alkenyl, sterol, polyphenol, flavonoid, and tocopherol.

11. The lipid nanoparticle composition of claim 10, wherein one or both of X and Y are cholesteryl esters.

12. 11. The lipid nanoparticle composition of claim 10, wherein one or both of X and Y are optionally alpha, beta, gamma, or delta tocopherol esters.

13. The lipid nanoparticle composition of claim 10, wherein one or both of X and Y are branched alkyl or alkenyl having from 8 to 30 carbon atoms, and optionally from 10 to 20 carbon atoms.

14. The lipid nanoparticle composition of claim 13, wherein one or both of X and Y are: 【Chemistry 4】

15. 11. The lipid nanoparticle composition of claim 10, wherein X and / or Y are flavonoids, optionally selected from quercetin, rutin, maclaxanthone, genistein, scopoletin, daidzein, taxifolin, naringenin, abyssinone, eriodictyol, fisetin, theaflavin, peonidin, diosmetin, tricin, biochanin, hesperidin, epicatechin, myricetin, kaempferol, luteolin, and apigenin.

16. X is a cholesteryl ester and Y is 【Chemistry 5】 The lipid nanoparticle composition of claim 10,

17. X is tocopherol, optionally alpha-tocopherol, and Y is 【Chemistry 6】 The lipid nanoparticle composition of claim 10,

18. The lipid nanoparticle composition of any one of claims 1 to 17, wherein t is 2 to 5, optionally 3.

19. The lipid nanoparticle composition of any one of claims 1 to 18, wherein n and p are each an integer in the range of 3 to 10, optionally 6, 7, or 8.

20. (propane-1,3-diylbis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (propane-1,3-diylbis(ethylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (octane-1,8-diylbis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (ethane-1,2-diylbis(benzylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), ((((1R,3S)-cyclohexane-1,3-diyl)bis(methylene))bis(azanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (((1s,4s)-cyclohexane-1,4-diyl)bis(azanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (cyclohexane-1,2-diylbis(azanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (((1R,2S)-cyclohexane-1,2-diyl)bis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (((1R,2R)-cyclohexane-1,2-diyl)bis(methylazanediyl))bis(pentane-5,1-diyl)bis(2-hexyldecanoate), (propane-1,3-diylbis(isopropylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (ethane-1,2-diylbis(tert-butylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), (bicyclo[1.1.1]pentane-1,3-diylbis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate), and The lipid nanoparticle composition of claim 1, wherein the lipid nanoparticle composition is selected from the group consisting of ((((1R,3S)-cyclohexane-1,3-diyl)bis(methylene))bis(azanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate).

21. The lipid nanoparticle composition of claim 1, wherein the ionizable lipid is (((1R,2S)-cyclohexane-1,2-diyl)bis(methylazanediyl))bis(heptane-7,1-diyl)bis(2-hexyldecanoate).

22. The lipid nanoparticle composition of any one of claims 1 to 21, further comprising one or more structured lipids.

23. 23. The lipid nanoparticle composition of claim 22, wherein the structured lipid is selected from one or more of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.

24. 24. The lipid nanoparticle composition of claim 23, wherein the structured lipid is cholesterol.

25. The lipid nanoparticle composition of any one of claims 1 to 24, further comprising one or more phospholipids.

26. The phospholipid may be 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OchemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. The lipid nanoparticle composition of claim 25,

27. 27. The lipid nanoparticle composition of any one of claims 1 to 26, further comprising one or more conjugated lipids that inhibit particle aggregation, optionally wherein the one or more conjugated lipids that inhibit particle aggregation comprise one or more PEG-lipids.

28. 28. The lipid nanoparticle composition of claim 27, wherein the one or more PEG lipids are selected from one or more of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols.

29. The lipid nanoparticle composition of claim 28, wherein the one or more PEG lipids is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG).

30. The lipid nanoparticle composition of any one of claims 1 to 29, comprising a structured lipid, a PEG lipid, and a phospholipid.

31. 31. The lipid nanoparticle composition of claim 30, wherein the molar ratio of the compound of formula 1 to the phospholipid is in the range of about 1:1 to about 10:1, or in the range of about 2:1 to about 9:1, or in the range of about 3:1 to about 8:1, or in the range of about 4:1 to about 7:1, or in the range of about 4:1 to about 6:1, and optionally about 5:

1.

32. 32. The lipid nanoparticle composition of claim 30 or 31, wherein the molar ratio of said phospholipid to said structured lipid ranges from about 1:1 to about 1:10, or from about 1:2 to about 1:9, or from about 1:3 to about 1:8, or from about 1:3 to about 1:7, or from about 1:3 to about 1:5, or from about 1:5 to about 1:6, optionally about 1:

4.

33. 33. The lipid nanoparticle composition of any one of claims 30 to 32, wherein the molar ratio of said structured lipid to said PEG lipid ranges from about 50:1 to about 1:0.025, or from about 40:1 to about 5:1, or from about 40:1 to about 10:1, or from about 34:1 to about 30:1, or from about 30:1 to about 15:1, or from about 30:1 to about 20:1, optionally about 50:1.

5.

34. The lipid nanoparticle composition of claim 30, wherein the molar ratio of the compound of formula 1, the phospholipid, the structured lipid, and the PEG lipid is about 50: about 10: about 38.5: about 1.5, respectively.

35. The lipid nanoparticle composition of claim 30, wherein the molar ratio of the compound of formula (I), the phospholipid, the structured lipid, and the PEG lipid is about 31.3: about 8.2: about 60: about 0.5, respectively.

36. The lipid nanoparticle composition of claim 30, wherein the molar ratio of the compound of formula (I), the phospholipid, the structured lipid, and the PEG lipid is about 23.4: about 16.1: about 60: about 0.5, respectively.

37. The lipid nanoparticle composition of claim 30, wherein the molar ratio of the compound of formula (I), the phospholipid, the structured lipid, and the PEG lipid is about 31.3: about 8.2: about 59: about 1.5, respectively.

38. 38. The lipid nanoparticle composition of any one of claims 1 to 37, wherein the lipid nanoparticles in the composition encapsulate one or more therapeutic, prophylactic, or diagnostic agents.

39. 40. The lipid nanoparticle composition of claim 38, wherein the lipid nanoparticle encapsulates one or more polynucleotides.

40. The lipid nanoparticle composition of claim 39, wherein the polynucleotide is RNA.

41. The lipid nanoparticle composition of claim 40, wherein the RNA is one or more selected from small RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA, double-stranded RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), double-stranded mRNA, circular RNA (oRNA), and self-amplifying mRNA (SAM).

42. The lipid nanoparticle composition of claim 41 , wherein the RNA is mRNA or modified mRNA (mmRNA).

43. The lipid nanoparticle composition of claim 42, wherein the mRNA or mmRNA encodes a polypeptide of an infectious organism, optionally a virus, optionally a betacoronavirus, influenza virus, or herpes virus.

44. The lipid nanoparticle composition of claim 42, wherein the mRNA or mmRNA encodes one or more cancer-associated polypeptides, epitopes, antigens, or neoantigens.

45. The lipid nanoparticle composition of claim 39, wherein the polynucleotide is an antisense oligonucleotide.

46. The lipid nanoparticle composition of claim 39, wherein the polynucleotide is DNA.

47. 47. The lipid nanoparticle composition of any one of claims 39 to 46, wherein the ratio of the amine of the ionizable lipid of formula 1 to the phosphate of the polynucleotide (N:P ratio) is from about 2:1 to about 50:1, or from about 2:1 to about 40:1, or from about 2:1 to about 20:1, or from about 2:1 to about 15:1, or from about 2:1 to about 12:1, or from about 35:1 to about 45:1, or from about 2:1 to about 10:1, or from about 3:1 to about 12:1, or from about 4:1 to about 12:1, or from about 5:1 to about 7:

1.

48. The lipid nanoparticle composition of claim 47, wherein the N:P ratio is about 6:1 or about 12:

1.

49. The lipid nanoparticle composition of any one of claims 30 to 48, wherein the molar ratio of the compound of formula 1, the phospholipid, the structured lipid, and the PEG lipid is about 23.4 to about 50: about 8.2 to about 10: about 38.5 to about 60: about 0.5 to about 1.

5.

50. 50. A method of delivering a therapeutic agent, comprising administering to a subject in need of the therapeutic agent a composition according to any one of claims 1 to 49.

51. 51. The method of claim 50, wherein the subject is a mammal, optionally a human.

52. 51. The method of claim 50, wherein the subject is a bird.

53. 53. The method of claim 50, 51, or 52, wherein the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

Images