Ionizable Cationic Lipids and Lipid Nanoparticles

Abstract

Disclosed are ionizable cationic lipids, methods for synthesizing them, and intermediates and methods for synthesizing intermediates that are useful for synthesizing these lipids.Ionizable cationic lipids are useful as components of lipid nanoparticles (LNPs), which in turn can be used for intracellular delivery of nucleic acid in vivo or ex vivo.Also disclosed are LNP compositions, including LNPs that contain functionalized lipids to allow conjugation of binding moieties, and targeted LNPs (tLNPs) (i.e., LNPs in which binding moieties are conjugated to functionalized lipids and can also function as targeting moieties to guide tLNPs to desired tissues or cell types).

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 489,381, filed March 9, 2023, U.S. Provisional Patent Application No. 63 / 366,462, filed June 15, 2022, and U.S. Provisional Patent Application No. 63 / 362,501, filed April 5, 2022, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] background Lipid formulations have been used in the laboratory to deliver nucleic acids into cells. Early formulations based on the cationic lipid 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and the ionizable fusogenic lipid dioleoylphosphatidylethanolamine (DOPE) had large particle sizes that were problematic when used in vivo, exhibiting rapid clearance, lung tropism, and toxicity. To address these issues, lipid nanoparticles (LNPs) containing ionizable cationic lipids have been developed, leading to regulatory approval and market entry of RNA-based products such as siRNA ONPATTRO® and two mRNA-based SARS-CoV-2 vaccines. There is limited ability to control which tissues or cells take up LNPs upon administration. Intravenously administered LNPs are primarily taken up by the liver, lungs, or spleen, depending largely on net charge and particle size. It is possible to target more than 90% of LNPs to the liver by a combination of formulation and intravenous administration. Intramuscular administration can provide clinically useful levels of local delivery and expression. LNPs can be redirected to other tissues or cell types by conjugating a binding moiety with specificity for the target tissue or cell type to the LNP, for example, by conjugating a polypeptide containing an antigen-binding domain from an antibody. Nevertheless, avoiding uptake by the liver remains difficult. Furthermore, with current systems, only a small fraction of the encapsulated nucleic acid is successfully delivered into the cells and cytoplasm of interest. Current formulations can only release 2-5% of the administered RNA into the cytoplasm (see, for example, Gilleron et al., Nat. Biotechnol. 31:638-646, 2013 and Munson et al., Commun. Biol. 4:211-224, 2021). Thus, problems remain with off-target delivery, low efficiency of nucleic acid release into the cytoplasm, and toxicity associated with accumulation of constituent lipids. Thus, the present disclosure provides ionizable lipids and lipid nanoparticles that fulfill a pressing need in the art. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Gilleron et al., Nat. Biotechnol. 31:638-646, 2013 [Non-Patent Document 2] Munson et al., Commun. Biol. 4:211-224, 2021 Summary of the Invention [Means for solving the problem]

[0004] overview Certain embodiments of the present disclosure relate to ionizable cationic lipids having a structure selected from the group consisting of Formula 1, Formula 2, and Formula 3.

[0005] Another aspect of the present disclosure relates to lipid nanoparticles (LNPs) comprising one or more ionizable cationic lipids each and independently having a structure selected from the group consisting of Formula 1, Formula 2, and Formula 3. In certain embodiments, the LNPs may further comprise one or more of a phospholipid, a sterol, a co-lipid, a PEG-lipid, or a combination thereof. Examples of phospholipids include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), and combinations thereof. Examples of sterols include, but are not limited to, cholesterol, campesterol, sitosterol, stigmasterol, and combinations thereof. Examples of co-lipids include, but are not limited to, cholesterol hemisuccinate (CHEMS) and quaternary ammonium head group-containing lipids. Examples of quaternary ammonium head group-containing lipids include, but are not limited to, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA), 3β-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), and combinations thereof. Examples of PEG-lipids include PEG moieties with molecular weights (MW) of 1000-5000 Da, and / or C 14 ~C 18Examples of PEG-lipids include, but are not limited to, DMG-PEG2000 (1,2-dimyristoyl-glycero-3-methoxypolyethylene glycol-2000), DPG-PEG2000 (1,2-dipalmitoyl-glycero-3-methoxypolyethylene glycol-2000), DSG-PEG2000 (1,2-distearoyl-glycero-3-methoxypolyethylene glycol-2000), DOG-PEG2000 (1,2-dioleoyl-glycero-3-methoxypolyethylene glycol-2000), DMPE-PEG200 (1,2-dimyristoyl-glycero-3-methoxypolyethylene glycol-2000), glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), DPPE-PEG2000 (1,2-dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), DSPE-PEG2000 (1,2-distearoyl-glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), DOPE-PEG2000 (1,2-dioleoyl-glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), and combinations thereof. In certain embodiments, the PEG-lipid comprises an optically pure glycerol moiety. In certain embodiments, the LNP further comprises a functionalized PEG-lipid. In certain embodiments, the LNP of embodiment 28, the functionalized PEG-lipid is conjugated to a binding moiety (e.g., an antibody). In certain embodiments, the functionalized PEG-lipid is a C 16 ~C 18 In certain embodiments, the functionalized PEG-lipid comprises a dipalmitoyl lipid or a distearoyl lipid.

[0006] In certain embodiments, the LNPs comprise 40-60 mol% ionizable cationic lipid. In certain embodiments, the LNPs comprise 7-30 mol% phospholipid. In certain embodiments, the LNPs comprise 20-45 mol% sterol. In certain embodiments, the LNPs comprise 1-30 mol% co-lipid. In certain embodiments, the LNPs comprise 0-5 mol% PEG-lipid. In certain embodiments, the LNPs comprise 0.1-5 mol% functionalized PEG-lipid.

[0007] In certain embodiments, the LNP further comprises a nucleic acid (e.g., mRNA). In certain embodiments, the weight ratio of total lipid to nucleic acid is between 10:1 and 50:1.

[0008] Another aspect of the present disclosure relates to a method of delivering a nucleic acid into a cell, the method comprising contacting the cell with one or more LNPs disclosed herein, at least some of the LNPs comprising a nucleic acid. [Brief description of the drawings]

[0009] [Figure 1A] 1A-1F show synthetic schemes for compounds having the structure of Formula 1. Figure 1A shows the synthesis up to intermediate I-fA, starting with readily available reagents. [Figure 1B] FIG. 1B shows a synthetic route from intermediate I-fA to compounds having the structure of Formula 1 (Y=O, NH or N-CH3). [Figure 1C] FIG. 1C shows a synthetic route from intermediate I-fA to compounds having the structure of formula 1 (Y=CH2). Unless otherwise specified, all substituents are defined as in formula 1. [Figure 1D] Specifically, FIG. 1D shows the synthesis of intermediates Ic, Id, Ie, and If, which are embodiments of formulae I-cA, I-dA, I-eA, and I-fA, respectively, where p is 1, n is 1, and R is a C9 alkyl straight chain. [Figure 1E]FIG. 1E shows a synthetic route from intermediate If to compounds A-1 through A-3, which are embodiments of Formula 1 where Y=O, NH, and N-CH, respectively, where p is 1, n is 1, X is N(Me) and R is a C alkyl straight chain. [Figure 1F] FIG. 1F shows a synthetic route from intermediate If to compound A-4, an embodiment of Formula 1 where Y=CH2, where p is 1, n is 1, X is N(Me)2, and R is a C9 alkyl straight chain.

[0010] [Figure 2A] Figures 2A-2F show synthetic schemes for compounds having the structure of Formula 2. Figure 2A shows the synthesis up to intermediate II-hA, starting with readily available reagents. [Figure 2B] FIG. 2B shows a synthetic route from intermediate II-hA to compounds having the structure of Formula 2 (Y=O, NH or N-CH3). [Figure 2C] FIG. 2F shows a synthetic route from intermediate II-hA to compounds having the structure of formula 2 (Y=CH2). Unless otherwise specified, all substituents are defined as in formula 2. [Figure 2D] Specifically, FIG. 2D depicts the synthesis of intermediates II-c, II-d, II-e, II-f, II-g, and II-h, which are embodiments of formulas II-cA, II-dA, II-eA, II-fA, II-gA, and II-hA, respectively, where p is 1, n is 1, and R is a C9 alkyl straight chain, where applicable. [Figure 2E] FIG. 2E shows a synthetic route from intermediate II-h to compounds A-5 through A-7, which are embodiments of Formula 2 where Y=O, NH, and N-CH, respectively, where p is 1, n is 1, and R is a C alkyl straight chain. [Figure 2F] FIG. 2F shows a synthetic route from intermediate II-g to compound A-8, an embodiment of Formula 2 where Y=CH2, where p is 1, n is 1, and R is a C9 alkyl straight chain.

[0011] [Figure 3A] Figures 3A-3D show synthetic schemes for compounds having the structure of Formula 3. Figure 3A shows the synthesis to compounds having the structure of Formula 3 (W=C=O) starting with readily available reagents. [Figure 3B] FIG. 3D shows a synthetic route from intermediate III-cA to compounds having the structure of formula 3 (W=CH2). Unless otherwise specified, all substituents are defined as in formula 3. [Figure 3C] FIG. 3C shows the synthesis starting with readily available reagents to intermediates III-a, III-b, III-c, and compound A-9, which are embodiments of formula III-aA, III-bA, III-cA, and formula 3, respectively, where p is 1, n is 2, and Rc is a C9 alkyl straight chain, where applicable. [Figure 3D] FIG. 3D shows the synthetic route from intermediate III-c to intermediates III-d, III-e, III-f, and compound A-10, which are embodiments of III-dA, III-eA, III-fA, and formula 3 (W=CH2), where p is 1, n is 2, and Rc is a C9 alkyl straight chain.

[0012] [Figure 4A] 4A-B show synthetic schemes for reagents that can be used to create polyethylene glycol-containing lipid head groups. Figure 4A shows the synthesis of an embodiment of XR125 where m is 2 and o is 3 (see Table 3). [Figure 4B] FIG. 4B shows the synthesis of an embodiment of XR126 where o is 3 (see Table 3).

[0013] [Figure 5A-B]Figures 5A-C show the viability (5A), frequency of transfection (5B), and level of expression as geometric mean fluorescence intensity (gMFI) of transfected cells (5C) of HEK293F cells transfected with mCherry mRNA encapsulated within LNPs, where the ionizable cationic lipid was one of compounds A-2, A-11, A-12, A-13, A-14, or A-15. [Figure 5C] Figures 5A-C show the viability (5A), frequency of transfection (5B), and level of expression as geometric mean fluorescence intensity (gMFI) of transfected cells (5C) of HEK293F cells transfected with mCherry mRNA encapsulated within LNPs, where the ionizable cationic lipid was one of compounds A-2, A-11, A-12, A-13, A-14, or A-15.

[0014] [Figure 6] FIG. 6 shows the frequency and expression levels of mCherry mRNA transfected in vitro into mouse splenic T cells by CD5-targeted lipid nanoparticles incorporating the indicated ionizable cationic lipids A-2, A-11, A-12, A-13, A-14, and A-15, respectively, as determined by flow cytometry. Expression levels are presented as the mean fluorescence intensity (MFI: geometric mean) of the positive peak in the flow cytometry histogram, and transfection rates are the percentage of CD3+ cells in the positive peak. *: 10a, 10f, and 10p are described in Journal of Medicinal Chemistry 63:12992-13012, 2020.

[0015] [Figure 7]FIG. 7 shows the frequency and expression levels of mCherry mRNA transfected in vivo into mouse splenic T cells by CD5-targeted lipid nanoparticles incorporating the indicated ionizable cationic lipids A-2, A-11, A-12, A-13, A-14, and A-15, respectively, as determined by flow cytometry. Expression levels are presented as the mean fluorescence intensity (MFI: geometric mean) of the positive peak in the flow cytometry histogram, and transfection rates are the percentage of CD3+ cells in the positive peak. *: 10a, 10f, and 10p are described in Journal of Medicinal Chemistry 63:12992-13012, 2020.

[0016] [Figure 8] 8 shows a conceptual biodegradation scheme of compound A-11 (above the line) and the starting compounds and end products of biodegradation (below the line). The disclosed ionizable cationic lipids, and in particular the compounds of formula 1, may undergo biodegradation according to such conceptual scheme, without being bound by any particular theory. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description The present disclosure provides ionizable cationic lipids, methods for synthesizing them, and intermediates useful for synthesizing these lipids, and methods for synthesizing intermediates.The present disclosure provides ionizable cationic lipids of the present disclosure as components of lipid nanoparticles (LNPs), which can be used to deliver nucleic acid into cells in vivo or ex vivo.Also disclosed herein are LNP compositions that include LNPs that include functionalized PEG lipids to allow conjugation of binding moieties to generate targeted LNPs (tLNPs), i.e., LNPs that include binding moieties that direct tLNPs to desired tissues or cell types.Also disclosed herein are methods of delivering nucleic acid into cells, comprising contacting cells with the LNPs or tLNPs of the present disclosure.

[0018] Before describing this disclosure in more detail, it may be helpful to provide abbreviations and definitions of certain terms used herein. Additional definitions are set forth throughout this disclosure. Abbreviation

[0019] Abbreviations used herein include the following:

[0020] BF3-OEt2- Boron trifluoride diethyl etherate

[0021] BOC - tert-butyloxycarbonyl

[0022] CDI - Carbonyldiimidazole

[0023] cLogD - Calculation LogD

[0024] cLogP - calculated LogP (distribution coefficient)

[0025] c-pKa - calculated pKa

[0026] DMF - Dimethylformamide

[0027] DMAP - 4-Dimethylaminopyridine

[0028] EDC-HCl - 1-Ethyl-3-(3'-dimethylaminopropyl)carbodiimide·HCl

[0029] Et3N - Triethylamine

[0030] MeOH - Methanol

[0031] MeOTf - Methyl trifluoromethanesulfonate

[0032] Pd / C - Palladium on carbon

[0033] PEG - Polyethylene glycol

[0034] PPTs - Pyridinium p-Toluenesulfonate

[0035] TFA - Trifluoroacetic acid definition

[0036] As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The terms "a" and "an," as used herein, should be understood to refer to "one or more" of the listed components.

[0037] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0038] The term "about" when used herein in reference to a number refers to a range centered around that number, ranging from 15% less than that number to 15% more than that number. The term "about" when used in reference to a range refers to an expanded range ranging from 15% less than the lowest number recited in the range to 15% more than the highest number recited in the range.

[0039] Throughout this disclosure, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and where appropriate, fractional values ​​thereof (e.g., tenths and hundredths of an integer), unless otherwise specified. Also, any numerical range in this disclosure relating to any physical characteristic, such as polymer subunits, size, or thickness, should be understood to include any integer within the recited range, unless otherwise specified. Throughout this disclosure, any numerical range includes its recited endpoints, unless otherwise specifically specified.

[0040] Unless the context requires otherwise, throughout this specification and claims, the term "comprise" and variations thereof, such as "comprises" and "comprising," should be interpreted in an open-ended inclusive sense, i.e., "including, but not limited to." As used herein, the terms "include" and "comprise" are used interchangeably.

[0041] The phrase "at least one of," when followed by a list of items or elements, refers to an open-ended set of one or more of the elements in the list, which may include more than one of the elements, but does not necessarily include more than one of the elements.

[0042] "Derivative" as used herein refers to a chemically or biologically modified version of a compound that is structurally similar to and derivable (actually or theoretically) from the parent compound. In general, a "derivative" differs from an "analog" in that while the parent compound may be the starting material for producing a "derivative," the parent compound may not necessarily be used as the starting material for producing an "analog." A derivative may have different chemical or physical properties than the parent compound. For example, a derivative may be more hydrophilic or hydrophobic, or may have altered reactivity compared to the parent compound.

[0043] Alkyl refers to a saturated hydrocarbon moiety that is an alkane that has lost one hydrogen leaving a bond that connects it to another part of an organic molecule. In some embodiments, the hydrogen is unsubstituted. In other embodiments, one or more hydrogens of an alkyl group may be replaced with the same or different substituents.

[0044] Alkenyl refers to a hydrocarbon moiety having one or more carbon-carbon double bonds, but which is otherwise saturated. In some embodiments, the hydrogen is unsubstituted. In other embodiments, one or more hydrogens of an alkenyl group may be replaced with the same or different substituents.

[0045] Alkynoic refers to a carboxylic acid moiety that contains one or more carbon-carbon triple bonds. In some embodiments, the hydrogen is unsubstituted. In other embodiments, one or more hydrogens of an alkynoic group may be replaced with the same or different substituents.

[0046] Amide refers to a carboxylic acid derivative that contains a carbonyl group of the carboxylic acid attached to an amine moiety.

[0047] Aryl refers to an aromatic or heteroaromatic ring that has lost a hydrogen, leaving a bond connecting it to another part of an organic molecule. Examples of aryl include, but are not limited to, phenyl, naphthalenyl, pyridine, pyrimidine, pyrazine, pyrrole, furan, thiophene, imidazole, thiazole, oxazole, etc.

[0048] Aryl-alkyl refers to a moiety that includes one or more aryl rings and one or more alkyl moieties. The position of one or more aryl rings can vary within the alkyl moiety of the moiety. For example, one or more aryl rings can be at the end of one or more alkyl moieties, can be fused into the carbon chain of one or more alkyl moieties, and can replace one or more hydrogens of one or more alkyl moieties; one or more alkyl moieties can replace one or more hydrogens of one or more aryl rings. In some embodiments, there is a single ring, while in other embodiments, it is multiple rings.

[0049] A branched alkyl is a saturated alkyl moiety in which the alkyl group is not linear. The alkyl moiety (e.g., methyl, ethyl, propyl, butyl, etc.) may be attached to variable positions on the main alkyl chain. In some embodiments, a single branch is present, while in other embodiments, multiple branches are present.

[0050] Branched alkenyl refers to an alkenyl group that includes at least one branch from the main chain, which may be formed by replacing one or more hydrogens of the main chain with the same or different alkyl groups (such as, but not limited to, methyl, ethyl, propyl, butyl, etc.) In some embodiments, a branched alkenyl is a single branched structure, while in other embodiments, a branched alkenyl may have multiple branches.

[0051] Straight chain alkyls are unbranched, acyclic versions of the alkyl moieties mentioned above.

[0052] Straight chain alkenyls are unbranched, acyclic versions of the alkenyl moieties mentioned above.

[0053] Cycloalkyl refers to a moiety that is a cycloalkyl ring of 3 to 12 carbons. In some embodiments, the cycloalkyl is a single ring structure, while in other embodiments, the cycloalkyl can have multiple rings.

[0054] Cycloalkyl-alkyl refers to a moiety that includes one or more cycloalkyl rings having 3-12 carbons and one or more alkyl moieties. The position of the cycloalkyl rings can vary within the alkyl moiety of the moiety. For example, one or more cycloalkyl rings can be at the terminus of one or more alkyl moieties, can be fused within the carbon chain of one or more alkyl moieties, and can replace one or more hydrogens of one or more alkyl moieties; one or more alkyl moieties can replace one or more hydrogens of one or more cycloalkyl rings. In some embodiments, the cycloalkyl ring is a single ring structure, while in other embodiments, the cycloalkyl-alkyl can have multiple rings.

[0055] Ester refers to a carboxylic acid derivative that contains a carbonyl group linked to an alkyloxy group to form an ester bond, -C(=O)-O-.

[0056] Ether refers to an oxygen atom bonded to the same or different two-carbon based moiety.

[0057] Head group refers to the hydrophilic or polar portion of the lipid.

[0058] Imide refers to a moiety that contains a nitrogen bond to two carbonyl groups.

[0059] Sterols refer to a subgroup of steroids that contain at least one hydroxyl (OH) group. Examples of sterols include, but are not limited to, cholesterol, ergosterol, β-sitosterol, stigmasterol, stigmastanol, 20-hydroxycholesterol, 22-hydroxycholesterol, etc. Ionizable Cationic Lipids

[0060] Disclosed are ionizable cationic lipids useful as components of lipid nanoparticles for delivery of nucleic acids, including DNA, mRNA, or siRNA, into cells. The ionizable cationic lipids have a c-pKa of 8-11 and a cLogD of 9-18 or 11-14. These ranges may result in a measured pKa in LNPs or tLNPs of 6-7, which promotes ionization in endosomes. In some embodiments, somewhat higher basicity may be desirable and may be obtained from ionizable cationic lipids having c-pKa and cLogD within the aforementioned ranges. In some embodiments, the c-pKa is about 8, about 9, about 10, or about 11, or within a range bounded by any pair of these values. In some embodiments, the cLogD is about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or within a range bounded by any pair of these values.

[0061] In certain embodiments, the ionizable cationic lipid is represented by the formula: [ka] (Wherein, Y is O, NH, N-CH3, or CH2; n is an integer from 0 to 4, X is [ka] [ka] and m is an integer from 1 to 3, o is an integer from 1 to 4, p is an integer from 1 to 4, When p=1, each R is independently C6 to C 16 Straight chain alkyl; C6~C 16 Branched alkyl; C6-C 16 Straight chain alkenyl; C6~C 16 Branched alkenyl; C9-C16 Cycloalkyl-alkyl (cycloalkyl is C3-C8 cycloalkyl located at either end or within the alkyl chain); or C8-C 18 aryl-alkyl, where aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; When p=2, each R is independently C6 to C 14 Straight chain alkyl; C6~C 14 Straight chain alkenyl; C6~C 14 Branched alkyl; C6-C 14 Branched alkenyl; C9-C 14 Cycloalkyl-alkyl (cycloalkyl is C3-C8 cycloalkyl located at either end or within the alkyl chain); or C8-C 16 aryl-alkyl, where aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; When p=3, each R is independently C6 to C 12 Straight chain alkyl; C6~C 12 Straight chain alkenyl; C6~C 12 Branched alkyl; C6-C 12 Branched alkenyl; C9-C 12 Cycloalkyl-alkyl (cycloalkyl is C3-C8 cycloalkyl located at either end or within the alkyl chain); or C8-C 14 aryl-alkyl, where aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; When p=4, each R is independently C6 to C 10 Straight chain alkyl; C6~C 10 Straight chain alkenyl; C6~C 10 Branched alkyl; C6-C 10 Branched alkenyl; C9-C 10 Cycloalkyl-alkyl (cycloalkyl is C3-C8 cycloalkyl located at either end or within the alkyl); or C8-C 12It has the structure aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain.

[0062] Some embodiments specifically include one or more species or subgenera based on specific selections of R, X, Y, m, n, o, p, and / or carbon chain length, structure, or saturation. Other embodiments specifically exclude one or more species or subgenera based on specific selections of R, X, Y, m, n, o, p, and / or carbon chain length, structure, or saturation. In some embodiments, when p is 1, each R is independently selected from C8 to C 12 , C 13 , or C 14 It is a straight chain alkyl. In some embodiments, each R from the nearest common branch point is the same. In some embodiments, each R is the same.

[0063] In some embodiments, the ionizable cationic lipid is represented by formula 1a [ka] (Wherein, each R is independently C6 to C 16 Straight chain alkyl; C6~C 16 Straight chain alkenyl; C6~C 16 Branched alkyl; C6-C 16 Branched alkenyl; C9-C 16 Cycloalkyl-alkyl (cycloalkyl is C3-C8 cycloalkyl located at either end or within the alkyl chain); or C8-C 18 aryl-alkyl, where aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; Y is O, NH, N-CH3, or CH2; n is an integer from 0 to 4, X is [ka] and m is an integer from 1 to 3, and o is an integer from 1 to 4.

[0064] In other embodiments, the ionizable cationic lipid is represented by the formula: [ka] (Wherein, Y is O, NH, N-CH3, or CH2; n is an integer from 0 to 4, X is [ka] and m is an integer from 1 to 3, o is an integer from 1 to 4, p is an integer from 1 to 4, When p=1, each R is independently C6 to C 16 Straight chain alkyl; C6~C 16 Straight chain alkenyl; C6~C 16 Branched alkyl; C6-C 16 Branched alkenyl; C9-C 16 Cycloalkyl-alkyl (cycloalkyl is C3-C8 cycloalkyl located at either end or within the alkyl chain); or C8-C 18 aryl-alkyl, where aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; When p=2, each R is independently C6 to C 14 Straight chain alkyl; C6~C 14 Straight chain alkenyl; C6~C 14 Branched alkyl; C6-C 14 Branched alkenyl; C9-C 14 Cycloalkyl-alkyl (cycloalkyl is C3-C8 cycloalkyl located at either end or within the alkyl chain); or C8-C 16aryl-alkyl, where aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; When p=3, each R is independently C6 to C 12 Straight chain alkyl; C6~C 12 Straight chain alkenyl; C6~C 12 Branched alkyl; branched C6-C 12 Alkenyl; C9~C 12 Cycloalkyl-alkyl (cycloalkyl is C3-C8 cycloalkyl located at either end or within the alkyl chain); or C8-C 14 aryl-alkyl, where aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; When p=4, each R is independently C6 to C 10 Straight chain alkyl; straight chain C6-C 10 Alkenyl; C6~C 10 Branched alkyl; C6-C 10 Branched alkenyl; C9-C 10 Cycloalkyl-alkyl (cycloalkyl is C3-C8 cycloalkyl located at either end or within the alkyl); or C8-C 12 It has the structure aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain.

[0065] Some embodiments include one or more species or subgenera based on specific selections of R, X, Y, m, n, o, p, and / or carbon chain length, structure, or saturation. Other embodiments specifically exclude one or more species or subgenera based on specific selections of R, X, Y, m, n, o, p, and / or carbon chain length, structure, or saturation. In some embodiments, each R from the nearest common branch point is the same. In some embodiments, each R is the same.

[0066] In some embodiments, the ionizable cationic lipid is represented by formula 2a [ka] (Wherein, R is C6 to C 16 Straight chain alkyl; C6~C 16 Straight chain alkenyl; C6~C 16 Branched alkyl; branched C6-C 16 Alkenyl; C9~C 16 Cycloalkyl-alkyl (cycloalkyl is C3-C8 cycloalkyl located at either end or within the alkyl chain); or C8-C 18 aryl-alkyl, where aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; Y is O, NH, N-CH3, or CH2; n is an integer from 0 to 4, X is [ka] and m is an integer from 1 to 3, and o is an integer from 1 to 4.

[0067] In a further embodiment, the ionizable cationic lipid is represented by formula 3 [ka] (Wherein, W is C=O or CH2; n is an integer from 0 to 4, X is [ka] and m is an integer from 1 to 3, o is an integer from 1 to 4, p is an integer from 1 to 4, If p=1, then each R c are independent, C8~C 18 Straight chain alkyl; C8~C18 Straight chain alkenyl; C8~C 18 Branched alkyl; C8-C 18 Branched alkenyl; C 11 ~C 18 Cycloalkyl-alkyl (cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl chain); or C 10 ~C 20 aryl-alkyl, where aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; If p=2, then each R c are independent, C8~C 16 Straight chain alkyl; C8~C 16 Straight chain alkenyl; C8~C 16 Branched alkyl; C8-C 16 Branched alkenyl; C 11 ~C 16 Cycloalkyl-alkyl (cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl chain); or C 10 ~C 18 aryl-alkyl, where aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; If p=3, then each R c are independent, C8~C 14 Straight chain alkyl; C8~C 14 Straight chain alkenyl; C8~C 14 Branched alkyl; C8-C 14 Branched alkenyl; C 11 ~C 14 Cycloalkyl-alkyl (cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl chain); or C 10 ~C 16 aryl-alkyl, where aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; If p=4, then each R c are independent, C8~C 12 Straight chain alkyl; C8~C12 Straight chain alkenyl; C8~C 12 Branched alkyl; C8-C 12 Branched alkenyl; C 11 ~C 12 Cycloalkyl-alkyl (cycloalkyl is C3-C8 cycloalkyl located at either end or within the alkyl); or C 10 ~C 14 It has the structure aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain.

[0068] Some embodiments include R c , W, X, m, n, o, p, and / or one or more species or subgenera based on specific choices of carbon chain length, structure or saturation. c In some embodiments, the distance between each R from the nearest common branch point is specifically excluded from one or more species or subgenera based on a particular selection of R, W, X, m, n, o, p, and / or carbon chain length, structure, or saturation. c In some embodiments, each R c is the same.

[0069] In some embodiments, the ionizable cationic lipid is represented by formula 3a [ka] (In the formula, R c is C8~C 18 Straight chain alkyl; C8~C 18 Straight chain alkenyl; C8~C 18 Branched alkyl; C8-C 18 Branched alkenyl; C 11 ~C 18 Cycloalkyl-alkyl (cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl chain); or C 10 ~C 20aryl-alkyl, where aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; W is C=O or CH2; n is an integer from 0 to 4, X is [ka] and m is an integer from 1 to 3, and o is an integer from 1 to 4.

[0070] For each of the above aspects, in some embodiments, all four R groups are the same. In other embodiments, the two R groups emanating from the first branch point are the same. c The groups are identical to each other, and the two R c The groups are identical to each other, but the R c The R group is different from the R group emanating from the second branch point.

[0071] For each of the above aspects, some embodiments include, where applicable, R c , W, X, Y, m, n, o, and / or p. Other embodiments are limited to one or a subset of the options R c , W, X, Y, m, n, o, p, and / or carbon chain length, structure or saturation options are specifically excluded. Each range of carbon chain lengths is intended to convey embodiments of all individual lengths and subranges thereof.

[0072] For each of the above aspects and embodiments, in some cases, R c is a straight chain alkyl, and in a further case, the chain is unsubstituted. In yet a further case, R c is C8 or C9 or C 10 ~C 12 It is.

[0073] For each of the above aspects and embodiments, in some cases, X is [ka] For each of the above aspects and embodiments, in some cases, Y is O, and in other cases, Y is NH or N-CH.

[0074] cLogD is a calculated measure of lipophilicity that takes into account the ionization state of a molecule at a particular pH and predicts the partitioning of lipids between water and octanol as a function of pH. More specifically, cLogD is calculated at a specified pH based on cLogP and c-pKa. (LogP is the partition coefficient of a molecule between the aqueous phase and the lipophilic phase, usually considered as octanol and water.) If a more basic ionizable lipid is desired, it should be balanced by a higher lipophilicity, as represented by a higher cLogD value. The balance of basicity and lipophilicity is used herein to maximize LNP function, including both LNP stability and release of cargo (e.g., nucleic acid) after uptake by cells. Thus, as m, n, or p increase, the overall lipophilicity of the ionizable cationic lipids disclosed herein, as represented by cLogD, can be balanced by a shorter chain length of R. Some embodiments of the ionizable cationic lipid species encompassed by Formulas 1-3 have a cLogD in the range of about 9 to about 18 or about 9 to about 22, calculated using ACD Labs Structure Designer v12.0, where cLogP was calculated using ACD Labs Version B, and cLogD was calculated at pH 7.4.

[0075] The measured pKa of 6-7 for the LNPs that carry nucleic acid load ensures that the ionizable cationic lipids in the LNPs remain essentially neutral in the bloodstream and interstitial spaces, but become ionized upon endosome acidification after uptake into cells. After acidification in the endosomal space, the lipids protonate and more strongly associate with the phosphate backbone of the nucleic acid, thereby destabilizing the structure of the LNP and facilitating nucleic acid release from the LNP into the cytoplasm (also referred to as endosomal escape). Thus, the ionizable cationic lipids disclosed herein constitute a means for destabilizing the LNP structure (when ionized) or for facilitating nucleic acid release or endosomal escape.

[0076] The ionizable cationic lipids of the present disclosure have a branched structure that gives the lipids a conical rather than cylindrical shape, which helps promote endosome-eluting activity. The higher the endosome-eluting activity, the more efficient the release of the nucleotide cargo.

[0077] To promote biodegradability and minimize accumulation of the ionizable cationic lipids of the present disclosure, the fatty acid tail is designed to contain esters in positions that minimize steric hindrance of ester cleavage. For example, a single fatty acid tail tends to extend away from the ester carbonyl, while the presence of two tails results in tails extending in the opposite direction, as this is an energetically favorable conformation. This means that one of the tails will extend toward the carbonyl, which may sterically inhibit ester cleavage. Thus, large branches immediately adjacent to the ester carbonyl were avoided. In positioning the ester(s) within the lipid, potential degradation products were also considered to avoid the generation of toxic compounds such as formaldehyde.

[0078] Another consideration that may contribute to the tolerability of lipids is the extent to which ester cleavage or other catabolism produces fragments or by-products, and whether such fragments or by-products can be eliminated from the body without the involvement of oxidative degradation in the liver. The ionizable cationic lipids of the present disclosure are expected to be readily biodegradable, and the fragments are expected to be easily eliminated. For example, FIG. 8 shows that esterase cleavage or other hydrolysis of compound A-11 is predicted to produce tetraalcohol B and 4 equivalents of nonanoic acid. Cyclization should then result in the production of 2 equivalents of butyrolactone C and 1 equivalent of diol D. Esterase hydrolysis of C results in the production of 2 equivalents of diol-acid E. The products of biodegradation of A-11 are shown collectively below the line in FIG. 8. All of these entities are small and polar, and are expected to be eliminated from the body without the need for hepatic oxidation or conjugation. These considerations take on added importance when tLNP LNPs are used in long-term dosing regimens.

[0079] The advantage of relying at least in part on the ionizable cationic lipid of the present disclosure is that it avoids the toxicity associated with quaternary ammonium cationic lipids. Some LNPs based on such lipids, which are effectively permanently cationic, have shown lethal hyperacute toxicity in experimental animals. By using the ionizable cationic lipid of the present disclosure in LNPs, the use of quaternary ammonium cationic lipids can be substantially reduced, and toxicity can be reduced or avoided. In certain embodiments, the use of the LNPs or tLNPs of the present disclosure does not cause detectable toxicity to cells or in subjects. In certain embodiments, the use of the LNPs or tLNPs of the present disclosure causes only weak toxicity to cells or in subjects, i.e., it is asymptomatic or induces only mild symptoms that do not require intervention. In certain embodiments, the use of the LNPs or tLNPs of the present disclosure causes only moderate toxicity to cells or in subjects that requires only minimal, local or non-invasive intervention, and can impair activities in daily life.

[0080] The relationship between the efficacy and toxicity of a drug is generally expressed in terms of therapeutic window and therapeutic index. The therapeutic window is the dose range from the lowest dose that shows a detectable therapeutic effect to the maximum tolerated dose (MTD), i.e., the highest dose that provides the desired therapeutic effect without producing unacceptable toxicity. The most typical therapeutic index is calculated as the ratio of LD50:ED50 when based on animal studies, or TD50:ED50 when based on human studies (although this calculation can also be derived from animal studies and is sometimes called the protection index), where LD50, TD50, and ED50 are the doses that are lethal, toxic, and effective, respectively, in 50% of the test population. These concepts can be applied whether the toxicity is based on the active agent itself or on some other component of the drug product, such as the LNP or its components. For any inherent level of toxicity of the disclosed lipids or LNPs themselves, an increase in the efficiency of delivering nucleic acid into the cytoplasm improves the therapeutic window or index, since an effective amount of nucleic acid can be delivered with a smaller dosage of the LNP (and its constituent lipids).

[0081] Toxicity and adverse events are sometimes graded according to a 5-point scale. Grade 1 or mild toxicity is asymptomatic or induces only mild symptoms; may be characterized by only clinical or diagnostic findings; no intervention is indicated. Grade 2 or moderate toxicity may impair activities of daily living (e.g., food preparation, shopping, money management, telephone use, etc.), but only minimal, localized or non-invasive intervention is indicated. Grade 3 toxicity is medically significant but not immediately life-threatening; hospitalization or extended hospitalization is indicated; and activities of daily living related to self-care (e.g., bathing, dressing, feeding oneself, using the toilet, taking medications, and not being bedridden) may be impaired. Grade 4 toxicity is life-threatening and urgent intervention is indicated. Grade 5 toxicity results in death related to the adverse event. Thus, in various embodiments, the use of the disclosed LNPs and tLNPs limits toxicity to grade 2 or less, grade 1 or less, or results in no observations of toxicity.

[0082] In some cases, the LNPs and tLNPs of the disclosure are used according to a specified schedule, provided in a particular dosage, or administered via a particular route of administration. Methods for Making Ionizable Cationic Lipids

[0083] The synthesis of ionizable lipids can be simplified using structural symmetry and convergent rather than linear synthetic routes.

[0084] In certain aspects, the disclosure provides a method of synthesizing an ionizable cationic lipid of formula 1. In some embodiments, the method includes converting an intermediate having the structure of I-fA to an ionizable cationic lipid of formula 1. In some embodiments, the method further includes synthesizing an intermediate having the structure of I-fA (e.g., FIG. 1A).

[0085] In certain embodiments of the method for synthesizing an ionizable cationic lipid of formula 1 (Y=O, NH, or N-CH), the method further comprises reacting I-fA with carbonyldiimidazole to produce I-gA. In certain embodiments, the method further comprises reacting I-gA and X-(CH) n+2 In certain embodiments, the method further comprises coupling I-gA and X-(CH2) n+2 The coupling reaction of -YH is carried out in the presence of an alkylating agent. In certain embodiments, the alkylating agent is MeOTf, as shown in FIG. 1B. In certain embodiments, the coupling reaction converts an intermediate having the structure I-hA to X-(CH2) n+2 -YH to generate an ionizable cationic lipid of formula 1, where Y=O, NH, or N-CH. In certain embodiments, I-hA and X-(CH) n+2 The coupling reaction with -YH is carried out in the presence of a base, such as, but not limited to, NaH or Et3N.

[0086] In certain embodiments of the method for synthesizing an ionizable cationic lipid of formula 1 (Y=CH2), the method comprises forming an intermediate having the structure I-fA into a compound represented by the formula X-(CH2) n+3 -COOH to provide an ionizable cationic lipid of formula 1. In certain embodiments, the coupling method is carried out in the presence of DMAP and Et3N, for example as shown in Figure 1C.

[0087] In certain embodiments of the method for the synthesis of I-fA, the method comprises converting the intermediate of I-dA into (HO-CH2-(CH2) p)2-N-PG to provide an amine-protected derivative of I-fA, where PG is a protecting group for the amine. In certain embodiments, PG is -CO2t-Bu, as shown in FIG. 1A. In certain embodiments of the method for the synthesis of I-fA, the amine-protected derivative of I-fA is deprotected to provide I-fA. For example, the deprotection reagent can be TFA in dimethyl chloride, as shown in FIG. 1A. In certain embodiments, the method further comprises the synthesis of I-dA.

[0088] In certain embodiments, the method of synthesis of I-dA includes preparing a carboxylic acid derivative of I-dA, where the carboxylic acid moiety of I-dA is protected with a protecting group that can be selectively deprotected in preference to hydrolysis of the R-COO- moiety. In certain embodiments, the carboxylic acid derivative of I-dA is I-cA, which is a t-butyl ester of I-dA. See, for example, FIG. 1A. In certain embodiments, the carboxylic acid derivative of I-dA is prepared by reacting the desired diol carboxylic acid derivative (e.g., Ib, where the carboxylic acid derivative is a t-butyl ester, in other embodiments the derivative can be in other forms) and R-COOH. In certain embodiments, the diol carboxylic acid derivative is prepared by hydrogenation of an alkenyl diol carboxylic acid derivative (e.g., Ia, where the carboxylic acid derivative is a t-butyl ester, in other embodiments the derivative can be in other forms). In certain embodiments, the alkenyl diol carboxylic acid derivative is prepared by reacting dihydroxyacetone and alkyloxycarbonylmethylenetriphenylphosphorane (e.g., alkyl can be t-butyl as shown in FIG. 1A).

[0089] In some embodiments, the method of synthesizing an ionizable cationic lipid of formula 1 proceeds according to the synthetic schemes of formulas 1D-F. In some embodiments, the method of synthesizing an ionizable cationic lipid of formula 1 proceeds according to Examples 5-16 and 24 (e.g., compounds A-11-A-15), 17-23 (e.g., compound A-2), or 25-33 (e.g., compound A-16), and analogs of these compounds having different m, n, o, p, R, X, and / or Y can be made by substituting reactants as described herein. In some embodiments, the method is a method of synthesizing an ionizable cationic lipid of formula 1a. In some cases, the method is a method of synthesizing compound A-1, compound A-2, compound A-3, compound A-4, compound A-11, compound A-12, compound A-13, compound A-14, compound A-15, or compound A-16. In some embodiments, the methods specify only a single step, or a subset of steps, shown in Figures 1D-F or Examples 5-16 and 24, 17-23, or 25-33, resulting in a final product.

[0090] Further embodiments are intermediates I-cA, I-dA, I-eA, I-fA, I-gA, I-hA, Id, I-d2, Ie, I-e2, If, I-f2, Ig, I-g2, Ih, and I-h2 of the synthetic schemes of Figures 1A-F, Examples 8-12, and Examples 18-22, where the substituents are defined as in Formula 1 unless otherwise specified.

[0091] A further aspect is a method of synthesizing an intermediate of the synthetic scheme of Figures 1D-F, where the intermediate is Id, Ie, If, Ig, or Ih. In some embodiments, the method specifies only the final steps to produce the intermediate shown in Figures 1D-F. In other embodiments, the method specifies all or a subset of the steps shown in Figures 1D-F until the intermediate is reached. Further embodiments relate to analogs of intermediates Id, Ie, If, Ig, or Ih suitable for final products with different X, n, or p, such as I-eA or I-gA, such as I-d2, I-e2, I-f2, I-g2, or I-h2 shown in Examples 8-12 and Examples 18-22.

[0092] In the synthetic schemes shown in Figures 1D-F, the value of p is 1, which results from the coupling of intermediate Id with a BOC-blocked diethanolamine. Compounds with p between 2 and 4 can be synthesized by substituting appropriately sized BOC-blocked dialkylamino alcohols, namely 3,3'-azanediylbis(propan-1-ol), 4,4'-azanediylbis(butan-1-ol), and 5,5'-azanediylbis(pentan-1-ol), respectively.

[0093] In the synthesis schemes shown in Figures 1D-F, the value of n is 1, resulting from the reaction of intermediate Ih with 3-dimethylamino-1-propanol, N,N-dimethyl-1,3-propanediamine, N,N,N'-trimethyl-1,3-propanediamine in the presence of a base to produce compounds A-1-A-3, respectively, where Y is O, NH, or N-CH3, respectively. Compounds where n is 0 or 2-4 can be synthesized by replacing the propanediamine moiety with the analogous C2, C4, C5, or C6 moiety. Compound A-4 where Y is CH2 can be obtained by reacting the salt of intermediate If with 5-dimethylamino-pentanoic acid. Compounds where n is 0 or 2-4 can be synthesized by replacing the pentanoic acid moiety with the analogous C4, C6, C7, or C8 moiety.

[0094] In the synthetic schemes shown in Figures 1D-F, R is C9, resulting from the use of decanoic acid in the conversion of intermediate Ib to intermediate Ic. Alternative -oic acids of corresponding chain length and structure are C6-C8 or C6-C8, as appropriate. 10 ~C 18 can be used to obtain R

[0095] In the synthetic schemes shown in Figures 1D-F, X is N(CH3)2. Compounds according to formula 1 with alternative definitions of X can be synthesized by reacting alternative headgroup pieces from Tables 1-3 with Ih to give analogs of compounds A-1-A-3, respectively, as disclosed in Example 1 (infra), or by reacting alternative headgroup pieces from Table 4 with If to give analogs of compound A-4. The synthesis of headgroup pieces not previously disclosed in the art can be made similarly to their shorter congeners, or, in the case of polyethylene glycol-containing headgroup pieces, can be made according to the synthetic schemes shown in Figures 4A-B and disclosed in Example 4, or described in Examples 25-32 (infra).

[0096] The synthesis of polyethylene glycol-containing headgroup pieces requires polyethylene glycol amines and related reagents not previously described. Thus, some embodiments are intermediates V-5, V-5a, V-6, V-6A, V-7, V-7a, V-8, V-8a, V-12, V-13, V-14, or V-15 and methods of synthesis thereof according to the synthetic schemes shown in Figures 4A-B and disclosed in Example 4 or described in Examples 25-32.

[0097] Intermediates Ie and I-eA: [ka] To synthesize analogs having the structure Ia, where p is an integer between 1 and 4, and R is defined as in formula 1, dihydroxyacetone is reacted with tert-butoxycarbonylmethylenetriphenylphosphorane to give alkene Ia. Hydrogenation in the presence of Pd / C, for example in ethyl acetate, gives Ib (Figure 1D). Coupling of Ib with an appropriate carboxylic acid for the desired R in the presence of EDC-HCl and DMAP in dichloromethane gives triester Ic (Figure 1D) or its analogs with different R, denoted I-cA. Hydrolysis of the t-butyl ester with TFA in dichloromethane gives key monoacids, Id (Figure 1D) or its analogs with different R. Coupling of Id or its analogs with commercially available BOC-blocked diethanolamines (Figure 1D) or appropriately sized BOC-blocked dialkylamino alcohols (n=2-4) in dichloromethane gives compounds having the structure I-eA.

[0098] Intermediate Ig and its analogs with different R and / or p, i.e., the structures: [ka] To synthesize intermediates with the structure I-eA (where p is an integer between 1 and 4 and R is defined as in formula 1), intermediates with the structure I-eA are treated with TFA in dichloromethane to remove the BOC protecting group to give the salt If (Figure 1D), or analogs thereof with different R and / or p (e.g., intermediates with the structure I-fA shown in Figure 1A). The products are then converted to acyl-imidazolides Ig (Figure 1E), or acyl-imidazolides with the structure I-gA (Figure 1B), after reaction with carbonyldiimidazole and triethylamine in dichloromethane.

[0099] To complete the synthesis of compounds A-1 to A-3, as well as their analogs with different R and / or p, the required reactive intermediate is obtained by reacting an intermediate having the structure of I-gA with methyl triflate to generate the acyl-imidazolium Ih (Figure 1E), or its analogs with different R, such as I-h2 (Example 12) or I-hA (Figure 1B) for R of linear C8. When p=1 and n=1, the acyl-imidazolium intermediate is then reacted with 3-dimethylamino-1-propanol in the presence of triethylamine to provide compound A-1 (FIG. 1E) or an analogue with a different p (e.g., FIG. 1B); with N,N-dimethyl-1,3-propanediamine and triethylamine to provide compound A-2 (FIG. 1E; see also Example 23) or an analogue with a different p (e.g., FIG. 1B); or with N,N,N'-trimethyl-1,3-propanediamine and triethylamine to provide compound A-3 (FIG. 1E) or an analogue with a different p (FIG. 1B), in each case in dichloromethane.

[0100] To complete the synthesis of compound A-4 (FIG. 1F), or an analog thereof having different R and / or p, the salt If (FIG. 1C), or an analog thereof having different R and / or p, is reacted with 5-dimethylamino-pentanoic acid in the presence of EDC-HCl, DMAP, and triethylamine in dichloromethane to provide compound A-4 (FIG. 1F), or an analog thereof having different R and / or p (FIG. 1C).

[0101] Alternatives to the reagents used to obtain analogs of compounds A-1 through A-4 with different X, Y, and / or n are described above and are applicable to the above synthesis to obtain analogs of compounds A-1 through A-4 (Figures 1A-C) with different R and / or p.

[0102] Such analogs include compounds A-11 to A-15, where R is linear C8 instead of linear C9 as in A-1 to A-4. Additionally, A-11 differs from A-1 in that n=0 instead of n=1. A-12 differs from A-1 only in R. Additionally, A-13 differs from A-2 in that n=0 instead of n=1. Additionally, A-14 differs from A-3 in that n=0 instead of n=1. A-15 differs from A-3 only in R.

[0103] To complete the synthesis of compound A-11, the acyl-imidazolium I-h2 is reacted with 2-dimethylamino-ethanol in the presence of tetramethylethylenediamine as in Example 13. Analogues of compound A-11 that retain p=1 but have other R are made by substituting the acyl-imidazolium generated from the appropriate species of I-gA.

[0104] To complete the synthesis of compound A-12, the acyl-imidazolium I-h2 is reacted with 3-dimethylamino-propanol in the presence of tetramethylethylenediamine as in Example 14. Analogues of compound A-12 that retain p=1 but have other R are made by substituting the acyl-imidazolium generated from the appropriate species of I-gA.

[0105] To complete the synthesis of compound A-13, the acyl-imidazolium I-h2 is reacted with 2-dimethylamino-ethanol in the presence of triethylamine as in Example 15. Analogues of compound A-13 that retain p=1 but have other R are made by substituting the acyl-imidazolium generated from the appropriate species of I-gA.

[0106] To complete the synthesis of compound A-14, the acyl-imidazolium I-h2 is reacted with N,N,N'-trimethylethylenediamine in the presence of triethylamine as in Example 16. Analogues of compound A-14 that retain p=1 but have other R are made by substituting the acyl-imidazolium generated from the appropriate species of I-gA.

[0107] To complete the synthesis of compound A-15, the acyl-imidazolium I-h2 is reacted with N,N,N'-trimethylpropylenediamine in the presence of triethylamine as in Example 24. Analogues of compound A-15 that retain p=1 but have other R are made by substituting the acyl-imidazolium generated from the appropriate species of I-gA.

[0108] Y is N-CH3 and X is [ka] In compound A-16, a species of formula 1a where n is 1, m is 2, and o is 1, the head group piece terminates in a small polyethylene glycol moiety. Compound A-16 can be made according to the synthetic scheme presented in Example 4, and has been synthesized as shown in Examples 25-33. In these latter examples, I-d2 is finally reacted with V-15 in the presence of DMAP and EDC-HCl in dichloromethane. Analogs of I-d2 with different hydrocarbon tails (e.g., I-dA in FIG. 1A) can be used to generate analogs of compound A-16 with different R.

[0109] To synthesize V-15, one can start with tert-butyl(3-hydroxypropyl)(methyl)carbamate by adding a cooled suspension of NaH in THF to the tert-butyl(3-hydroxypropyl)(methyl)carbamate. A solution of 2-methoxyethyl methanesulfonate in THF is then added slowly and the mixture is stirred at elevated temperature for an extended period of time. After cooling to room temperature, the reaction is quenched by careful addition of saturated aqueous NH4Cl. The mixture is placed in ethyl acetate, the organic phase is separated, the aqueous phase is extracted with ethyl acetate, and the combined organic phase is washed with brine and dried over Na2SO4. The filtrate is concentrated to produce crude V-5a, which is dissolved in dichloromethane and dried over silica gel. The silica gel is placed in a column and V-5a is eluted with dichloromethane and concentrated to a yellow oil.

[0110] To synthesize V-6a, V-5a in dioxane is exposed to slowly added acid, e.g., HCl, stirred for several hours, and the solvent is removed. Crude V-6a is dissolved in dichloromethane and tert-butyl methyl (3-oxopropyl) carbamate is added. After stirring and incubation, NaBH(OAc)3 is added in portions at certain time intervals and further incubated. Water is then added and the pH is adjusted to 8 with Na2CO3. The mixture is extracted with dichloromethane, the organic phases are combined, dried over Na2SO4, and the solids are removed by filtration. Silica gel is added to the filtrate and concentrated to dryness. The silica gel is then added to a column and V-7a is eluted using a gradient of dichloromethane:methanol and dried to a yellow oil. V-7a is dissolved in dioxane and exposed to slowly added acid, e.g., HCl. After incubation, the solvent is removed to give crude V-8a as a white solid.

[0111] To synthesize V-12, imidazole is added to a solution of diethanolamine in dichloromethane, stirred, and a solution of t-butyldimethylsilyl chloride is slowly added. The resulting solution is incubated, and then the reaction is quenched by adding 10% aqueous NH4OH. The organic phase is separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is washed successively with saturated NH4Cl and brine, and dried. Filtration and concentration give V-12 as a clear, colorless oil.

[0112] CDI and Et3N are added in turn to a solution of V-12 in dichloromethane, the resulting solution is incubated with stirring and then poured into water. The organic phase is separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is washed successively with saturated NH4Cl and 5% aqueous NaHCO3 and dried. Filtration and concentration give V-13 as a pale yellow oil.

[0113] To a cold solution of V-13 in dichloromethane, MeOTf is added slowly. After stirring and cold incubation, a solution of Et3N and V-8a in dichloromethane is added slowly. Once the addition is complete, the solution is warmed and incubated for several hours. The reaction mixture is then poured into water and the organic layer is removed. The aqueous layer is extracted with dichloromethane and the combined organic phase is concentrated. The resulting crude V-14 is dissolved in heptane and the solution is extracted with MeOH / H2O. The combined aqueous phase is then extracted with heptane and the combined organic phase is washed with brine and dried over MgSO4. After filtration, silica gel is added to the filtrate and the mixture is concentrated to dryness. The silica gel is then added to a column and V-14 is eluted using a gradient of dichloromethane:methanol and the fractions containing V-14 are concentrated to give V-14 as a yellow oil.

[0114] To complete the synthesis of V-15, BF3-OEt2 is slowly added to a solution of V-14 in THF. The mixture is incubated with stirring for several hours and poured into water. The pH is adjusted to 8.0 with saturated aqueous NaHCO3 and the solvent is removed to approximately one-fifth of the original volume. The remaining solution is purified by flash chromatography using a water:acetonitrile gradient. Fractions containing V-14 are pooled and concentrated to give V-14 as an off-white oil.

[0115] In certain aspects, the disclosure provides a method of synthesizing an ionizable cationic lipid of formula 2. In some embodiments, the method comprises converting an intermediate having the structure of II-gA to an ionizable cationic lipid of formula 2. In some embodiments, the method further comprises synthesizing an intermediate having the structure of II-gA (FIG. 2A).

[0116] In certain embodiments of the method for synthesizing an ionizable cationic lipid of formula 2 (Y=O, NH, or N-CH), the method further comprises reacting II-gA with carbonyldiimidazole to provide II-hA. In certain embodiments, the method further comprises reacting II-hA and X-(CH) n+2 In certain embodiments, the method further comprises coupling II-hA and X-(CH2) n+2 The coupling reaction of -YH is carried out in the presence of an alkylating agent. In certain embodiments, the alkylating agent is MeOTf, as shown in FIG. 2B. In certain embodiments, the coupling reaction converts an intermediate having the structure II-iA to X-(CH2) n+2 -YH to provide an ionizable cationic lipid of formula 2, where Y=O, NH, or N-CH. In certain embodiments, II-iA and X-(CH) n+2 The coupling reaction with -YH is carried out in the presence of a base, such as, but not limited to, NaH or Et3N. See Figure 2B.

[0117] In certain embodiments of the method for synthesizing an ionizable cationic lipid of formula 2 (Y=CH2), the method comprises forming an intermediate having the structure II-gA into a mixture of X-(CH2) n+3 -COOH to provide an ionizable cationic lipid of formula 2. In certain embodiments, the coupling method is carried out in the presence of DMAP and Et3N, for example as shown in Figure 2C.

[0118] In certain embodiments of the method for synthesizing II-gA, the method includes coupling the intermediate of II-eA with R-COOH to provide an amine-protected derivative of II-gA, also called II-fA. In certain embodiments, the amine-protecting group is -CO2t-Bu, as shown in FIG. 2A. In certain embodiments of the method for synthesizing II-gA, II-fA, the amine-protected derivative of II-gA, is deprotected to provide II-gA. For example, the deprotecting reagent can be TFA in dimethyl chloride, as shown in FIG. 2A. In certain embodiments, the method further includes synthesizing II-eA.

[0119] In certain embodiments, the method for synthesizing II-eA includes preparing a derivative of II-eA, in which the hydroxyl group of II-eA is protected (i.e., OH-protected II-eA). For example, as shown in FIG. 2A, OH-protected II-eA can be prepared by reacting BOC-N((CH2) p+1 In another example, as shown in FIG. 2A, the OH-protected II-eA can be prepared by reacting the sodium salt of BOC-N((CH2) p+1 The compound may be II-dA, which may be prepared by reacting the sodium salt of (CH2-OH)2 with II-b.

[0120] In some embodiments, the method of synthesizing an ionizable cationic lipid of formula 2 proceeds according to the synthesis schemes of Figures 2A-F. In some embodiments, the method is a method of synthesizing an ionizable cationic lipid of formula 2a. In some cases, the method is a method of synthesizing compound A-5, compound A-6, compound A-7, or compound A-8. In some embodiments, the method specifies only a single step, or a subset of steps, shown in Figures 2A-F.

[0121] In a further aspect, the disclosure provides methods of synthesizing an intermediate of the synthetic scheme of Figures 2D-F, wherein the intermediate is II-e, II-f, II-g, II-h, or II-i. In some embodiments, the method includes only the final steps to produce the intermediate shown in Figures 2D-F. In other embodiments, the method includes all or a subset of the steps shown in Figures 2D-F until the intermediate is reached.

[0122] In the synthetic schemes shown in Figures 2D-F, the value of p is 1, resulting from the reaction of a BOC-blocked diethanolamine with intermediates II-a or II-b to produce intermediates II-c or II-d, respectively. Compounds with p between 2 and 4 can be synthesized by substituting appropriately sized BOC-blocked dialkylamino alcohols, namely 3,3'-azanediylbis(propan-1-ol), 4,4'-azanediylbis(butan-1-ol), and 5,5'-azanediylbis(pentan-1-ol), respectively.

[0123] In the synthesis schemes shown in Figures 2D-F, the value of n is 1, resulting from the reaction of intermediate II-i with the sodium salts of 3-dimethylamino-1-propanol, N,N-dimethyl-1,3-propanediamine, and N,N,N'-trimethyl-1,3-propanediamine to produce compounds A-5-A-7 (where Y is O, NH, or N-CH3, respectively). Compounds where n is 0 or 2-4 can be synthesized by replacing the propanediamine moiety with the analogous C2, C4, C5, or C6 moiety. Compound A-8 where Y is CH2 can be obtained by reacting the salt of intermediate II-g with 5-dimethylamino-pentanoic acid. Compounds where n is 0 or 2-4 can be synthesized by replacing the pentanoic acid moiety with the analogous C4, C6, C7, or C8 moiety.

[0124] In the synthetic schemes shown in Figures 2D-F, R is C9, which results from the use of decanoic acid in the conversion of intermediate II-e to intermediate II-f. -oic acids of alternative corresponding chain lengths and structures are C6-C8 or C6-C8, as appropriate. 10 ~C 18 can be used to obtain R

[0125] In the synthetic schemes shown in Figures 2D-F, X is N(CH3)2. Compounds according to formula 2 with alternative definitions of X can be synthesized by reacting alternative headgroup pieces from Tables 1-3 with II-i to give analogs of compounds A-5-A-7, respectively, as disclosed in Example 2 (infra), or by reacting alternative headgroup pieces from Table 4 with II-g to give analogs of compound A-8. The synthesis of headgroup pieces not previously disclosed in the art can be made similarly to their shorter congeners, or, in the case of polyethylene glycol-containing headgroup pieces, can be made according to the synthetic schemes shown in Figures 4A-B and disclosed in Example 4, or described in Examples 25-32 (infra).

[0126] Intermediate II-f and its analogs with different R and / or p, i.e., the structures: [ka] To synthesize intermediates with II-II, where p is an integer between 1 and 4 and R is defined as in formula 2, the sodium salt of a BOC-blocked diethanolamine or an appropriately sized dialkylamino alcohol (p=2-4) is reacted with 5-(2-bromoethyl)-2,2-dimethyl-1,3-dioxane (II-a) or 5-(2-bromoethyl)-2-phenyl-1,3-dioxane (II-b) in DMF to give II-c and II-d (Figure 2D) or their analogs with p=2-4 (Figure 2A), respectively. II-c and its analogs can be deblocked with mild acid in the presence of PPTs in MeOH to give diol II-e (Figure 2D) and its analogs with p=2-4 (Figure 2A). Alternatively, benzylidene acetal II-d and its analogs can be deblocked with hydrogen and Pd / C in ethyl acetate to similarly afford II-e (Figure 2D) or its analogs with p = 2-4 (Figure 2A). Coupling of II-e with the appropriate carboxylic acid for the desired R in the presence of EDC-HCl and DMAP in dichloromethane affords II-f (Figure 2D) or its analogs with different R and / or p (Figure 2A).

[0127] Intermediate II-h and its analogs with different R and / or p, i.e., the structures: [ka] To synthesize an intermediate having the structure II-fA, where p is an integer between 1 and 4 and R is defined as in formula 2, an intermediate having the structure II-fA is treated with TFA in dichloromethane to remove the BOC blocking group to give the amine salt II-g (Figure 2D), or its analogs having different R and / or p (Figure 2A). The amine salt II-g or its analogs are reacted with carbonyldiimidazole and triethylamine in dichloromethane to give the acylimidazole II-h or its analogs II-hA (Figure 2A).

[0128] To complete the synthesis of compounds A-5 through A-7, as well as their analogs with different R and / or p, the required reactive intermediate is obtained by reacting an intermediate having the structure II-hA with methyl triflate to generate the acyl-imidazolium II-i (Figure 2E), or its analogs with different R and / or p (Figure 2B). The acyl-imidazolium intermediate is then reacted with 3-dimethylamino-1-propanol in the presence of triethylamine, in each case in dichloromethane, to provide compound A-5 (Figure 2E) or analogs with different R and / or p (Figure 2B); with N,N-dimethyl-1,3-propanediamine and triethylamine to provide compound A-6 (Figure 2E) or analogs with different R and / or p (Figure 2B); or with N,N,N'-trimethyl-1,3-propanediamine and triethylamine to provide compound A-7 (Figure 2E) or analogs with different R and / or p (Figure 2B).

[0129] To complete the synthesis of compound A-8, or an analog thereof with different R and / or p (Figure 2C), salt II-g (Figure 2F), or an analog thereof with different R and / or p (Figure 2C) is reacted with 4-dimethylamino-butanoic acid in the presence of EDC-HCl, DMAP, and triethylamine in dichloromethane to provide compound A-8 (Figure 2F), or an analog thereof with different R and / or p (Figure 2C).

[0130] Alternatives to the reagents used to obtain analogs of compounds A-5 to A-8 having different X, Y, and / or n are described above and are applicable to the above synthesis to obtain analogs of compounds A-5 to A-8 having different R and / or p.

[0131] In certain aspects, the present disclosure provides a method for synthesizing an ionizable cationic lipid of formula 3. In some embodiments, the method comprises converting an intermediate having the structure of III-cA to an ionizable cationic lipid of formula 3. In some embodiments, the method further comprises synthesizing an intermediate having the structure of III-cA.

[0132] In certain embodiments of the method for synthesizing an ionizable cationic lipid of formula 3 (W=C=O), the method further comprises reacting III-cA with I-dA to provide an ionizable cationic lipid of formula 3. See, e.g., FIG. 3A.

[0133] In certain embodiments of the method for synthesizing an ionizable cationic lipid of formula 3 (W=CH2), the method further comprises converting III-cA to III-fA, and reacting III-fA with R-COOH to provide an ionizable cationic lipid of formula 3. For example, see FIG. 3B. In certain embodiments, the method further comprises preparing a derivative of III-fA, in which the hydroxyl group of III-fA is protected (i.e., OH-protected III-fA). For example, as shown in FIG. 3B, OH-protected III-fA can be III-dA, which can be prepared by reacting the sodium salt of III-cA with II-a. In another example, as shown in FIG. 3B, OH-protected III-fA can be III-eA, which can be prepared by reacting the sodium salt of III-cA with II-b.

[0134] In certain embodiments, III-cA is prepared by reduction of the carbonyl group of III-bA, for example with LiAlH, as shown in FIG. 3A. In certain embodiments, III-bA can be prepared by reducing III-aA with HN((CH) p In certain embodiments, III-aA and HN((CH) p The reaction of -CHOH) in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, for example as shown in Figure 3A. The embodiment of III-aA where n=2 can be prepared by reacting glutaric anhydride with dimethylamine, for example as shown in Figure 3A.

[0135] In some embodiments, the method of synthesizing an ionizable cationic lipid of formula 3 proceeds according to the synthesis scheme of Figures 3A-D. In some embodiments, the method is a method of synthesizing an ionizable cationic lipid of formula 3a. In some cases, the method is a method of synthesizing compound A-9 or compound A-10. In some embodiments, the method includes only a single step, or a subset of the steps, shown in Figures 3A-D.

[0136] Further aspects are intermediates III-aA, III-bA, III-cA, III-dA, III-eA, III-fA, III-d, III-e, and III-f of the synthetic scheme of Figures 3A-D, as well as methods of synthesizing each of intermediates III-d, III-e, and III-f. Further embodiments relate to analogs of intermediates III-d, III-e, and III-f suitable for final products with different X, n, or p.

[0137] In a further aspect, a method is provided for synthesizing an intermediate of the synthetic scheme of Figures 3C-D, where the intermediate is III-d, III-e, or III-f. In some embodiments, the method specifies only the final steps to produce the intermediate shown in Figures 3C-D. In other embodiments, the method specifies all or a subset of the steps shown in Figures 3C-D until the intermediate is reached.

[0138] In the synthetic scheme shown in Figure 3C-D, the value of p is 1, which results from the reaction of glutaric anhydride with dimethylamine to form III-a, which then reacts with diethanolamine. Compounds with p between 2 and 4 can be synthesized by substituting appropriately sized dialkylamino alcohols, namely 3,3'-azanediylbis(propan-1-ol), 4,4'-azanediylbis(butan-1-ol), and 5,5'-azanediylbis(pentan-1-ol), respectively.

[0139] In the synthetic scheme shown in Figure 3C-D, the value of n is 2, which results from the coupling of intermediate III-a with diethanolamine (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) and subsequent reduction. Compounds with n = 0-1 or 3-4 can be synthesized by substituting malonic acid, maleic anhydride, 1,6-hexanedioic acid, 1,7-heptanedioic acid in the coupling reaction with dimethylamine, followed by addition of the amide-acid with the amino alcohol.

[0140] In the synthesis of compound A-9 shown in Figure 3C, W is C=O. In the synthesis of compound A-10 shown in Figure 3D, W is CH2.

[0141] In the synthesis scheme shown in FIG. 3C-D, R cis C9, resulting from the use of decanoic acid in the conversion of intermediates III-c or III-f to compounds 9 or 10, respectively. Alternative -oic acids of corresponding chain length and structure are C6-C8 or C10, as appropriate. 10 ~C 20 R c can be used to obtain

[0142] In the synthetic scheme shown in Figures 3C-D, X is N(CH3)2. Compounds according to formula 3 with alternative definitions of X can be synthesized by reacting alternative headgroup pieces from Table 4 (instead of III-a) with diethanolamine, as disclosed in Example 3 (infra), to give analogs of compounds A-9 and A-10. The synthesis of headgroup pieces not previously disclosed in the art can be made similarly to their shorter congeners, or, in the case of polyethylene glycol-containing headgroup pieces, can be made according to the synthetic schemes shown in Figures 4A-B and disclosed in Example 4, or described in Examples 25-32 (infra).

[0143] To complete the synthesis of compound A-9, as defined for formula 3, and its analogs with different R and p, first glutaric anhydride is reacted with dimethylamine in THF to give 5-(dimethylamino)-5-oxopentanoic acid III-a. Coupling of III-a with diethanolamine in the presence of (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) or with appropriately sized dialkylamino alcohols (p=2-4) gives N1,N1-bis(2-hydroxyethyl)-N5,N5-dimethyl-pentanediamide III-b (Figure 3C) or its analogs (Figure 3A), which is reduced with LiAlH4 in THF to provide diol III-c (Figure 3C) or its analogs with p=2-4 (Figure 3A). Intermediate Id, or its analogs with different R, the synthesis of which is disclosed above, is then coupled with III-c in dichloromethane to give compound A-9 (FIG. 3C), as well as analogs with different R cand / or its analogs having p (Figure 3A).

[0144] To complete the synthesis of compound A-10, and its analogs with different R and p, as defined for formula 3, the sodium salt of III-c, or its analogs with p=2-4, is reacted with either bromide II-a or II-b in the presence of NaH in DMF to give diamines III-d and III-e (Figure 3D), or its analogs with p=2-4 (Figure 3B). Deprotection of III-d by PPTs in methanol, or III-e by hydrogen and Pd / C in ethyl acetate, provides tetraol III-f, or its analogs with p=2-4. Coupling of tetraol III-f with decanoic acid, or other carboxylic acid appropriate to generate analogs with different R, in the presence of EDC-HCl and DMAP in dichloromethane, leads to compound A-10 (Figure 3D), or its analogs with different R and / or p (Figure 3B).

[0145] Alternatives to the reagents used to obtain analogs of compounds A-9 to A-10 having different X and / or n are described above and are applicable to the above synthesis to obtain analogs of compounds A-9 to A-10 having different R and / or p.

[0146] Although the synthesis is described using a specific solvent, in all cases alternative solvents are known to those skilled in the art. THF can be replaced with, for example, but not limited to, DMF, diethyl ether, methyl t-butyl ether, dioxane, or 2-methyl THF. Ethyl acetate can be replaced with, for example, but not limited to, isopropyl acetate, THF, 2-methyl THF, dioxane, or methyl t-butyl ether. Dichloromethane can be replaced with, for example, but not limited to, ethyl acetate, isopropyl acetate, THF, methyl t-butyl ether, 2-methyl THF, dioxane, or heptane. Methanol can be replaced with, for example, but not limited to, ethanol, or aqueous THF. Lipid Nanoparticles (LNPs) and Targeted LNPs (tLNPs)

[0147] As used herein, "lipid nanoparticle" (LNP) refers to a solid particle, as distinct from a liposome, which has an aqueous lumen. The core of an LNP, like the lumen of a liposome, is surrounded by a layer of lipid, which may be, but is not necessarily, a continuous lipid monolayer, a bilayer as found in liposomes, or a multilayer having three or more lipid layers.

[0148] In certain aspects, the present disclosure provides lipid nanoparticles (LNPs) comprising ionizable cationic lipids of formula 1, formula 2 or formula 3, or combinations thereof. In some embodiments, the LNPs comprise ionizable cationic lipids of formula 1, formula 2 or formula 3, or combinations thereof, and phospholipids, sterols, co-lipids or PEGylated lipids, or combinations thereof. In certain embodiments, the PEG-lipids are not functionalized PEG-lipids. In certain embodiments, the LNPs comprise at least one PEG-lipid that is functionalized and at least one PEG-lipid that is not functionalized.

[0149] In a further aspect, the present disclosure provides targeted lipid nanoparticles (tLNPs) comprising ionizable cationic lipids of formula 1, formula 2 or formula 3 or combinations thereof. In some embodiments, the above-mentioned tLNPs may further comprise one or more of phospholipids, sterols, co-lipids and PEG-lipids or combinations thereof, and functionalized PEG-lipids. As used herein, "functionalized PEG-lipids" refers to PEG-lipids whose PEG moieties are derivatized with chemically reactive groups that can be used to conjugate targeting moieties to the PEG-lipids. The functionalized PEG-lipids can react with binding moieties after the LNPs are formed, so that the binding moieties are conjugated to the PEG moieties of the lipids. Thus, the conjugated binding moieties can function as targeting moieties of tLNPs.

[0150] In various embodiments of the LNPs or tLNPs of the present disclosure, the phospholipids include dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC) or 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), or combinations thereof. Phospholipids can contribute to the formation of the membrane surrounding the core of the LNPs or tLNPs, whether monolayer, bilayer or multilayer. In addition, phospholipids such as DSPC, DMPC, DPPC, DAPC, etc., impart stability and rigidity to the membrane structure. Phospholipids such as DOPE impart fusogenicity. Additional phospholipids such as DMPG, which achieve a negative charge at physiological pH, facilitate charge regulation. Thus, phospholipids constitute a means for promoting membrane formation, for imparting membrane stability and rigidity, for imparting fusogenicity, and for charge regulation.

[0151] In various embodiments of the LNPs or tLNPs of the present disclosure, the sterol is cholesterol, 20-hydroxycholesterol, 22-hydroxycholesterol, or a plant sterol. In further embodiments, the plant sterol comprises campesterol, sitosterol, or stigmasterol, or a combination thereof. In a preferred embodiment, the cholesterol is not of animal origin, but is obtained synthetically using a plant sterol as a starting point. LNPs incorporating C-24 alkyl (e.g., methyl or ethyl) plant sterols have been reported to provide improved gene transfection. The length of the alkyl tail, the flexibility of the sterol ring, and the polarity associated with the retained C-3-OH group are important to obtain high transfection efficiency. β-sitosterol and stigmasterol worked well, but vitamins D2, D3, and calcipotriol (analogs that lack the complete body of cholesterol), as well as betulin, lupeol ursolic acid, and oleanolic acid (containing the fifth ring) should be avoided. Sterols function to fill the space between other lipids in LNPs or tLNPs and affect LNP or tLNP shape. Sterols also control the fluidity of lipid composition and reduce temperature dependency. Thus, sterols such as cholesterol, 20-hydroxycholesterol, 22-hydroxycholesterol, campesterol, fucosterol, β-sitosterol, and stigmasterol constitute a means to control LNP shape and fluidity or to increase transfection efficiency.

[0152] With respect to the LNPs or tLNPs of the present disclosure, in some embodiments, the co-lipid is absent or comprises an anionic or cationic ionizable lipid. The co-lipid may be used to modulate various properties of the LNPs or tLNPs, such as surface charge, fluidity, rigidity, size, stability, etc. In some embodiments, the co-lipid is an ionizable lipid, such as cholesterol hemisuccinate (CHEMS), or an ionizable lipid of the present disclosure. In some embodiments, the co-lipid is a charged lipid, such as a quaternary ammonium head group-containing lipid. In some cases, the quaternary ammonium head group-containing lipid comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA), or 3β-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or a combination thereof. In certain embodiments, the compounds are chlorides, bromides, mesylates, or tosylates.

[0153] When the disclosed ionizable lipids of formulas 1, 2, and 3 have a measured pKa of 6-7, they can impart substantial endosomal release activity to LNPs or tLNPs containing the ionizable lipids. The more acidic or basic ionizable lipids of formulas 1, 2, and 3 can impart a surface charge and thus function as co-lipids as described immediately above. In such cases, it may be advantageous to incorporate another lipid with fusogenic activity into the LNPs or tLNPs of the present disclosure. Surface charge is known to affect the tissue tropism of LNPs or tLNPs, for example, positively charged LNPs or tLNPs have demonstrated spleen and lung tropism.

[0154] With respect to the LNPs or tLNPs of the present disclosure, in some embodiments, the PEG-lipid (i.e., lipid conjugated to polyethylene glycol (PEG)) is a C 14 ~C 20 It is a lipid. 14PEG-lipids with fatty acid chain lengths less than 100 nm were lost too rapidly from the (t)LNPs, whereas C 20PEG-lipids with chain lengths greater than 1,2-dimyristoyl-glycero-3-methoxypolyethylene glycol-2000), DPG-PEG2000 (1,2-dipalmitoyl-glycero-3-methoxypolyethylene glycol-2000), DSG-PEG2000 (1,2-distearoyl-glycero-3-methoxypolyethylene glycol-2000), DOG-PEG2000 (1,2-dioleoyl-glycero-3-methoxypolyethylene glycol-2000), DMPE-PEG200 (1,2-dimyristoyl-glycero-3-methoxypolyethylene glycol-2000), DMPE-PEG200 (1,2-dimyristoyl-glycero-3-methoxypolyethylene glycol-2000), DMPE-PEG200 (1,2-dioleo ... glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), DPPE-PEG2000 (1,2-dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), DSPE-PEG2000 (1,2-distearoyl-glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), DOPE-PEG2000 (1,2-dioleoyl-glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), or combinations thereof. In some embodiments, the PEG unit has a MW of 2000 Da.In some cases, the PEG-lipid of MW2000 is DMrG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DPrG-PEG2000 (1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DSrG-PEG2000 (1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DOrG-PEG2000 (1,2-dioleoyl-glycero-3-methoxypolyethylene-rac-glycol-2000), DMPer-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), The optically pure enantiomer of the glycerol moiety may be used, i.e., the glycerol moiety is homochiral. Optically pure as used herein with respect to the glycerol moiety means 95% or more of a single enantiomer (D or L). In some embodiments, the enantiomeric excess is 98% or more. In some embodiments, the enantiomeric excess is 99% or greater. Additional PEG-lipids, including achiral PEG-lipids constructed on a symmetric dihydroxyacetone backbone, a symmetric 2-(hydroxymethyl)butane-1,4-diol, or a symmetric glycerol backbone, are disclosed in U.S. Provisional Application No. 63 / 362,502, filed April 5, 2022, entitled PEG-Lipids and Lipid Nanoparticles, and PCT Application No. Docket No. 146758-8002.WO00, filed April 5, 2023, both of which are incorporated by reference in their entireties.

[0155] PEG moieties provide a hydrophilic surface on the LNP, inhibiting aggregation or coalescence of the LNP, thus contributing to its stability and reducing polydispersity. In addition, PEG moieties can prevent binding by LNP, including binding to plasma proteins. These plasma proteins include apoE, which is understood to mediate LNP uptake by the liver, such that inhibition of binding can result in an increase in the percentage of LNP that reaches other tissues. These plasma proteins also include opsonins, such that inhibition of binding reduces recognition by the reticuloendothelial system. PEG moieties can also be functionalized to serve as attachment points for targeting moieties. By conjugating cell or tissue specific binding moieties to the PEG moiety, tLNPs can avoid the liver and bind to their target tissue or cell type, greatly increasing the percentage of LNP that reaches the targeted tissue or cell type. Thus, PEG-lipids can function as a means to inhibit LNP binding, and PEG-lipids conjugated to binding moieties can function as a means for LNP targeting.

[0156] In some embodiments, a "binding moiety" or a "targeting moiety" refers to a protein, polypeptide, oligopeptide, peptide, carbohydrate, nucleic acid, or combination thereof that can specifically bind to a target or targets. A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner to a biological molecule of interest or another target. Exemplary binding moieties of the present disclosure include antibodies, Fab', F(ab')2, Fab, Fv, rIgG, scFv, hcAb (heavy chain antibody), single domain antibodies, VHH, VNAR, sdAb, nanobodies, receptor extracellular domains or ligand binding portions thereof, or ligands (e.g., cytokines, chemokines). A "Fab" (fragment antigen binding) is the portion of an antibody that binds to an antigen and includes the variable region and CH1 of the heavy chain linked to the light chain via an interchain disulfide bond. Various assays are known for identifying binding moieties of the present disclosure that specifically bind to a particular target, including Western blot, ELISA, and Biacore® analysis. Binding moieties, e.g., binding moieties comprising immunoglobulin light and heavy chain variable domains (e.g., scFvs), can be incorporated into various protein scaffolds or structures described herein, e.g., antibodies or antigen-binding fragments thereof, scFv-Fc fusion proteins, or fusion proteins comprising two or more such immunoglobulin binding domains.

[0157] The antibody or other binding moiety (or fusion protein thereof) is capable of detecting 10 5 M -1 A binding domain "specifically binds" to a target if it binds to the target with an affinity or Ka (i.e., the equilibrium association constant of a particular binding interaction in units of 1 / M) equal to or greater than 10. A binding domain (or fusion protein thereof) may be classified as a "high affinity" binding domain (or fusion protein thereof) and a "low affinity" binding domain (or fusion protein thereof). A "high affinity" binding domain has an affinity of at least 10 8 M -1 , at least 10 9 M -1, at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 , preferably at least 10 8 M -1 Or at least 10 9 M -1 "Low affinity" binding domains refer to those binding domains that have a Ka of up to 10 8 M -1 , up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 Alternatively, affinity is expressed in units of M (e.g., 10 -5 M~10 -13 The affinity of a particular binding interaction can be defined as the equilibrium dissociation constant (Kd) of the specific binding interaction (M). The affinity of binding domain polypeptides and fusion proteins according to the present disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents).

[0158] Some embodiments of the disclosed ionizable cationic lipids have head groups with small (less than 250 Da) PEG moieties. These lipids are not what the term PEG-lipids refers to when used herein. These small PEG moieties are generally too small to prevent binding to the same extent as the larger PEG moieties of the PEG-lipids disclosed above, but they do affect the lipophilicity of the ionizable cationic lipids. In addition, it is understood that the PEG-lipids are primarily located in the lamellae facing the outside, while most of the ionizable cationic lipids are located in the interior of the LNP.

[0159] In various embodiments, the binding moiety of the tLNP comprises an antigen-binding domain of an antibody, an antigen, a ligand-binding domain of a receptor, or a receptor ligand. In some embodiments, the binding moiety comprising an antigen-binding domain of an antibody comprises a complete antibody, a F(ab)2, a Fab, a minibody, a single-chain Fv (scFv), a diabody, a VH domain, or a nanobody, such as a VHH or a single-domain antibody. In some embodiments, the receptor ligand is a carbohydrate, such as a carbohydrate comprising a terminal galactose or N-acetylgalactosamine unit, bound to an asialoglycoprotein receptor. These binding moieties constitute a means for LNP targeting. Some embodiments specifically include one or more of these binding moieties. Other embodiments specifically exclude one or more of these binding moieties.

[0160] As used herein, "antibody" refers to a protein that contains an immunoglobulin domain with hypervariable regions, so-called complementarity determining regions (CDRs), that determine the specificity of the antibody when it binds to an antigen. Thus, the term antibody can refer to complete or whole antibodies, as well as antibody fragments and constructs that contain the antigen-binding portion of a whole antibody. Standard natural antibodies have a pair of heavy and light chains, but camelids (camels, alpacas, llamas, etc.) produce both antibodies with standard structures and antibodies that contain only heavy chains. The variable regions of camelid heavy chain-only antibodies have a different structure with elongated CDR3, called VHH, or nanobodies when produced as fragments. Antigen-binding fragments and constructs of antibodies include F(ab)2, F(ab), minibodies, Fv, single chain Fv (scFv), diabodies, and VH. Such elements can be combined to generate bi- and multispecific reagents, such as BiTEs. The term "monoclonal antibody" arose from hybridoma technology, but is now used to refer to any single species of antibody, regardless of its origin or how it was generated. Antibodies can be obtained through immunization, selection from naive or immunized libraries (e.g., by phage display), modification of isolated antibody coding sequences, or any combination thereof. Many antibodies that may be used as binding moieties are known in the art. A good source of information for antibodies with suggested or recommended International Nonproprietary Names (INNs), including sequence information, is Wilkinson & Hale, MAbs 14(1):2123299, 2022 (including its supplemental tables), which is incorporated herein by reference for all that it teaches about individual antibodies and various antibody formats that may be constructed. US11,326,182, particularly its Table 9, Cancer, Inflammation and Immune System Antibodies, is a source of sequence and other information for a wide range of antibodies, including many that do not have INNs, and is incorporated herein by reference for all that it teaches about individual antibodies.

[0161] The functionalized PEG-lipids of tLNPs contain one or more fatty acid tails, each of which, in the case of a linear fatty acid, is C 16 Above and C 20 For branched chain fatty acids, the tail is C 14 Fatty acids or more and C 20 The following may be permitted: In some embodiments, the fatty acid tail is C 16 In some embodiments, the fatty acid tail is 18 In some embodiments, the functionalized PEG-lipid comprises a dipalmitoyl lipid. In some embodiments, the functionalized PEG-lipid comprises a distearoyl lipid. The fatty acid tail serves as a means to anchor the PEG-lipid within the tLNP, reducing or eliminating the PEG-lipid from falling off the tLNP. This is a useful property of PEG-lipids, whether functionalized or not, but has greater significance in the case of functionalized PEG-lipids, since a targeting moiety is attached to it, and if the PEG-lipid (with a conjugated binding moiety) falls off the tLNP, the targeting function may be compromised.

[0162] Any suitable chemistry can be used to conjugate the binding moiety to the PEG of the PEG-lipid, including maleimide (see Parhiz et al., Journal of Controlled Release 291:106-115, 2018) and click (see Kolb et al., Angewandte Chemie International Edition 40(11):2004-2021, 2001; and Evans, Australian Journal of Chemistry 60(6):384-395, 2007) chemistries. Reagents for such reactions include lipid-PEG-maleimide, lipid-PEG-cysteine, lipid-PEG-alkyne, lipid, PEG-dibenzocyclooctyne (DBCO), and lipid-PEG-azide. Additional conjugation reactions utilize lipid-PEG-bromomaleimide, lipid-PEG-alkylnoic amide, PEG-alkynoic imide, and lipid-PEG-alkyne reactions, as disclosed in U.S. Provisional Application No. 63 / 362,502, filed April 5, 2022, both entitled PEG-Lipids and Lipid Nanoparticles, and PCT Application No. 146758-8002.WO00, filed April 5, 2023, which are incorporated by reference in their entireties. On the attachment side of the reaction, an existing cysteine ​​sulfhydryl can be used, or the protein can be derivatized, for example by adding a sulfur-containing carboxylic acid to the epsilon amino of lysine, and reacted with a maleimide, bromomaleimide, alkylnoic amide, or alkynoic imide. Alternatively, alkynes can be added to sulfhydryls or the epsilon amino of lysine to participate in click chemistry reactions.

[0163] With respect to the LNPs or tLNPs of the present disclosure, in some embodiments, the molar ratio of lipids is about 40 to about 60 mol% ionizable cationic lipid. In some embodiments of the LNPs or tLNPs, the molar ratio of lipids is about 7 to about 30 mol% phospholipid. In some embodiments of the LNPs or tLNPs, the molar ratio of lipids is about 20 to about 45 mol% sterol. In some embodiments of the LNPs or tLNPs, the molar ratio of lipids is 1 to 30 mol% co-lipid. In some embodiments of the LNPs or tLNPs, the molar ratio of lipids is 0 to 5 mol% PEG-lipid. In some embodiments of the LNPs or tLNPs, the molar ratio of lipids is 0.1 to 5 mol% functionalized PEG-lipid. In some embodiments, the functionalized PEG-lipid is conjugated to a binding moiety.

[0164] Due to physiological and manufacturing constraints of LNPs or tLNPs for in vivo use, particles having a hydrodynamic diameter of about 50 to about 150 nm are desirable. Thus, in some embodiments, the LNPs or tLNPs have a hydrodynamic diameter of 50 to 150 nm, and in some cases, the hydrodynamic diameter is 120 nm or less, 110 nm or less, 100 nm or less, or 90 nm or less. Particle size uniformity is also desirable, and a polydispersity index (PDI) of 0.2 (on a scale of 0 to 1) or less may be acceptable. Both the hydrodynamic diameter and the polydispersity index are determined by dynamic light scattering (DLS). Particle diameters as assessed from cryogenic transmission electron microscopy (Cryo-TEM) may be smaller than those determined by DLS.

[0165] The LNP or tLNP of the present disclosure further comprises a nucleic acid. In various embodiments, the nucleic acid is an mRNA, a self-replicating RNA, an siRNA, an miRNA, a DNA, a gene editing component (e.g., guide RNA, tracr RNA, sgRNA), a gene rewriting component, an mRNA encoding a gene or a base editing protein, a zinc finger nuclease, a Talen, a CRISPR nuclease, e.g., Cas9, a DNA molecule that serves as a template for repair or is inserted), etc., or a combination thereof. In some embodiments, the mRNA encodes a chimeric antigen receptor (CAR). In other embodiments, the mRNA encodes a gene editing or base editing or gene rewriting protein. In some embodiments, the nucleic acid is a guide RNA. In some embodiments, the LNP or tLNP comprises both a gene or base editing or gene rewriting protein encoding mRNA and one or more guide RNAs. The CRISPR nuclease may have an altered activity, for example, the nuclease may be modified to be a nickase instead of creating a double-strand break, or to bind to the sequence specified by the guide RNA but not have enzymatic activity. Base editing proteins are often fusion proteins that contain a deaminase domain and a sequence-specific DNA binding domain (e.g., an inactive CRISPR nuclease).

[0166] For LNPs or tLNPs of the present disclosure, in some embodiments, the total lipid to nucleic acid ratio is about 10:1 to about 50:1 by weight. In some embodiments, the total lipid to nucleic acid ratio is about 10:1, about 20:1, about 30:1, or about 40:1 to about 50:1, or 10:1 to 20:1, 30:1, 40:1, or 50:1, or any range bounded by these ratio pairs.

[0167] In some aspects, the disclosure provides a method of making LNPs or tLNPs, comprising mixing an aqueous solution of nucleic acid with an alcoholic solution of lipids. In certain embodiments, the mixing is rapid. The aqueous solution is buffered to a pH of about 3 to about 5, for example, but not limited to, with citrate or acetate. In various embodiments, the alcohol can be ethanol, isopropanol, t-butanol, or a combination thereof. In some embodiments, the rapid mixing is achieved by pumping the two solutions through a T-junction or with an impinging jet mixer. Microfluidic mixing through a zigzag-arranged herringbone mixer (SHM) or hydrodynamic mixer (microfluidic hydrodynamic focusing), microfluidic branching mixer, and microfluidic baffle mixer may also be used. After the LNPs are formed, they are diluted with a buffer, such as phosphate, HEPES, or Tris, to reduce the alcohol (ethanol) concentration within a pH range of 6 to 8.5. The diluted LNPs are purified by either dialysis or ultrafiltration or diafiltration using tangential flow filtration (TFF) against a buffer (e.g., phosphate, HEPES, or Tris) in the pH range of 6-8.5 to remove the alcohol. Alternatively, size exclusion chromatography can be used. Once the alcohol is completely removed, the buffer is exchanged for a similar buffer containing a cryoprotectant (e.g., glycerol or a sugar, such as sucrose, trehalose, or mannose). The LNPs are concentrated to the desired concentration, followed by filtration, e.g., through a 0.2 μm polyethersulfone (PES) or modified PES filter, filled into glass vials, stoppered, capped, and stored frozen. In an alternative embodiment, a cryoprotectant is used and the LNPs are lyophilized for storage rather than as a frozen liquid. Additional methodologies for making LNPs can be found, for example, in US20200297634, US20130115274, and WO2017 / 048770, each of which is incorporated by reference for all content that teaches the production of LNPs.

[0168] One aspect is a method for making tLNP, which comprises rapid mixing of an aqueous solution of nucleic acid and an alcoholic solution of lipid disclosed for LNP.In some embodiments, the lipid mixture comprises functionalized PEG-lipid for subsequent conjugation to targeting moiety.As used herein, functionalized PEG-lipid refers to a PEG-lipid whose PEG moiety is derivatized with a chemically reactive group (e.g., maleimide, NHO ester, Cys, azide, alkyne, etc.) that can be used to conjugate targeting moiety to PEG-lipid and thus to LNP containing PEG-lipid.In other embodiments, functionalized PEG-lipid is inserted into LNP after LNP is first formed from other components.In either type of embodiment, targeting moiety is conjugated to functionalized PEG-lipid after functionalized PEG-lipid containing LNP is formed. Protocols for conjugation can be found, for example, in Parhiz et al. J. Controlled Release 291:106-115, 2018, and Tombaccz et al., Molecular Therapy 29(11):3293-3304, 2021, each of which is incorporated by reference for all content that teaches conjugation of PEG-lipids to binding moieties. Alternatively, the targeting moiety can be conjugated to the PEG-lipid prior to insertion into the preformed LNP.

[0169] In certain embodiments of the method for preparing tLNPs, the method comprises: i) forming an initial LNP by mixing all components of a tLNP except for one or more functionalized PEG-lipids and one or more targeting moieties; ii) forming pre-conjugated tLNPs by mixing the initial LNPs with one or more functionalized PEG-lipids; iii) conjugating the pre-conjugated tLNP with one or more targeting moieties to form a tLNP; Includes.

[0170] In certain embodiments of the method for preparing tLNPs, the method comprises: i) forming a pre-conjugated tLNP by mixing all components of the tLNP, including one or more functionalized PEG-lipids, except for one or more targeting moieties; ii) conjugating the pre-conjugated tLNP with one or more targeting moieties to form a tLNP; Includes.

[0171] In certain embodiments of the method for preparing tLNPs, the method comprises: i) conjugating one or more functionalized PEG-lipids with one or more targeting moieties to form one or more conjugated functionalized PEG-lipids; ii) forming a tLNP by mixing all the components of the tLNP, including one or more conjugated functionalized PEG-lipids; Includes.

[0172] In certain embodiments of the method for preparing tLNPs, the method comprises: i) conjugating one or more functionalized PEG-lipids with one or more targeting moieties to form one or more conjugated functionalized PEG-lipids; ii) forming LNPs by mixing all components of the tLNPs except for one or more conjugated functionalized PEG-lipids; iii) forming tLNPs by mixing the initial LNPs with one or more conjugated functionalized PEG-lipids; Includes.

[0173] After conjugation, the tLNPs are purified by dialysis, tangential flow filtration, or size exclusion chromatography and stored as disclosed above for LNPs.

[0174] The encapsulation efficiency of nucleic acid by LNP or tLNP is typically determined using a nucleic acid-binding fluorescent dye added to a complete aliquot and a dissolved aliquot of the final LNP or tLNP preparation to determine the amount of unencapsulated and total nucleic acid, respectively. Encapsulation efficiency is typically expressed as a percentage and calculated as 100×(TU) / T, where T is the total amount of nucleic acid and U is the amount of unencapsulated nucleic acid. In various embodiments, the encapsulation efficiency is 80% or more, 85% or more, 90% or more, or 95% or more.

[0175] In another aspect, disclosed herein is a method of delivering nucleic acid into a cell, comprising contacting the cell with the LNP or tLNP of any of the above aspects. In some embodiments, the contacting step is performed ex vivo. In some embodiments, the contacting step is performed in vivo. In some cases, the in vivo contacting includes intravenous, intramuscular, subcutaneous, intranodal or intralymphatic administration. In some embodiments, toxicity is limited (or nearly limited) to grade 0 or 1 or 2, as discussed above.

[0176] The following examples are intended to illustrate various embodiments of the present invention.Therefore, the specific embodiments discussed should not be interpreted as limitations on the scope of the present invention.It is clear to those skilled in the art that various equivalents, changes and modifications may be made without departing from the scope of the present invention, and it is understood that such equivalent embodiments should be included herein.Furthermore, all references cited in this disclosure are incorporated herein by reference in their entirety, unless they are inconsistent with this disclosure, as if fully set forth herein. EXAMPLES

[0177] Example 1 Synthesis of Compounds Having the Structure of Formula 1 Dihydroxyacetone can be reacted with tert-butoxycarbonylmethylene)triphenylphosphorane to provide the alkene Ia. Hydrogenation of Ia provides Ib, and coupling of Ib with decanoic acid (EDC-HCl, DMAP) gives the tri-ester Ic. Hydrolysis of the t-butyl ester (CF3CO2H, CH2Cl2) gives the monoacid Id. Coupling of Id with BOC-blocked diethanolamine gives Ie. BOC removal (CF3CO2H, CH2Cl2) gives the salt If (see FIG. 1D), which is converted to the acyl-imidazolide Ig after reaction with carbonyldiimidazole. Reaction of Ig with methyl triflate produces the acyl-imidazolium Ih, an intermediate that is converted to compounds A-1 to A-3. Reaction of Ih with 3-dimethylamino-1-propanol in the presence of base affords carbamate compound A-1, reaction of Ih with N,N-dimethyl-1,3-propanediamine provides NH-urea compound A-2, and reaction of Ih with N,N,N'-trimethyl-1,3-propanediamine affords compound A-3 (see Figure 1E).

[0178] Compound A-4 can be obtained from the reaction of salt If with 5-dimethylamino-pentanoic acid (EDC-HCl, DMAP, Et3N) (see Figure 1F).

[0179] The head group in compound A-1, i.e., X in formula 1, is derived from 3-dimethylamino-1-propanol. To obtain analogs of compound A-1 with the disclosed alternative head groups X and various values ​​of n, the compounds in Table 1 can be used in the transformation of Ih to replace 3-dimethylamino-1-propanol. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0180] Reagents XR1-XR9, XR12-XR18, XR21-XR27, XR30-XR38, and XR41-49 are known in the art as reported by the Chemical Abstract Society's SciFinder®, and XR1-XR5, XR7, XR12-XR15, XR21-XR25, XR30-XR31, XR33, and XR41 are commercially available. Polyethylene glycol-containing reagents can be synthesized as described in Example 4, as shown below.

[0181] The head group in compound A-2, i.e., X in formula 1, is derived from N,N-dimethyl-1,3-propanediamine. To obtain analogs of compound A-2 with the disclosed alternative head groups X and various values ​​of n, the compounds in Table 2 can be used in the transformation of Ih to replace N,N-dimethyl-1,3-propanediamine. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0182] Reagents XR52-XR60, XR63-XR70, XR73-XR81, XR84-XR92, and XR95-XR103 are known in the art as reported by the Chemical Abstract Society's SciFinder®, and XR52-XR57, XR63-XR66, XR73-XR77, XR84, XR86-XR87, and XR95 are commercially available. Polyethylene glycol-containing reagents can be synthesized as described in Example 4, as shown below.

[0183] The head group in compound A-3, i.e., X in formula 1, is derived from N,N,N'-trimethyl-1,3-propanediamine. To obtain analogs of compound A-3 with the disclosed alternative head groups X and various values ​​of n, the compounds in Table 3 can be used in the transformation of Ih to replace N,N,N'-trimethyl-1,3-propanediamine. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0184] Reagents XR106-XR114, XR117-XR124, XR127-XR131, XR134, XR138-XR142, XR149-XR153, and XR156 are known in the art as reported by the Chemical Abstract Society's SciFinder®, and XR106-XR110, XR117-XR120, and XR127 are commercially available. XR132-XR133, XR135, XR143-XR146, XR154-XR154, and XR156 are prepared similarly to their shorter congeners. Polyethylene glycol-containing reagents are synthesized as disclosed in Example 4, as shown below.

[0185] The head group in compound A-4, i.e., X in formula 1, is derived from 4-dimethylamino-butanoic acid. To obtain analogs of compound A-4 with the disclosed alternative head groups X and various values ​​of n, the compounds in Table 4 can be used in the conversion of If to replace 4-dimethylamino-butanoic acid. [Table 4-1] [Table 4-2] [Table 4-3]

[0186] Reagents XR160-XR168, XR171-XR178, XR181-XR189, XR192-XR196, and XR203-XR206 are known in the art as reported by the Chemical Abstract Society's SciFinder®, and XR160, XR162-XR164, and XR181 are commercially available. XR196-XR199 and XR207-XR211 can be prepared as well as their shorter congeners. Polyethylene glycol-containing reagents can be synthesized as described in Example 4, as shown below. Example 2 Synthesis of compounds having the structure of formula 2

[0187] Reaction of sodium salts of BOC-blocked diethanolamine with 5-(2-bromoethyl)-2,2-dimethyl-1,3-dioxane II-a or 5-(2-bromoethyl)-2-phenyl-1,3-dioxane II-b leads to II-c and II-d, respectively. Different deprotection options are available, for example as exemplified by II-c and II-d, respectively. II-c can be deprotected with weak acids (PPTs, MeOH) to generate diol II-e. Benzylidene acetal II-d can be deprotected with hydrogen and Pd / C to provide II-e. Coupling of II-e with decanoic acid (EDC-HCl, DMAP) provides II-f, which after deprotection of the BOC-protected amine by exposure to CF3CO2H generates amine salt II-g. Reaction of amine salt II-g with carbonyldiimidazole affords II-h, followed by reaction of acylimidazole II-h with methyl triflate to provide intermediates that can be used in the synthesis of compounds A-5 to A-7, acyl-imidazolium II-i (Figure 2D). Reaction of II-i with 3-dimethylamino-1-propanol in the presence of base affords carbamate compound A-5, reaction of II-i with N,N-dimethyl-1,3-propanediamine affords NH-urea compound A-6, and reaction of II-i with N,N,N'-trimethyl-1,3-propanediamine affords compound A-7 (Figure 2E).

[0188] The amide compound A-8 can be obtained from the reaction of salt II-g with 4-dimethylamino-butanoic acid (EDC-HCl, DMAP, EtN) (Figure 2F).

[0189] The head group in compound A-5, i.e., X in formula 2, is derived from 3-dimethylamino-1-propanol. To obtain analogs of compound A-5 with the disclosed alternative head groups X and various values ​​of n, the compounds of Table 1 (above) can be used in the transformation of II-i to replace 3-dimethylamino-1-propanol.

[0190] The head group in compound A-6, i.e., X in formula 2, is derived from N,N-dimethyl-1,3-propanediamine. To obtain analogs of compound A-6 with the disclosed alternative head groups X and various values ​​of n, the compounds of Table 2 (above) can be used in the transformation of II-i to replace N,N-dimethyl-1,3-propanediamine.

[0191] The head group in compound A-7, i.e., X in formula 2, is derived from N,N,N'-trimethyl-1,3-propanediamine. To obtain analogs of compound A-7 with the disclosed alternative head groups X and various values ​​of n, the compounds of Table 3 (above) can be used in the transformation of II-i to replace N,N,N'-trimethyl-1,3-propanediamine.

[0192] The head group in compound A-8, i.e., X in formula 2, is derived from 4-dimethylamino-butanoic acid. To obtain analogs of compound A-8 with the disclosed alternative head groups X and various values ​​of n, the compounds in Table 4 (above) can be used in the conversion of II-g to replace 4-dimethylamino-butanoic acid.

[0193] The polyethylene glycol-containing reagent is synthesized as disclosed in Example 4 below. Example 3 Synthesis of compounds having the structure of formula 3

[0194] Reaction of glutaric anhydride with dimethylamine gives 5-(dimethylamino)-5-oxopentanoic acid III-a. Coupling of III-a with diethanolamine in the presence of (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) gives N1,N1-bis(2-hydroxyethyl)-N5,N5-dimethyl-pentanediamide III-b, which upon reduction (LiAlH4) provides diol III-c. Coupling of diol III-c with acid Id then gives compound A-9 (Figure 3C).

[0195] Reaction of the sodium salt of III-c (NaH, DMF) with either bromide II-a or II-b gives the diamines III-d and III-e. Deprotection of III-d with PPTs in methanol or III-e with hydrogen and palladium on carbon provides the tetraol III-f. Coupling of the tetraol III-f with decanoic acid (EDC-HCl, DMAP) affords compound A-10.

[0196] The head group in compounds A-9 and A-10, i.e., X in formula 3, is derived from 5-(dimethylamino)-5-oxopentanoic acid (III-a) reacted with diethanolamine. To obtain analogs of compounds A-9 and A-10 with the disclosed alternative head groups X and various values ​​of n, the carboxylic acids in Table 4 can be used to replace 5-(dimethylamino)-5-oxopentanoic acid (III-a) according to the following scheme: [ka] The reaction is completed according to Figures 3C-D, as appropriate.

[0197] The polyethylene glycol-containing reagent is synthesized as disclosed in Example 4 below. Example 4 Synthesis of polyethylene glycol-containing head groups

[0198] Reaction of commercially available 1,1-dimethylethyl N-(3-hydroxypropyl)-N-methylcarbamate sodium salt (NaH, DMF) with commercially available mesylate, 2-(2-(2-methoxyethoxy)ethoxy)ethyl methanesulfonate, provides V-5, which then gives amine V-6 after BOC removal (TFA) and neutralization. Reductive amination utilizing commercially available 1,1-dimethylethyl N-methyl-N-(3-oxopropyl)carbamate then gives BOC-protected amine V-7. Targeted PEG-containing headgroup piece V-8 (XR125 in Table 3) is then obtained after BOC removal (TFA) and amine neutralization. See Figure 4A.

[0199] Similarly, reaction of commercially available BOC-blocked 4-hydroxypiperidine sodium salt (NaH, DMF) with commercially available 2-(2-(2-methoxyethoxy)ethoxy)ethyl methanesulfonate provides V-1, which then gives amine V-2 after BOC removal (TFA) and neutralization. Reductive amination utilizing commercially available 1,1-dimethylethyl N-methyl-N-(3-oxopropyl)carbamate then gives BOC-protected amine V-3. Target PEG-containing headgroup piece V-4 (XR126 in Table 3) then gives after BOC removal (TFA) and amine neutralization. See Figure 4B.

[0200] Shorter chain PEG-containing head group entities can be obtained by substituting known / commercially available shorter chain mesylates V-9 (known) and V-10 (commercially available) in place of the 2-(2-(2-methoxyethoxy)ethoxy)ethyl methanesulfonate utilized in the above scheme. [ka]

[0201] To synthesize variants of V-4 with different values ​​of n or alternative definitions of Y, appropriate analogs of 1,1-dimethylethyl N-methyl-N-(3-oxopropyl)carbamate are used to form the desired PEG-containing head group piece. For variants of V-8, appropriate analogs of 1,1-dimethylethyl N-(3-hydroxypropyl)-N-methylcarbamate are used to introduce different values ​​of m, and analogs of 1,1-dimethylethyl N-methyl-N-(3-oxopropyl)carbamate are used to introduce different values ​​of n or definitions of Y to form the desired PEG-containing head group piece. Example 5 Synthesis of 1,1-dimethylethyl 4-hydroxy-3-(hydroxymethyl)-2-butenoate (Ia) [ka]

[0202] To a solution of dihydroxyacetone (150.0 g, 1.67 mol) in anhydrous dichloromethane (3.0 L) under nitrogen, tert-butoxycarbonylmethylene-triphenylphosphorane (627 g, 1.67 mol) was added in portions over 1 h. The mixture was stirred at room temperature for 18 h, then silica gel (750 g, type: ZCX-2, 100-200 mesh) was added to the solution and the solvent was removed in vacuo to give crude 1 impregnated onto the silica gel. The dried silica gel was placed onto a gravity column of silica gel (3700 g, type: ZCX-2, 100-200 mesh, packed with petroleum ether) and the resulting column was eluted with a gradient of petroleum ether:ethyl acetate (100:0 to 50:50). Compound Ia was eluted with petroleum ether:ethyl acetate 50:50, and the Ia fraction was concentrated in vacuo to give 1 (235.0 g) containing Ph3PO (73.8% purity by HNMR, 55% yield of Ia).

[0203] 1H NMR (400 MHz, DMSO-d6, ppm): δ 5.77 (m, 1H), 5.00 (t, J = 5.6 Hz, 1H), 4.80 (t, J = 5.7 Hz, 1H), 4.49 (dd, J = 5.8, 1.5 Hz, 2H), 4.17 (dd, J = 5.6, 2.0 Hz, 2H), 1.42 (s, 9H);LCMS(+mode):C9H 16 O4+H + Calculated value: 189.11, actual value: 189.10. Example 6 Synthesis of tert-butyl 4-hydroxy-3-(hydroxymethyl)butanoate (Ib) [ka]

[0204] To a solution of Ia (100.00 g, 73.8% purity, 0.53 mol) in anhydrous EtOH (1.0 L) in a 2.0 L round bottom flask under nitrogen, Et3N (8.10 g, 0.080 mol) was added followed by 10% Pd / C (20.0 g). The mixture was placed under a hydrogen balloon at room temperature for 16 h. HPLC analysis showed that the hydrogenation was not complete, so the hydrogen balloon was recharged and the reaction was continued for another 16 h. The mixture was filtered through a pad of Celite®, the filter cake was rinsed with anhydrous EtOH (200 mL), and the combined filtrate was concentrated in vacuo to give Ib (198.0 g) containing P3PO as a yellow oil (67.5% purity by HNMR, 66% yield of Ib).

[0205] 1 H NMR (400MHz, DMSO-D6, ppm): d 4.47 (t, J = 5.10Hz, 2H), 3.44-3.29 (5H), 2.17 (d, J = 7.00Hz, 2H), 1.93 (m, 1H), 1.39 (s, 9H). Example 7 Synthesis of 2-(2-(tert-butoxy)-2-oxoethyl)propane-1,3-diyldinonanoate (I-c2) [ka]

[0206] To a solution of Ib (120.00 g, 67.5% purity, 0.63 mol) in CHCl (1.2 L) in a 4 L flask under nitrogen, nonanoic acid (199.60 g, 1.26 mol, 2.0 equiv.) and DMAP (77.10 g, 0.63 mol) were added in sequence. The mixture was stirred at room temperature for 10 min, then EDCl (266.8 g, 1.39 mol, 2.20 equiv.) was added in one portion. The mixture was stirred at room temperature for 16 h, then the solvent was removed in vacuo to give crude I-c2 as a sticky yellow oil. Crude I-c2 was dissolved in heptane / methyl tert-butyl ether (MTBE) (9:1, 2.0 L) and the solution was washed with 10% aqueous citric acid (2×1.2 L). The organic phase was washed with brine (2.0 L), MeOH:H2O (5:1, 3×1.20 L), and then dried over anhydrous Na2SO4. The solids were removed by filtration, and silica gel (400 g, type: ZCX-2, 100-200 mesh) was added to the solution. The solvent was removed in vacuum to give crude I-c2 impregnated onto the silica gel. The dried silica gel was placed onto a gravity column of silica gel (4000 g, type: ZCX-2, 100-200 mesh, packed with petroleum ether), and the resulting column was eluted with a gradient of petroleum ether:THF (100:0 to 95:5). Compound I-c2 was eluted with petroleum ether:THF 98:2, and the fraction of I-c2 was concentrated in vacuum to give I-c2 as a colorless oil (208.0 g, 97.2% purity by HPLC, 68% yield).

[0207] 1 H NMR (400MHz, CDCl3, ppm): d 4.11 (m, 4H), 2.52 (m, 1H), 2.31 (t, J = 7.40Hz, 4H), 1.62 (m, 4H), 1.46 (s, 9H), 1.35-1.27 (20H), 0.87 (m, 6H).

[0208] When used to synthesize lipids according to formula 1 or 3 according to the synthetic schemes disclosed herein, I-c2 results in lipids where R is a linear C8, while Ic results in lipids where R is a linear C9. Example 8 Synthesis of 4-(nonanoyloxy)-3-((nonanoyloxy)methyl)butanoic acid (I-d2) [ka]

[0209] To a solution of I-c2 (110.0 g, 0.234 mol) in CHCl (1.0 L) at room temperature under nitrogen, trifluoroacetic acid (TFA) (330 mL, 4.31 mol) was added over 30 min. The resulting solution was stirred at room temperature for 16 h and then concentrated in vacuo to give crude I-d2 as a yellow oil. Crude I-d2 was dissolved in CHCl (1.10 L) and the solution was washed with water (2×0.50 L). The combined aqueous phase was extracted with CHCl (0.5 L) and the combined organic layers were dried over anhydrous MgSO. Filtration and concentration in vacuo gave I-d2 as a light brown oil (96.0 g, 0.232 mol, 99%, 98.5% pure by HPLC).

[0210] 1 H NMR (400MHz, CDCl3, ppm): d 4.13 (m, 4H), 2.58 (m, 1H), 2.49 (d, II = 6.90Hz, 2H), 2.33 (t, J = 7.60Hz, 4H), 1.63 (m, 4H), 1.36-1.22 (20H), 0.89 (t, J = 6.60Hz, 6H);LCMS(+mode):C 23 H 42 O6+H + Calculated value: 415.31. Measured value: 415.30.

[0211] When used to synthesize lipids according to formula 1 or 3 according to the synthetic schemes disclosed herein, I-d2 results in lipids where R is a linear C8, while Id results in lipids where R is a linear C9. Example 9 Synthesis of (((((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl))bis(oxy))bis(2-oxoethane-2,1-diyl))bis(propane-2,1,3-triyl)tetranonanoate (I-e2) [ka]

[0212] To a solution of I-d2 (142.8 g, 0.344 mol) in CHCl (1.0 L) at room temperature under nitrogen, tert-butyl bis(2-hydroxyethyl)carbamate (33.6 g, 0.164 mol) and DMAP (20.0 g, 0.164 mol) were added in sequence. The mixture was stirred for 10 min, then EDCl (78.7 g, 0.410 mol) was added over 10 min, and the resulting solution was stirred at room temperature for 16 h. The reaction mixture was washed with water (2×1.2 L), the aqueous phase was extracted with CHCl (2×1.2 L), and the combined organic phase was dried over anhydrous MgSO. After filtration to remove solids, the solution was concentrated in vacuum to a volume of approximately 1.5 L, silica gel (350 g, type: ZCX-2, 100-200 mesh) was added, and the mixture was concentrated in vacuum to dryness. The dried silica gel was placed on a gravity column of silica gel (2100 g, type: ZCX-2, 100-200 mesh, packed with heptane) and the resulting column was eluted with a gradient of heptane:THF (100:0 to 90:10). Compound I-e2 was eluted with heptane:THF 95:5 and the I-e2 fraction was concentrated in vacuo to give I-e2 as a yellow oil (143.5 g, 96.3% purity by HPLC, 83% yield).

[0213] 1H NMR (400MHz, CDCl3, ppm): d 4.21 (m, 4H), 4.11 (m, 8H), 3.49 (m, 4H), 2.58 (m, 2H),2.43 (d, J = 6.90Hz, 4H), 2.31 (t, J = 7.60Hz, 8H), 1.66-1.59 (8H), 1.48 (s, 9H), 1.20-1.39 (40H), 0.89 (t, J = 6.60Hz, 12H).

[0214] When used in the synthesis of lipids according to formula 1 or 3 according to the synthetic schemes disclosed herein, I-e2 results in lipids where R is a linear C8, while Ie results in lipids where R is a linear C9. Example 10 Synthesis of bis(2-((4-(nonanoyloxy)-3-((nonanoyloxy)methyl)butanoyl)oxy)ethyl)amine trifluoroacetate (I-f2) [ka]

[0215] To a solution of I-e2 (143.0 g, 0.137 mol) in CH2Cl2 (850 mL) at room temperature under nitrogen was added TFA (286 mL, 3.74 mol) over 30 min. After the addition was complete, the mixture was stirred for 5 h, then the solution was poured into 10% aqueous K2HPO4 (1.40 L) and water (0.70 L). The organic phase was dried (MgSO4), filtered, and concentrated in vacuo to give the ammonium salt 6 as a yellow oil (140.5 g, 89% purity by HPLC, 87% yield).

[0216] 1H NMR (400MHz, CDCl3, ppm): d 4.79 (m, 4H), 4.20 (m, 4H), 4.10 (m, 4H), 3.46 (m, 4H), 2.55 (m,2H), 2.42 (d, J = 7.10Hz, 4H), 2.34 (t, J = 7.60Hz, 8H), 1.62 (m, 8H), 1.21-1.38 (40H), 0.90 (m, 12H);LCMS(+mode):C 50 H 92 NO 12 Calculated value: 898.66. Measured value: 899.0.

[0217] When used in the synthesis of lipids according to formula 1 or 3 according to the synthetic schemes disclosed herein, I-e2 results in lipids where R is a linear C8, while Ie results in lipids where R is a linear C9. Example 11 Synthesis of ((((1H-imidazole-1-carbonyl)azanediyl)bis(ethane-2,1-diyl))bis(oxy))bis(2-oxoethane-2,1-diyl))bis(propane-2,1,3-triyl)tetranonanoate (I-g2) [ka]

[0218] To a solution of I-f2 (130.0 g, 0.123 mol) in CHCl (1.30 L) at room temperature under nitrogen, carbonyldiimidazole (CDI) (79.8 g, 0.493 mol) and EtN (25.2 g, 0.246 mol) were added in sequence. The resulting solution was stirred at room temperature for 3 h. HPLC analysis indicated the reaction was not complete, and additional CDI (39.9 g, 0.246 mol) and EtN (14.6 g, 0.123 mol) were added. The solution was stirred at room temperature for 14 h, and then the mixture was poured into aqueous HCl (0.8 M, 1.30 L). The organic phase was separated and the aqueous phase was extracted with CHCl (1.30 L). The combined organic phase was concentrated in vacuo to give crude I-g2 as a yellow oil, which was dissolved in heptane (1.30 L). The heptane solution was washed with MeOH-HO (5:1, 2×0.65 L) and brine (0.65 L). The heptane solution was dried (MgSO), filtered to remove solids, and the filtrate was concentrated in vacuo to give 7 (122.0 g, 83.3% purity by HPLC, 83% yield) as a viscous yellow oil.

[0219] 1 H NMR (400MHz, CDCl3, ppm): d 7.99 (s,1H), 7.31 (s, 1H), 7.14 (s, 1H), 4.32 (m,4H), 4.12 (m, 8H), 3.77 (m, 4H), 2.55 (m, 2H), 2.42 (d, J = 6.90Hz, 4H), 2.31 (m,8H), 1.62 (m,8H), 1.21-1.33 (40H), 0.90 (m, 12H);LCMS(+mode):C 54 H 93 N3O 13 Calculated value: 991.67. Measured value: 993.0. Example 12 Synthesis of 1-(bis(2-((4-(nonanoyloxy)-3-((nonanoyloxy)methyl)butanoyl)oxy)ethyl)carbamoyl)-3-methyl-1H-imidazol-3-ium trifluoromethanesulfonate (I-h2) [ka]

[0220] Acyl-imidazole I-g2 (18.0 g, 18.2 mmol) was dissolved in CHCl (270 mL) under nitrogen and cooled in an ice-water bath. To this cooled solution of I-g2, methyl trifluoromethanesulfonate (MeOTf) (3.30 g, 20.0 mmol) was added over 15 min. The resulting solution was stirred at 0° C. for 1 h and then continued towards the target lipid (see below). HPLC and LCMS showed complete consumption of I-g2. Example 13 Synthesis of Compound A-11 [ka]

[0221] To a solution of I-h2, prepared from I-g2 (20.2 mmol) as described above, cooled in an ice-water bath under nitrogen, tetramethylethylenediamine (11.70 g, 100.8 mmol) and 2-dimethylamino-ethanol (3.60 g, 40.3 mmol) were added in sequence. The mixture was stirred at 0 °C for 1 h, then warmed to room temperature and stirred for 18 h. The solution was concentrated in vacuo and the residue was dissolved in ethyl acetate (300 mL). The solution was washed with 10% aqueous citric acid (2 × 300 mL), the organic phase was separated, and the aqueous phase was extracted with ethyl acetate (300 mL). The combined organic phase was washed with 5% aqueous NaHCO3 (300 mL), brine (300 mL), and dried (MgSO4). Solids were removed by filtration, silica gel (40 g, type: ZCX-2, 100-200 mesh) was added to the solution, and the mixture was concentrated to dryness in vacuo. The dried silica gel was placed on a silica gel gravity column (200 g, type: ZCX-2, 100-200 mesh, packed with CHCl) and the resulting column was eluted with a gradient of CHCl:MeOH (100:0 to 90:10). Compound A-11 was eluted with CHCl:MeOH 95:5 and the fractions of compound A-11 were concentrated in vacuo to give compound A-11 as a yellow oil (12.0 g, HPLC purity 88%). Compound A-11 was further purified by reversed-phase flash chromatography (WelFlash XSelect CSH Prep C18, 5 mm OBD, regular 30 × 150 mm column; solvent: A: 0.1% formic acid in water, B: acetonitrile, gradient 50-80%, 20 min, flow rate 55 mL / min). The fractions containing compound A-11 were pooled and concentrated in vacuo, and the residue was dissolved in heptane (150 mL). The heptane solution was washed with MeOH / water (75:25, 100 mL) and brine (100 mL). The organic phase was dried (Na2SO4), filtered to remove solids, and the filtrate was concentrated in vacuo to give compound A-11 (10.15 g, 97.8% purity by HPLC, 49% yield) as a yellow oil.

[0222] 1H NMR (400MHz, CDCl3, ppm): d 4.20 (m, 6H), 4.09 (m, 8H), 3.54 (m, 4H), 2.55 (m, 4H), 2.41 (d, J = 6.90Hz, 4H), 2.25-2.32 (14H), 1.61 (m, 8H), 1.20-1.34 (40H), 0.88 (t, J = 6.70Hz, 12H);LCMS(+mode):C 55 H 100 N2O 14 +H + Calculated value: 1013.72. Measured value: 1013.80 [M+H + ]. Example 14 Synthesis of compound A-12 [ka]

[0223] To a solution of I-h2, prepared from I-g2 (18.2 mmol) as described above, cooled in an ice-water bath under nitrogen, tetramethylethylenediamine (6.30 g, 54.4 mmol) and 3-dimethylamino-propanol (2.20 g, 21.8 mmol) were added in sequence. The mixture was stirred at 0 °C for 1 h, then warmed to room temperature and stirred for 18 h. The solution was concentrated in vacuo and the residue was dissolved in ethyl acetate (300 mL). The solution was washed with 10% aqueous citric acid (2 × 300 mL), the organic phase was separated, and the aqueous phase was extracted with ethyl acetate (300 mL). The combined organic phase was washed with 5% aqueous NaHCO3 (300 mL), brine (300 mL), and dried (MgSO4). Solids were removed by filtration, silica gel (36 g, type: ZCX-2, 100-200 mesh) was added to the solution, and the mixture was concentrated to dryness in vacuo. The dried silica gel was placed on a silica gel gravity column (180 g, type: ZCX-2, 100-200 mesh, packed with CHCl) and the resulting column was eluted with a gradient of CHCl:MeOH (100:0 to 90:10). Compound A-12 was eluted with CHCl:MeOH 95:5 and the compound A-12 fraction was concentrated in vacuo to give compound A-12 as a yellow oil (11.2 g, HPLC purity 85%). Compound A-12 was further purified by reversed-phase flash chromatography (WelFlash XSelect CSH Prep C18, 5 mm OBD, regular 30 × 150 mm column; solvent: A: 0.1% formic acid in water, B: acetonitrile, gradient 50-80%, 20 min, flow rate 55 mL / min). The fractions containing compound A-12 were pooled and concentrated in vacuo, and the residue was dissolved in heptane (150 mL). The heptane solution was washed with 5% aqueous Na2CO3 (100 mL), MeOH / water (75:25, 100 mL) and brine (100 mL). The organic phase was dried (Na2SO4), filtered to remove solids, and the filtrate was concentrated in vacuo to give compound A-12 (10.22 g, 96.8% purity by HPLC, 48% yield) as a yellow oil.

[0224] 1H NMR (400MHz, CDCl3, ppm): d 4.06-4.23 (14H), 3.53 (m, 4H), 2.55 (m, 2H), 2.41 (d, J = 6.90Hz, 6H), 2.27-2.32 (14H), 1.85 (m, 2H), 1.57-1.62 (8H), 1.20-1.35 (40H), 0.88 (t, J = 6.80Hz, 12H);LCMS(+mode):C 56 H 102 N2O 14 +H + Calculated value: 1027.74. Measured value: 1027.90 [M+H + ]. Example 15 Synthesis of compound A-13 [ka]

[0225] To a solution of I-h2, prepared from I-g2 (22.0 mmol) as described above, cooled in an ice-water bath under nitrogen, triethylamine (6.72 g, 66.0 mmol) and 2-dimethylamino-ethylamine (2.34 g, 26.0 mmol) were added in sequence. The mixture was stirred at 0°C for 1 h, then warmed to room temperature and stirred for 18 h. The solution was concentrated in vacuo and the residue was dissolved in ethyl acetate (600 mL). The solution was washed with 5% aqueous Na2CO3 (2 x 300 mL), brine (300 mL), and dried (MgSO4). Solids were removed by filtration, silica gel (40 g, type: ZCX-2, 100-200 mesh) was added to the solution, and the mixture was concentrated in vacuo to dryness. The dried silica gel was placed on a silica gel gravity column (250 g, type: ZCX-2, 100-200 mesh, packed with CHCl) and the resulting column was eluted with a gradient of CHCl:MeOH (100:0 to 90:10). Compound A-13 was eluted with CHCl:MeOH 97:3 and the compound A-13 fraction was concentrated in vacuo to give compound A-13 as a yellow oil (18.0 g, HPLC purity 83%). Compound A-13 was further purified by reversed-phase flash chromatography (WelFlash XSelect CSH Prep C18, 5 mm OBD, regular 30 × 150 mm column; solvent: A: 0.1% formic acid in water, B: acetonitrile, gradient 50-80%, 20 min, flow rate 55 mL / min). The fractions containing compound A-13 were pooled and concentrated in vacuo, and the residue was dissolved in heptane (150 mL). The heptane solution was washed with saturated aqueous NaHO3 (200 mL), MeOH / water (80:20, 2 x 200 mL) and brine (200 mL). The organic phase was dried (Na2SO4), filtered to remove solids, and the filtrate was concentrated in vacuo to give compound A-13 (10.58 g, 95.5% purity by HPLC, 47% yield) as a yellow oil.

[0226] 1H NMR (300MHz, CDCl3, ppm): d 5.50 (brs, 1H), 4.22 (t, J = 6.00Hz, 4H), 4.03-4.20 (8H), 3.52 (m, 4H), 3.30 (m, 2H), 2.56 (m, 2H), 2.32-2.46 (6H), 2.20-2.32 (14H), 2.50-2.65 (8H), 1.15-1.32 (40H), 0.88 (m, 12H);LCMS(+mode):C 54 H 101 N3O 13 +H + Calculated value: 1012.74. Measured value: 1012.80 [M+H + ]. Example 16 Synthesis of compound A-14 [ka] To a solution of I-h2, prepared from I-g2 (22.0 mmol) as described above, cooled in an ice-water bath under nitrogen, triethylamine (6.72 g, 66.0 mmol) and N,N,N'-trimethylethylenediamine (2.71 g, 26.0 mmol) were added in sequence. The mixture was stirred at 0 °C for 1 h, then warmed to room temperature and stirred for 18 h. The solution was concentrated in vacuo and the residue was dissolved in ethyl acetate (600 mL). The solution was washed with 10% aqueous citric acid (2 x 300 mL), 5% aqueous Na2CO3 (2 x 300 mL), brine (300 mL), and dried (MgSO4). Solids were removed by filtration, silica gel (40 g, type: ZCX-2, 100-200 mesh) was added to the solution, and the mixture was concentrated in vacuo to dryness. The dried silica gel was placed on a silica gel gravity column (250 g, type: ZCX-2, 100-200 mesh, packed with CHCl) and the resulting column was eluted with a gradient of CHCl:MeOH (100:0 to 90:10). Compound A-14 was eluted with CHCl:MeOH 97:3 and the fractions of compound A-14 were concentrated in vacuo to give compound A-14 as a yellow oil (18.0 g, HPLC purity 88%). Compound A-14 was further purified by reversed-phase flash chromatography (WelFlash XSelect CSH Prep C18, 5 mm OBD, regular 30 × 150 mm column; solvent: A: 0.1% formic acid in water, B: acetonitrile, gradient 50-80%, 20 min, flow rate 55 mL / min). Fractions containing compound A-14 were pooled and concentrated in vacuo, and the residue was dissolved in heptane (500 mL). The heptane solution was washed with saturated aqueous NaHO3 (500 mL), MeOH / water (80:20, 2 x 200 mL) and brine (200 mL). The organic phase was dried (Na2SO4), filtered to remove solids, and the filtrate was concentrated in vacuo to give compound A-14 (10.37 g, 96.6% purity by HPLC, 46% yield) as a yellow oil.

[0227] 1H NMR (300MHz, CDCl3, ppm): d 4.21 (t, J = 6.00Hz, 4H), 4.04-4.17 (8H), 3.42 (t, J = 6.00Hz, 4H), 3.30 (t, J = 6.90Hz, 2H), 2.87 (s, 3H), 2.30-2.60 (8H), 2.10-2.27 (14H), 1.59 (m, 8H), 1.14-1.30 (40H), 0.88 (t, J = 6.90Hz, 12H);LCMS (+ mode):C 56 H 103 N3O 13 +H + Calculated value: 1026.76. Measured value: 1027.00 [M+H + ]. Example 17 Synthesis of 2-(2-(tert-butoxy)-2-oxoethyl)propane-1,3-diylbis(decanoate) (Ic) [ka]

[0228] To a solution of Ib (30.0 g, 0.16 mol) in CH2Cl2 (300 mL) under nitrogen, decanoic acid (54.3 g, 0.32 mol) and DMAP (77.10 g, 0.63 mol) were added in sequence. The mixture was stirred at room temperature for 10 min, then EDCl (66.7 g, 0.36 mol) was added in one portion. The mixture was stirred at room temperature for 16 h, then the solvent was removed in vacuo to give crude Ic as a sticky yellow oil. Crude Ic was dissolved in MTBE (450 mL) and the organic phase was extracted with 1% aqueous citric acid (2 x 300 mL). The combined aqueous phase was extracted with MTBE (2 x 300 mL) and the combined organic phase was washed with brine (450 mL) and dried (Na2SO4). The solids were removed by filtration and the filtrate was concentrated in vacuo to give crude Ic as a yellow oil. Crude Ic was dissolved in ethyl acetate (250 mL) and silica gel (90 g, type: ZCX-2, 100-200 mesh) was added to the solution. The solvent was removed in vacuum to give crude Ic that was infiltrated onto the silica gel. The dried silica gel was placed onto a gravity column of silica gel (900 g, type: ZCX-2, 100-200 mesh, packed with petroleum ether) and the resulting column was eluted with a gradient of petroleum ether:ethyl acetate (100:0 to 95:5). Compound Ic was eluted with petroleum ether:ethyl acetate 98:2 and the fraction of Ic was concentrated in vacuum to give Ic as a pale pink oil (49.8 g, 98.6% purity by HPLC, 92% yield).

[0229] 1 H NMR (400MHz, CDCl3, ppm): d 4.11 (m, 4H), 2.53 (m, 1H), 2.32 (m, 6H), 1.63 (m, 4H), 1.49 (s, 9H), 1.20-1.30 (24H), 0.90 (m, 6H). Example 18 Synthesis of 4-(decanoyloxy)-3-((decanoyloxy)methyl)butanoic acid Id [ka]

[0230] To a solution of Ic (49.0 g, 98.2 mmol) in CHCl (400 mL) at room temperature under nitrogen, trifluoroacetic acid (TFA) (147 mL, 1.92 mol) was added over 30 min. The resulting solution was stirred at room temperature for 16 h and then concentrated in vacuo to give crude Id as a yellow oil. Crude Id was dissolved in CHCl (500 mL) and the solution was washed with water (2×0250 L). The combined aqueous phase was extracted with CHCl (0.25 L) and the combined organic layers were washed with 2% aqueous NaHCO (250 mL) and dried over anhydrous MgSO. Filtration and concentration in vacuo gave Id as an ivory solid (42.0 g, 95.6 mol, 99% yield, 98.5% purity by HPLC).

[0231] 1 H NMR (400MHz, CDCl3, ppm): d 4.13 (m, 4H), 2.58 (m, 1H), 2.49 (d, J = 6.90Hz, 2H), 2.32 (t, J = 7.60Hz, 4H), 1.63 (m, 4H), 1.36-1.22 (24H), 0.89 (t, J = 6.70Hz, 6H);LCMS(+mode):C 25 H 46 O6+H + Calculated value: 443.34. Measured value: 443.30. Example 19 Synthesis of (((((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl))bis(oxy))bis(2-oxoethane-2,1-diyl))bis(propane-2,1,3-triyl)tetrakis(decanoate) Ie [ka]

[0232] To a solution of tert-butyl bis(2-hydroxyethyl)carbamate (7.50 g, 36.6 mmol) in CHCl (225 mL) at room temperature under nitrogen, Id (34.00 g, 76.8 mmol) and DMAP (4.50 g, 36.6 mmol) were added in sequence. The mixture was stirred for 10 min, then EDCl (17.60 g, 91.5 mmol) was added over 10 min, and the resulting solution was stirred at room temperature for 16 h. The reaction mixture was washed with water (2×300 mL), the aqueous phase was extracted with CHCl (2×300 mL), and the combined organic phase was dried over anhydrous MgSO. After filtration to remove solids, silica gel (187.5 g, type: ZCX-2, 100-200 mesh) was added to the filtrate, and the mixture was concentrated to dryness in vacuo. The dried silica gel was placed on a gravity column of silica gel (1125 g, type: ZCX-2, 100-200 mesh, packed with heptane) and the resulting column was eluted with a gradient of heptane:THF (100:0 to 90:10). Compound Ie was eluted with heptane:THF 95:5 and the Ie fraction was concentrated in vacuo to give Ie as a yellow oil (25.50 g, 91% purity by HPLC, 60% yield).

[0233] 1 H NMR (400MHz, CDCl3, ppm): d 4.20 (m, 4H), 4.12 (m, 8H), 3.49 (m, 4H), 2.58 (m, 2H), 2.43 (d, J = 7.00Hz, 4H), 2.31 (t, J = 7.60Hz, 8H), 1.66-1.59 (8H), 1.48 (s, 9H), 1.21-1.39 (48H), 0.89 (m, 12H). Example 20 Synthesis of bis(2-((4-(decanoyloxy)-3-((decanoyloxy)methyl)butanoyl)oxy)ethyl)ammonium trifluoroacetate (If) [ka]

[0234] To a solution of Ie (27.00 g, 23.3 mmol) in CH2Cl2 (150 mL) at room temperature under nitrogen was added TFA (54.0 mL, 0.71 mol) over 30 min. After the addition was complete, the mixture was stirred for 5 h, then the solution was washed with 10% aqueous K2HPO4 (270 mL) and water (2 x 135 mL). The organic phase was dried (MgSO4), filtered, and concentrated in vacuo to give the ammonium salt If as a yellow oil (25.50 g, 88% purity by HPLC, 90% yield).

[0235] 1 H NMR (400MHz, CDCl3, ppm): d 4.43 (m, 4H), 4.12 (m, 8H), 3.40 (m, 4H), 2.54 (m, 2H), 2.42 (m, 4H), 2.32 (m, 8H), 1.61 (m, 8H), 1.20-1.37 (48H), 0.89 (t, J = 6.70Hz, 12H); LCMS(+mode):C 54 H 100 NO 12 Calculated value: 954.72. Measured value: 955.00. Example 21 Synthesis of (((((1H-imidazole-1-carbonyl)azanediyl)bis(ethane-2,1-diyl))bis(oxy))bis(2-oxoethane-2,1-diyl))bis(propane-2,1,3-triyl)tetrakis(decanoate)Ig [ka]

[0236] To a solution of If (25.00 g, 22.1 mmol) in CHCl (250 mL) at room temperature under nitrogen, carbonyldiimidazole (CDI) (14.40 g, 88.6 mol) and EtN (4.50 g, 44.3 mol) were added in sequence. The resulting solution was stirred at room temperature for 3 h. HPLC analysis indicated the reaction was not complete, and additional CDI (14.4 g, 88.6 mmol) and EtN (4.50 g, 44.3 mol) were added. The solution was stirred at room temperature for 14 h, and then the mixture was poured into aqueous HCl (0.8 M, 250 mL). The organic phase was separated and the aqueous phase was extracted with CHCl (250 mL). The combined organic phase was concentrated in vacuo to give crude Ig as a yellow oil, which was dissolved in heptane (250 mL). The heptane solution was washed with MeOH-HO (5:1, 2×125 mL) and brine (125 mL). The heptane solution was dried (MgSO), filtered to remove solids, and the filtrate was concentrated in vacuo to give Ig (23.40 g, 79.8% purity by HPLC, 80% yield) as a viscous pale yellow oil.

[0237] 1 H NMR (400MHz, CDCl3, ppm): d 7.98 (s,1H), 7.30 (s, 1H), 7.13 (s, 1H), 4.32 (m,4H), 4.11 (m, 8H), 3.76 (m, 4H), 2.54 (m, 2H), 2.41 (d, J = 6.90Hz, 4H), 2.30 (m,8H), 1.60 (m,8H), 1.18-1.32 (48H), 0.89 (m, 12H);LCMS(+mode):C 58 H 101 N3O 13 +H + Calculated value: 1048.74. Measured value: 1049.10. Example 22 Synthesis of 1-(bis(2-((4-(decanoyloxy)-3-((decanoyloxy)methyl)butanoyl)oxy)ethyl)carbamoyl)-3-methyl-1H-imidazol-3-ium trifluoromethanesulfonate (Ih) [ka]

[0238] Acyl-imidazole Ig (20.0 g, 18.2 mmol) was dissolved in CH2Cl2 (300 mL) under nitrogen and cooled in an ice-water bath. To this cooled solution of Ig, MeOTf (4.50 g, 27.3 mmol) was added over 15 min. The resulting solution was stirred at 0°C for 1 h and then continued towards the target lipid (see below). HPLC and LCMS showed complete consumption of Ig.

[0239] LCMS(+mode):C 59 H 104 N3O 13 Calculated value: 1062.76. Measured value: 1063.00. Example 23 Synthesis of Compound A-2 [ka]

[0240] To a solution of Ih prepared from Ig (18.2 mmol) as described above cooled in an ice-water bath under nitrogen, triethylamine (5.50 g, 54.7 mmol) and 3-dimethylaminopropylamine (2.80 g, 27.3 mmol) were added in sequence. The mixture was stirred at 0° C. for 1 h, then warmed to room temperature and stirred for 18 h. The solution was concentrated in vacuo and the residue was dissolved in heptane (600 mL). The solution was washed with MeOH / H2O (80:20, 2×150 mL), brine (150 mL), and dried (MgSO4). The solids were removed by filtration and the filtrate was concentrated in vacuo to give crude compound A-2 as a viscous yellow oil. The crude product was dissolved in ethyl acetate (300 mL), washed with 5% aqueous Na2CO3 (2×300 mL), brine (300 mL), and dried (MgSO4). The solids were removed by filtration, silica gel (40 g, type: ZCX-2, 100-200 mesh) was added to the solution, and the mixture was concentrated to dryness in vacuo. The dried silica gel was placed on a gravity column of silica gel (200 g, type: ZCX-2, 100-200 mesh, packed with CHCl) and the resulting column was eluted with a gradient of CHCl:MeOH (100:0 to 90:10). Compound A-2 was eluted with CHCl:MeOH 95:5, and the compound A-2 fraction was concentrated in vacuo to give compound A-2 as a yellow oil (12.5 g). Compound A-2 was dissolved in heptane (150 mL), washed with MeOH / HO (80:20, 2×150 mL), brine (150 mL), and dried (MgSO). The solids were removed by filtration and the filtrate was concentrated in vacuo to give compound A-2 as a pale yellow oil (11.96 g, 94% purity by HPLC, 57% yield).

[0241] 1H NMR (400MHz, CDCl3, ppm): d 6.61 (m, 1H), 4.20 (t, J = 6,.40Hz, 4H), 4.10 (m, 8H),3.49 (t, J = 6.00Hz, 4H), 3.32 (m, 2H), 2.46-2.56 (4H), 2.41 (m, 4H), 2.23-2.32 (14H), 1.73 (m, 2H), 1.59 (m, 8H), 1.19-1.34 (48H), 0.89 (t, J = 6.70Hz, 12H60 LCMS (+ mode):C 56 H 111 N3O 13 +H + Calculated value: 1082.82. Measured value: 1083.00 [M+H + ]. Example 24 Synthesis of compound A-15 [ka]

[0242] To a solution of I-h2, prepared from I-g2 (15.12 mmol) as described above, cooled in an ice-water bath under nitrogen, triethylamine (4.60 g, 45.36 mmol) and N,N,N'-trimethylpropylenediamine (2.63 g, 22.68 mmol) were added in sequence. The mixture was stirred at 0 °C for 1 h, then warmed to room temperature and stirred for 18 h. The mixture was poured into water (750 mL) and the organic phase separated. The aqueous phase was extracted with CHCl (2 × 300 mL) and the combined organic phase was washed with brine (300 mL) and dried (NaSO). Solids were removed by filtration, silica gel (30 g, type: ZCX-2, 100-200 mesh) was added to the filtrate, and the mixture was concentrated to dryness in vacuo. The dried silica gel was placed on a gravity column of silica gel (150 g, type: ZCX-2, 100-200 mesh, packed with heptane) and the resulting column was eluted with a gradient of heptane:ethyl acetate (100:0 to 0:100). Compound A-15 was eluted with heptane:ethyl acetate 70:30 and the fraction of compound A-15 was concentrated in vacuo to give compound A-15 as a yellow oil (12.0 g, HPLC purity 88%). Compound A-15 was further purified by reversed-phase flash chromatography (WelFlash XSelect CSH Prep C18, 5 mm OBD, regular 30 × 150 mm column; solvent: A: 0.05% formic acid in water, B: acetonitrile, gradient 50-80%, 20 min, flow rate 55 mL / min). Fractions containing compound A-15 were pooled and concentrated in vacuo, and the residue was dissolved in heptane (150 mL). The heptane solution was washed with MeOH / water (80:20, 2×100 mL) and brine (100 mL). The organic phase was dried (MgSO4), filtered to remove solids, and the filtrate was concentrated in vacuo to give compound A-15 (10.33 g, 93% purity by HPLC, 66% yield) as a pale yellow oil.

[0243] 1H NMR (300MHz, CDCl3, ppm): d 4.20 (t, J = 6.00Hz, 4H), 4.04-4.14 (8H), 3.41 (t, J = 6.00Hz, 4H), 3.20 (t, J = 7.20Hz, 2H), 2.83 (s, 3H), 2.52 (m,2H), 2.20-2.40 (20H), 1.74 (m, 2H), 1.60 (m, 8H), 1.14-1.32 (40H), 0.90 (m, 12H);LCMS (+ mode):C 57 H 105 N3O 13 +H + Calculated value: 1040.77. Measured value: 1040.90 [M+H + ]. Example 25 Synthesis of tert-butyl(3-(2-methoxyethoxy)propyl)(methyl)carbamate (V-5a) [ka]

[0244] To a suspension of NaH (62% in oil, 10.1 g, 6.22 g, 0.259 mol) in THF (160 mL) cooled in an ice-water bath under nitrogen was added a solution of tert-butyl (3-hydroxypropyl) (methyl) carbamate (40.0 g, 0.216 mol) in THF (160 mL) over 1 h. After the addition was complete, the mixture was stirred for 1 h and then a solution of 2-methoxyethyl methanesulfonate (39.10 g, 0.254 mol) in THF (280 mL) was added over 1 h. After the addition was complete, the mixture was warmed to 80° C. and stirred for 18 h. The mixture was then cooled to room temperature and the reaction was quenched by careful addition of saturated aqueous NH4Cl (500 mL) over 1 h. The mixture was poured into ethyl acetate (500 mL) and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (2×500 mL) and the combined organic phase was washed with brine (1.5 L) and dried (Na2SO4). Filtration and concentration of the filtrate in vacuo afforded crude V-5a (27.0 g) as a yellow liquid. The crude material was dissolved in CHCl (200 mL), silica gel (50 g, type: ZCX-2, 100-200 mesh) was added to the filtrate, and the mixture was concentrated to dryness in vacuo. The dried silica gel was placed on a gravity column of silica gel (500 g, type: ZCX-2, 100-200 mesh, packed and eluted with CHCl). The fractions containing V-5a were concentrated in vacuo to afford V-5a as a yellow oil (10.0 g, 41.0 mmol, 19%).

[0245] 1 H NMR (400MHz, CD3OD, ppm): d 3.33-3.73 (6H), 3.39 (s, 3H), 3.29 (t, J = 7.00Hz, 2H), 2.85 (s, 3H), 1.82 (m, 2H), 1.46 (s, 9H);LCMS (+ mode):C 12 H 25 NO4+H + Calculated value: 248.19. Found value: 248.20 [M+H + ]. Example 26 Synthesis of 3-(2-methoxyethoxy)-N-methylpropane-1-aminium chloride (V-6a) [ka]

[0246] To a solution of V-5a (17.0 g, 68.7 mmol) in dioxane (350 mL) at room temperature under nitrogen was added a solution of HCl in dioxane (2 M, 350 mL) over 30 min. The solution was stirred at room temperature for 10 h and then the solvent was removed in vacuo to give crude V-6a (13.0 g) as a yellow oil. Crude V-6a was used without purification.

[0247] LCMS (+ mode): Calculated for C7185NO2: 148.13. Found: 148.30. Example 27 Synthesis of tert-butyl (3-((3-(2-methoxyethoxy)propyl)(methyl)amino)propyl)(methyl)carbamate (V-7a) [ka]

[0248] To a solution of V-6a (15.00 g, 81.7 mmol) in dichloromethane (180 mL) under nitrogen was added tert-butyl methyl (3-oxopropyl)carbamate (20.99 g, 112 mmol) in one portion. The mixture was stirred at room temperature for 30 min, then NaBH(OAc)3 (43.26 g, 204 mmol) was added in portions over 20 min. The solution was stirred at room temperature for 2 h, then water (200 mL) was added and the pH of the solution was adjusted to pH = 8 by addition of saturated aqueous Na2CO3. The mixture was extracted with CHCl2 (3 x 200 mL) and the combined organic phase was dried (Na2SO4). The solids were removed by filtration, silica gel (40 g, type: ZCX-2, 100-200 mesh) was added to the filtrate, and the mixture was concentrated to dryness in vacuo. The dried silica gel was placed onto a gravity column of silica gel (500 g, type: ZCX-2, 100-200 mesh, packed with CHCl and eluted with a gradient of CHCl:MeOH 100:0 to 90:10). The fractions containing V-7a (CHCl:MeOH 95:5) were concentrated in vacuo to give V-7a as a yellow oil (8.0 g, 25.1 mmol, 31%).

[0249] 1 H NMR (400MHz, CDCl3, ppm): d 3.20-3.65 (14H), 2.86 (m, 4H), 1.62-1.85 (6H), 1.48 (s, 9H); LCMS (+ mode): C 16 H 34 N2O4+H + Calculated value: 319.26. Found value: 319.40 [M+H + ]. Example 28 Synthesis of 3-((3-(2-methoxyethoxy)propyl)(methyl)amino)-N-methylpropan-1-aminium chloride (V-8a) [ka]

[0250] To a solution of V-7a (1.00 g, 3.14 mmol) in dioxane (20 mL) at room temperature under nitrogen was added HCl in dioxane (2 M, 20 mL) over 5 min. The resulting solution was stirred at room temperature for 10 h, then the solvent was removed in vacuo to give crude V-8a (800 mg) as a white solid. Crude 18 was used without further purification.

[0251] 1 H NMR (400MHz, CDCl3, ppm): d 10.92 (brs, 1H), 9.75 (brs, 2H), 3.00-3.62 (15H), 2.94 (brs, 3H), 2.76 (brs, 3H), 2.53 (brm, 2H), 2.17 (brm, 2H);LCMS(+mode):C 11 H 27 Calculated N2O2: 219.21. Measured: 219.20. Example 29 Synthesis of bis(2-((tert-butyldimethylsilyl)oxy)ethyl)amine (V-12) [ka]

[0252] To a solution of diethanolamine (100.00 g, 0.952 mol) in dichloromethane (1.0 L) at room temperature under nitrogen, imidazole (194.0 g, 2.86 mol) was added and the resulting solution was stirred for 5 min. To this mixture, a solution of t-butyldimethylsilyl chloride (316.3 g, 2.10 mol) in dichloromethane (1.0 L) was added over 30 min. The resulting solution was stirred at room temperature for 2 h and then the reaction was quenched by adding 10% aqueous NH4OH (400 mL). The organic phase was separated, the aqueous phase was extracted with dichloromethane (2 x 600 mL) and the combined organic phase was washed with saturated aqueous NH4Cl (5 x 800 mL), brine (800 mL) and dried (Na2SO4). Filtration and concentration in vacuo afforded V-12 (300.0 g, 0.899 mol, 94%) as a clear, colorless oil.

[0253] 1 H NMR (400MHz, CDCl3, ppm): d 3.75 (t, J = 5.30Hz, 4H), 2.74 (t, J = 5.30Hz, 4H), 2.03 (brs, 1H), 0.91 (s, 18H), 0.07 (s, 12H);LCMS (+ mode):C 16 H 19 NO2Si2+H + Calculated value: 334.26. Measured value: 334.40. Example 30 Synthesis of N,N-bis(2-((tert-butyldimethylsilyl)oxy)ethyl)-1H-imidazole-1-carboxamide (V-13) [ka]

[0254] To a solution of V-12 (230.0 g, 0.690 mol) in dichloromethane (2.30 L) at room temperature under nitrogen, CDI (446.20 g, 2.75 mol) and Et3N (139.10 g, 1.38 mol) were added in sequence. The resulting solution was stirred at room temperature for 16 h, and then the mixture was poured into water (2.30 L). The organic phase was separated, the aqueous layer was extracted with dichloromethane (1.15 L), and the combined organic phase was washed with saturated aqueous NH4Cl (2 x 4.6 L), 5% aqueous NaHCO3 (4.6 L), and dried (Na2SO4). Filtration and concentration in vacuo afforded crude V-13 as a yellow oil, which was dissolved in heptane (250 mL). The solution was washed with MeOH / H2O (80:20, 1.15 L) and dried (MgSO4). Filtration and concentration in vacuo afforded V-13 (270.0 g, 0.631 mol, 91%) as a pale yellow oil.

[0255] 1H NMR (400MHz, CDCl3, ppm): d 8.06 (t, J = 1.10Hz, 1H), 7.42 (t, J = 1.40Hz, 1H), 7.07 (dd, J = 1.40, 1.10Hz, 1H), 3.85 (t, J = 5.30Hz, 4H), 3.64 (t, J = 5.30Hz, 4H), 0.89 (s, 18H), 0.08 (s, 12H);LCMS(+mode):C 20 H 41 N3O3Si2+H + Calculated value: 428.28. Measured value: 428.30. Example 31 Synthesis of 1,1-bis(2-((tert-butyldimethylsilyl)oxy)ethyl)-3-(3-((3-(2-methoxyethoxy)propyl)(methyl)amino)propyl)-3-methylurea (V-14) [ka]

[0256] To a solution of V-13 (20.0 g, 48 mmol) in dichloromethane (200 mL) cooled in an ice-water bath under nitrogen, MeOTf (8.40 g, 51.0 mmol) was added over 5 min. The resulting mixture was stirred at 0° C. for 1 h, then a solution of Et3N (14.0 g, 140 mmol) and V-8a (17.80 g, 70 mmol) in dichloromethane (200 mL) was added to the solution over 30 min. After the addition was complete, the mixture was warmed to room temperature and stirred for 16 h. The reaction mixture was poured into water (200 mL) and the organic phase was removed. The aqueous layer was extracted with dichloromethane (2×200 mL) and the combined organic phase was concentrated in vacuo. The resulting crude V-14 was dissolved in heptane (300 mL) and the solution was extracted with MeOH / H2O (75:25, 2×100 mL). The combined aqueous phase was extracted with heptane (6×200 mL) and the combined organic phase was washed with brine (400 mL). The organic phase was dried (MgSO4). After filtration, silica gel (60 g, type: ZCX-2, 100-200 mesh) was added to the filtrate and the mixture was concentrated to dryness in vacuo. The dried silica gel was placed onto a gravity column of silica gel (330 g, type: ZCX-2, 100-200 mesh, packed with CHCl2, eluted with a gradient of CHCl2 / MeOH 100:0 to 90:10). The fractions containing V-14 (CHCl2 / MeOH 93:7) were concentrated in vacuo to give V-14 as a yellow oil (11.15 g, 22.0 mmol, 46%).

[0257] 1 H NMR (400MHz, CDCl3, ppm): d 3.72 (t, J = 6.20Hz, 4H), 3.48-3.60 (6H), 3.40 (s, 3H), 3.33 (t, J = 6.20Hz, 4H), 3.17 (t, J = 7.50Hz, 2H), 2.83 (s, 3H), 2.31-2.50 (4H), 2.26 (brs, 3H), 1.70-1.83 (4H), 0.90 (s, 18H), 0.06 (s, 12H);LCMS (+ mode):C 28 H 63 N3O5Si2+H +Calculated value: 578.44. Measured value: 578.30. Example 32 Synthesis of 1,1-bis(2-hydroxyethyl)-3-(3-((3-(2-methoxyethoxy)propyl)(methyl)amino)propyl)-3-methylurea (V-15) [ka]

[0258] To a solution of V-14 (11.15 g, 22.0 mmol) in THF (125 mL) at room temperature under nitrogen was added BF3-OEt2 (8.20 mL, 9.40 g, 66.0 mmol) over 10 min. The mixture was stirred at room temperature for 16 h and then poured into water (100 mL). The pH of the solution was adjusted to pH = 8.0 by addition of saturated aqueous NaHCO3 and the solvent was removed in vacuo to a volume of about 25 mL. The resulting solution was purified by reversed-phase flash chromatography (WelFlash AQ-C18 gel, typically 120 g, A: water, B: acetonitrile, gradient 0-30% over 15 min, flow rate: 80 mL / min). The target V-15 was eluted with 30% acetonitrile, and the fractions containing V-15 were pooled and concentrated in vacuo to give V-15 as an off-white oil (7.38 g, 21.12 mmol, 96% yield).

[0259] 1 H NMR (400MHz, CDCl3, ppm): d 4.50 (brs, 2H), 3.72 (m, 4H), 3.41-3.57 (6H), 3.28-3.38 (9H), 2.82 (s,3H), 2.48 (m, 2H), 2.39 (t, J = 6.60Hz, 2H), 2.25 (s, 3H), 1.70-1.85 (4H);LCMS(+mode):C 16 H 35 N3O5+H + Calculated value: 350.27. Measured value: 350.40. Example 33 Synthesis of compound A-16 [ka]

[0260] To a solution of V-15 (6.50 g, 18.6 mmol) in dichloromethane (130 mL) at room temperature under nitrogen, I-d2 (17.00 g, 40.9 mmol), DMAP (2.30 g, 18.6 mmol) and EDCl (8.20 g, 42.7 mmol) were added in sequence. The resulting solution was stirred at room temperature for 16 h and then poured into water (100 mL). The organic phase was separated, the aqueous phase was extracted with dichloromethane (2×100 mL) and the combined organic phase was concentrated in vacuo. The resulting crude compound A-16 was dissolved in heptane (150 mL) and the resulting solution was washed with MeOH / water (80:20, 100 mL), brine (100 mL) and dried (Na2SO4). The solids were removed by filtration, silica gel (25 g, type: ZCX-2, 100-200 mesh) was added to the filtrate, and the mixture was concentrated to dryness in vacuo. The dried silica gel was placed on a gravity column of silica gel (175 g, type: ZCX-2, 100-200 mesh, packed with CHCl and eluted with a gradient of CHCl / MeOH 100:0 to 90:10). The fractions containing compound A-16 (CHCl / MeOH 95:5) were concentrated in vacuo to give compound A-16 (94% purity by HPLC) as a yellow oil. Compound A-16 was further purified by reversed-phase flash chromatography (WelFlash XSelect CSH Prep C18, 5 mm OBD, regular 30 × 150 mm column; solvent: A: 0.1% formic acid in water, B: acetonitrile, gradient 50-80%, 20 min, flow rate 55 mL / min). The fractions containing compound A-16 were pooled and concentrated in vacuo, and the residue was dissolved in heptane (150 mL). The heptane solution was washed with 5% aqueous Na2CO3 (2×100 mL), MeOH / water (75:25, 2×100 mL), and brine (100 mL). The organic phase was dried (Na2SO4), filtered to remove solids, and the filtrate was concentrated in vacuo to give compound A-16 (12.08 g, 96.4% purity by HPLC, 51% yield) as a yellow oil.

[0261] 1H NMR (400MHz, CDCl3, ppm): d 4.19 (t, J = 6.00Hz, 4H), 4.08 (m, 8H), 3-46-3.60 (6H), 3.41 (t, J = 6.00Hz, 4H), 3.37 (s, 3H), 3.17 (m, 2H), 2.82 (s, 3H), 2.53 (m, 2H), 2.35-2.42 (6H), 2.24-2.33 (10H), 2.19 (s, 3H), 1.66-1.80 (4H), 1.54-1.65 (8H), 1.20-1.36 (40H), 0.89 (m, 12H);LCMS(+mode):C 62 H 115 N3O 15 +H + Calculated value: 1142.84. Measured value: 1142.90. Example 34 Biophysical and biochemical characterization

[0262] Biophysical and biochemical properties of clogD, c-pKa, pKa, and ex vivo stability in mouse plasma were determined for compounds A-2 and A-11 through A-15, as well as for three benchmark lipids 10a, 10f, and 10p that are known to successfully deliver nucleic acids into cells (see Journal of Medicinal Chemistry 63:12992-13012, 2020). [Table 5]

[0263] cLogD and c-pKa were calculated as described above. The measured pKa of lipids was determined as formulated into lipid nanoparticles using the TNS assay as described in the Examples below.

[0264] From past experience, the difference between c-pKa and measured pKa in LNPs (ΔpKa) is estimated to be 2-3 units, but compounds A-2 and A-11-A-15 all surprisingly had ΔpKa less than 2. The activity of ionizable amino lipids to promote endosomal escape of nucleic acid cargo is typically greatest for lipids with a pKa of 6-7. Of the disclosed lipids tested in this example, only compound A-11 had an observed pKa in this range. One approach to reduce the measured basicity of these lipids towards and within the range preferred for good endosomal escape activity is to increase the chain length of the fatty acid tail (R in formula I) by 1-4 carbons, respectively. Table 6 shows the structures of analogs of compounds A-2 and A-12-A-15 with the R group lengthened, along with their calculated cLogD and c-pKa (C for compound A-2). 10 ~C 13 , Compounds A-12 to A-15: C9 to C 12 ). In each case, cLogD increased with increasing R length, reflecting increased lipophilicity, while c-pKa remained the same. However, increased lipophilicity results in a decrease in the measured pKa of the lipid when incorporated within an LNP, resulting in an increase in ΔpKa. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6]

Table 6-7

[0265] To determine the stability of the disclosed lipids in mouse plasma, lipid stock solutions were prepared by dissolving lipids in isopropanol at a concentration of 5 mg / mL. The required volume of lipid-isopropanol solution was then diluted to a 100 μM concentration in a total volume of 1.0 mL with 50:50 (v / v) ethanol / water. Ten microliters of this 100 μM solution was injected into 1.0 mL of mouse plasma (BioIVT, Lot MSE394920, CD-1 mouse, anticoagulant: sodium heparin, unfiltered) pre-warmed to 37 °C and stirred at 50 rpm with a magnetic stir bar. Thus, the starting concentration of lipid in plasma was 1 μM. Aliquots (50 μL) were taken after 0, 15, 30, 45, 60, and 120 min, transferred to microcentrifuge tubes, and quenched with 3 volumes (150 μL) of ice-cold acetonitrile / methanol (4:1). Positive control incubations utilized the same plasma, using Benfluorex (1 μM) as substrate and Labetalol (1.0 μg / l) as in situ quenching. The quenched solutions were vortexed and centrifuged at 13,000 rpm for 5 min, and the supernatant (100 μL) was transferred to a 96-well plate and diluted with water (200 μL, 0.1% FA). After filtration through a 0.45 μm 96-well filtration plate, the filtrate was analyzed by LC-MS (ThermoScientific Vanquish UPLC system consisting of a Waters AQUITY I-class UPLC system, binary pump, autosampler, and column compartment, Waters Xevo G2-XS QTof mass spectrometer; Phenomenex F5, 1.7 μm, 2.1 × 50 mm column). Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The flow rate was 0.6 mL / min. The elution gradient was as follows: time, 0.5 min: 20% B; 0.5–2 min: 20–100% B; 2–4.8 min: 100% B; 4.8–5.45 min: 100–20% B. Mass spectrometry was in positive scan mode from 600 to 1100 m / z. Lipid molecular ion peaks were integrated by extracted ion chromatography (XIC) using Xcalibur software (Thermo Fisher).The relative peak areas compared to T=0 after normalization with the peak area of ​​the internal standard were used as the percentage of lipid remaining at each time point. 1 / 2 Values ​​were calculated using a first-order decay model.

[0266] Compounds A-2 and A-11-A-15 were all clearly degradable in mouse plasma comparable to the benchmark 10f, but not to the rapidly degraded 10a and extremely stable 10p. Even compound A-11, the slowest degrading compound tested, had a half-life of less than 10 hours, suggesting that it could be administered several times a week without problematic accumulation. Although these data only documented the disappearance of the initial compound, these lipids were designed to have multiple ester linkages to facilitate further degradation to species that could be easily eliminated from the body without the need for oxidative metabolism in the liver. Example 35 LNP encapsulation of mRNA

[0267] Using mRNA encoding the fluorescent marker mCherry, the ability of various disclosed ionizable cationic lipids to be incorporated into LNPs that encapsulate mRNA was evaluated.

[0268] mCherry mRNA was synthesized by T7 RNA polymerase-mediated in vitro transcription (IVT) of a linearized DNA template using full substitution of uridine with N1-methylpseudouridine. A Cap1 structure was cotranscriptionally added to the 5' end of the mRNA, and a 3' polyadenosine tail was encoded by the DNA template. After IVT, the mRNA was purified using a two-step chromatographic process using OligoDT affinity chemistry for bulk capture and ion-pair reversed-phase chemistry to remove residual impurities.

[0269] The mRNA was encapsulated in LNPs using a self-assembly process in which an aqueous solution of mRNA at pH=3.5 was rapidly mixed with a solution of lipids dissolved in ethanol, followed by stepwise phosphate and Tris buffer dilutions and tangential flow filtration (TFF) purification. The LNP composition in this study was ionizable cationic lipid / distearoylphosphatidylcholine / cholesterol / DMG-PEG2000 (50:10:38.5:1.5 mol / mol) encapsulated at an N / P ratio (ratio of positively chargeable lipid amine (N=nitrogen) groups to negatively charged nucleic acid phosphate (P) groups) of 6. The LNPs were frozen at -80°C. LNPs were made in which the ionizable cationic lipid was one of compounds A-2, A-11, A-12, A-13, A-14, or A-15. The diameter of the nanoparticles was measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) instrument. Size measurements were performed in associated disposable capillary cells in Tris buffer at pH 7.4 at 25 °C. A non-invasive backscattering system (NIBS) with a scattering angle of 173° was used for size measurements. [Table 7]

[0270] All of these ionizable cationic lipids formed LNPs with generally acceptable sizes and good encapsulation efficiencies. Compound A-13 LNPs showed the highest size uniformity. Example 36 Transfection of HEK293F cells

[0271] The ability of the LNPs formed in the previous examples to transfect HEK293F cells, a human embryonic kidney cell-derived cell line, with mCherry mRNA was evaluated. Viral Production Cells (Gibco Catalog Number: A35347), a chemically defined serum- and protein-free medium (LV-MAX TMA derivative of the HEK 293F cell line adapted to the Production Medium (Gibco Catalog Number: A3583401) was grown in suspension and allowed to settle, resulting in approximately 1 × 10 6 Cells were resuspended at 100 cells / mL and 200 μL was distributed into wells of a 96-well U-bottom plate. Frozen LNPs were thawed and diluted to 100 μg mRNA / mL with sterile water for injection. An appropriate volume of LNPs was added to provide 0, 0.3, 0.6, or 2 μg RNA per well in duplicate and mixed by repipetting. Cells were then incubated at 37°C in a CO2 incubator for 1 hour, washed three times with phosphate buffered saline, and resuspended in 400 μL medium in a deep-bottom 96-well plate and incubated at 37°C in a CO2 incubator on an orbital shaker at 900 RPM.

[0272] 24 hours after adding LNPs to cells, they were stained with Aqua Live / Dead (Thermo: Cat. L34965) to evaluate cell viability. Transfection rate and expression level in transfected cells were evaluated by flow cytometry based on mCherry fluorescence. As seen in Figure 5A, all LNPs, except for compound A-11, caused a reduction in cell number compared to non-transfected cells even at the lowest dose tested, and some caused almost complete cell death at higher doses. Compound A-11 caused only a slight reduction in cell number even at the highest dose tested.

[0273] As seen in Figure 5B, all LNPs achieved stable transfection frequencies of 80% or more in live cells, except for compound A-11, which only approached a 50% transfection rate at the highest dose tested. Expression levels were variable and had no defined pattern (Figure 12C). LNPs containing compounds A-2, A-12, A-13, A-14, and A-15 were all more basic (more positively charged) than A-11, which correlates with their differential ability to transfect HEK293F cells. Example 37 Incorporation of ionizable lipids into tLNPs

[0274] Compounds A-2 and A-11 to A-15, as well as benchmark lipids 10a, 10f, and 10p, were each incorporated into tLNPs that packaged mRNA encoding the fluorescent protein mCherry, and the physicochemical properties were measured. The results are shown in Table 6 below.

[0275] CleanCap® mCherry 5-methoxyuridine (5moU) mRNA (L-7203) was purchased from TriLink. The mRNA was encapsulated in LNPs using a self-assembly process in which an aqueous solution of mRNA at pH=3.5 was rapidly mixed with a solution of lipids dissolved in ethanol, followed by stepwise phosphate and Tris buffer dilutions and TFF purification. The LNP composition in this study was ionizable cationic lipid / distearoylphosphatidylcholine / cholesterol / DMG-PEG2000 / distearoylphosphatidylethanolamine (DSPE)-PEG2000-maleimide (50:10:38.5:1.4:0.1 mol / mol), encapsulated with an N / P ratio of 6 (ratio of positively chargeable polymeric amine (N=nitrogen) groups to negatively charged nucleic acid phosphate (P) groups). The hydrodynamic diameter of the nanoparticles was measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) instrument.

[0276] Anti-CD5 mAb was then conjugated to the above LNPs to generate tLNPs. Purified rat anti-mouse CD5 antibody, clone 53-7.3 (BioLegend), was coupled to LNPs via N-succinimidyl S-acetylthioacetate (SATA)-maleimide conjugation chemistry. Briefly, LNPs incorporating DSPE-PEG(2000)-maleimide were formulated and stored at 4°C on the day of conjugation. The antibody was modified with SATA (Sigma-Aldrich) to introduce sulfhydryl groups to available lysine residues, allowing conjugation to maleimide. SATA was deprotected using 0.5 M hydroxylamine, followed by removal of unreacted components by G-25 Sephadex Quick Spin Protein column (Roche Applied Science, Indianapolis, IN). The reactive sulfhydryl group on the antibody was then conjugated to the maleimide moiety on the LNP using thioether conjugation chemistry. Purification was performed using a Sepharose CL-4B gel filtration column (Sigma-Aldrich). tLNPs (LNPs conjugated with targeting antibodies) were frozen at -80°C. Others conjugated the antibody to free functionalized PEG-lipids and then incorporated the conjugated lipids into preformed LNPs. However, we found that this procedure was more controllable and produced more consistent results.

[0277] Quant-iT TM RiboGreen RNA Assay Kit (Invitrogen TM The mRNA content was determined using the Fluorescence Intensity (F) Quantitative Analysis of the mRNA by RiboGreen Reagent. The encapsulation efficiency was calculated by determining the non-encapsulated mRNA content by measuring the fluorescence intensity (F) after addition of RiboGreen reagent to the LNPs and comparing this value with the total fluorescence intensity (F) of the RNA content obtained after lysis of the LNPs with 1% Triton® X-100 (% encapsulation = (F-F) / Fx100).

[0278] Dynamic light scattering (DLS) was used on a Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) to determine the particle size (hydrodynamic diameter) and polydispersity index of targeted lipid nanoparticles. Size measurements were performed at 25 °C in Tris buffer at pH 7.4 in associated disposable capillary cells. A non-invasive backscattering system (NIBS) with a scattering angle of 173° was used for size measurements.

[0279] 6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt (TNS salt, Toronto Research Chemicals, Toronto, ON, Canada) was used to determine the apparent pKa of ionizable lipids in lipid nanoparticles. Lipid nanoparticles were diluted in 1x Dulbecco's PBS to a concentration of 1 mM total lipid. TNS salt was prepared as a stock solution of 1 mg / mL in DMSO and then further diluted to a working solution of 60 μg / mL (179 mM) using distilled water. The diluted lipid nanoparticle samples were further diluted to 90 μM total lipid in 165 μL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, and to a final TNS concentration of 1.33 μg / mL (4 μM) at pH ranging from 3.5 to 12.2. After pipette mixing and incubation in the dark for 15 min at room temperature, the fluorescence intensity was measured at room temperature on a BioTek Synergy H1 plate reader using excitation and emission wavelengths of 321 nm and 445 nm, respectively. The fluorescence signal was blank-subtracted, plotted as a function of pH, and then analyzed using nonlinear (Boltzmann) regression analysis to determine the apparent pKa as the pH that yielded half-maximal fluorescence intensity as calculated by the Henderson-Hasselbalch equation.

[0280] The tLNPs produced in this example are based on a reasonably conventional lipid composition and functionalized PEG-lipids for conjugation of targeting moieties and ionizable cationic lipids as disclosed herein. The conventional composition provides a good platform for evaluating the contribution of ionizable lipids to the properties of tLNPs, and a baseline for evaluating further optimization of the overall composition. As seen in Table 7, tLNPs incorporating compounds A-2 or A-11 through A-15 all had hydrodynamic diameters and polydispersity indices within the acceptable ranges of 50-150 nm and 0.2 or less for PDI. Encapsulation efficiency of 80% or greater is acceptable, with 85% or greater and 90% or greater being preferred. All compounds tested exceeded the 90% or greater threshold (except one of the benchmark lipids, 10a, which did not). [Table 8] Example 38 Targeted transfection of T cells in vitro

[0281] To evaluate the performance of the tLNPs described in Example 35, they were used to transfect mouse T cells in tissue culture. Mouse splenic T cells were isolated from mechanically dissociated mouse spleens using a standard T cell isolation kit (Stem Cell Technologies #19851). Isolated T cells were cultured for 3 days in the presence of CD3 / CD28 T cell activation beads (Gibco #11453D) in complete RPMI medium supplemented with mouse interleukin-2. After activation, T cells were magnetically separated from the activation beads and plated at 2 x 10 per well in 100 μL of complete RPMI medium. 5The cells were transferred to a 96-well plate at a concentration of 100 μg / mL. The tLNP formulation described in Example 35 (above) was diluted to 100 μg / mL and 6 μL (0.6 μg) of tLNP was added to each well of cells to be tested. The cells were incubated with tLNP for 1 hour at 37° C., and then the tLNP was washed off by centrifuging the plate, removing the supernatant, and replacing it with fresh medium. The transfected cells were then returned to the incubator overnight. The next day, the cells were washed and resuspended in staining buffer containing fluorescently tagged antibodies against T cell markers for 30 minutes, followed by a final wash. After washing, the cells were resuspended in staining buffer and run through a Novocyte Quanteon flow cytometer to detect mCherry expression and mouse T cell markers. CD3 + The results for T cells are shown in FIG.

[0282] As seen in FIG. 6, tLNPs incorporating compound A-11 and benchmark lipid 10p showed stable and comparable results, with transfection rates of about 80% or higher, indicating high levels of expression. Transfection with tLNPs incorporating compounds A-12-A-15 or benchmark lipids 10a and 10f all resulted in similar levels of expression, lower than A-11 and 10p but still substantial. Transfection rates varied from about 20% to about 60%. In comparison, the results for tLNPs incorporating compound A-2 were lower but still positive. Of the disclosed compounds tested herein, the superior performance of compound A-11 correlates with it being the only one of those compounds with a measured pKa of 6-7. However, the performance of the other compounds does not correlate with the magnitude of their deviation from the preferred range of measured pKa, indicating that outside this range other factors dominate. Example 39 Targeted transfection of T cells in vivo

[0283] To evaluate the performance of the tLNPs described in Example 35, they were used to transfect mouse T cells by injecting the tLNPs into live mice and evaluated for their ability to generate mouse T cells expressing the mCherry reporter gene in vivo. All tLNP test articles were thawed at room temperature for 30 minutes and then diluted 1:2 with sterile water for injection to achieve a final dose concentration of 100 μg / mL. 100 μL (10 μg) of each test article was then injected into 8-week-old female C57Bl / 6 mice via the tail vein. All post-treatment mice were then sacrificed 24 hours after treatment and their spleens were harvested. Each spleen was then dissociated to a single cell suspension and stained with antibodies to identify T cells, B cells, monocytes, and non-hematopoietic cells. The stained samples were then analyzed by flow cytometry for expression of mCherry in immune cell subsets and non-hematopoietic cells. Data analysis was performed using FlowJo (version 10.8.1) and GraphPad Prism (9.4.1.).

[0284] As seen in Figure 7, both the transfection rate and the level of mCherry expression were significantly reduced compared to in vitro. This is inferred from the lower effective dose following administration of tLNPs to live animals compared to the addition of tLNPs to wells of tissue culture plates. The tLNPs incorporating compound A-11 performed significantly better than any of the others, with a transfection rate of approximately 7% and an MFI that was clearly greater than that achieved with the other tLNPs. The tLNPs incorporating the three benchmark lipids performed comparable to each other, with transfection rates of approximately 2%, while the tLNPs incorporating the other tested compounds were not clearly distinguishable from the background. Of the compounds tested herein, it was surprising that only compound A-11 has a measured pKa of 6-7, but in light of that, the lower performance of compounds A-2 and A-12-A-15 was not unexpected. tLNPs incorporating compound A-11 performed substantially better than tLNPs incorporating benchmark lipids with measured pKas of 6-7, supporting the idea that pKa is not the only determinant of performance. Example 40 Further embodiments

[0285] Further embodiments are now disclosed.

[0286] Embodiment 1. Formula 1 [ka] An ionizable cationic lipid having the structure: wherein Y is O, NH, N-CH3, or CH2; n is an integer from 0 to 4, X is [ka] [ka] and m is an integer from 1 to 3, o is an integer from 1 to 4, p is an integer from 1 to 4, When p=1, each R is independently C6 to C 16 Straight chain alkyl; C6~C 16 Branched alkyl; C6-C 16 Straight chain alkenyl; C6~C 16 Branched alkenyl; C9-C 16 cycloalkyl-alkyl (wherein the cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl chain); or 18 aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=2, each R is independently C6 to C 14 Straight chain alkyl; C6~C 14 Straight chain alkenyl; C6~C 14 Branched alkyl; C6-C 14 Branched alkenyl; C9-C 14 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl chain); 16 aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=3, each R is independently C6 to C 12 Straight chain alkyl; C6~C 12 Straight chain alkenyl; C6~C 12 Branched alkyl; C6-C 12 Branched alkenyl; C9-C 12 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl chain); 14aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=4, each R is independently C6 to C 10 Straight chain alkyl; C6~C 10 Straight chain alkenyl; C6~C 10 Branched alkyl; C6-C 10 Branched alkenyl; C9-C 10 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl); C8-C 12 aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; Ionizable cationic lipids.

[0287] Embodiment 2. Formula 2 [ka] An ionizable cationic lipid having the structure: wherein Y is O, NH, N-CH3, or CH2; n is an integer from 0 to 4, X is [ka] and m is an integer from 1 to 3, o is an integer from 1 to 4, p is an integer from 1 to 4, When p=1, each R is independently C6 to C 16 Straight chain alkyl; C6~C 16 Straight chain alkenyl; C6~C 16 Branched alkyl; C6-C 16 Branched alkenyl; C9-C 16cycloalkyl-alkyl (wherein the cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl chain); or 18 aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=2, each R is independently C6 to C 14 Straight chain alkyl; C6~C 14 Straight chain alkenyl; C6~C 14 Branched alkyl; C6-C 14 Branched alkenyl; C9-C 14 cycloalkyl-alkyl (wherein the cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl chain); or 16 aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=3, each R is independently C6 to C 12 Straight chain alkyl; C6~C 12 Straight chain alkenyl; C6~C 12 Branched alkyl; branched C6-C 12 Alkenyl; C9~C 12 cycloalkyl-alkyl (wherein the cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl chain); or 14 aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=4, each R is independently C6 to C 10 Straight chain alkyl; straight chain C6-C 10 Alkenyl; C6~C 10 Branched alkyl; C6-C 10 Branched alkenyl; C9-C 10Cycloalkyl-alkyl (wherein the cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl); or C8-C 12 aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; Ionizable cationic lipids.

[0288] Embodiment 3. Formula 3 [ka] An ionizable cationic lipid having the structure: Wherein W is C=O or CH2; n is an integer from 0 to 4, X is [ka] and m is an integer from 1 to 3, o is an integer from 1 to 4, p is an integer from 1 to 4, If p=1, then each R c are independent, C8~C 18 Straight chain alkyl; C8~C 18 Straight chain alkenyl; C8~C 18 Branched alkyl; C8-C 18 Branched alkenyl; C 11 ~C 18 cycloalkyl-alkyl (wherein the cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl chain); or C 10 ~C 20 aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; If p=2, then each R c are independent, C8~C 16Straight chain alkyl; C8~C 16 Straight chain alkenyl; C8~C 16 Branched alkyl; C8-C 16 Branched alkenyl; C 11 ~C 16 cycloalkyl-alkyl (wherein the cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl chain); or C 10 ~C 18 aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; If p=3, then each R c are independent, C8~C 14 Straight chain alkyl; C8~C 14 Straight chain alkenyl; C8~C 14 Branched alkyl; C8-C 14 Branched alkenyl; C 11 ~C 14 cycloalkyl-alkyl (wherein the cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl chain); or C 10 ~C 16 aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; If p=4, then each R c are independent, C8~C 12 Straight chain alkyl; C8~C 12 Straight chain alkenyl; C8~C 12 Branched alkyl; C8-C 12 Branched alkenyl; C 11 ~C 12 cycloalkyl-alkyl (wherein the cycloalkyl is a C3-C8 cycloalkyl located at either end or within the alkyl); or 10 ~C 14 aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either end or within the alkyl chain; Ionizable cationic lipids.

[0289] Embodiment 4. The ionizable cationic lipid of embodiment 1 or 2, wherein Y is O.

[0290] Embodiment 5. The ionizable cationic lipid of embodiment 1 or 2, wherein Y is NH.

[0291] Embodiment 6. The ionizable cationic lipid of embodiment 1 or 2, wherein Y is N-CH3.

[0292] Embodiment 7. The ionizable cationic lipid of embodiment 1 or 2, wherein Y is CH2.

[0293] Embodiment 8. X is [ka] 3. The ionizable cationic lipid of embodiment 1 or 2, wherein

[0294] Embodiment 9. The ionizable cationic lipid of embodiment 3, wherein W is C=O.

[0295] Embodiment 10. R or R is a linear alkyl. c 10. The ionizable cationic lipid according to any one of the preceding embodiments, comprising:

[0296] Embodiment 11. R or R is a linear alkenyl. c 10. The ionizable cationic lipid according to any one of the preceding embodiments, comprising:

[0297] Embodiment 12. R or R is a branched alkyl c 10. The ionizable cationic lipid according to any one of the preceding embodiments, comprising:

[0298] Embodiment 13. The ionizable cationic lipid of any one of embodiments 1 to 9, comprising R which is branched alkenyl.

[0299] Embodiment 14. R or R is cycloalkyl-alkyl c 10. The ionizable cationic lipid according to any one of the preceding embodiments, comprising:

[0300] Embodiment 15. R or R is aryl-alkyl c 10. The ionizable cationic lipid according to any one of the preceding embodiments, comprising:

[0301] Embodiment 16. Each R or R c 16. The ionizable cationic lipid according to any one of the preceding embodiments, wherein the groups are the same.

[0302] Embodiment 17. R or R arising from a first branch point c Both of the groups are the same, and the R or R arising from the second branch point c Both R or R groups are the same but originating from the first branch point. c The group is an R or R c 16. The ionizable cationic lipid according to any one of the preceding embodiments, which is different from the group

[0303] Embodiment 18. A lipid nanoparticle (LNP) comprising an ionizable cationic lipid according to any one of embodiments 1 to 17.

[0304] Embodiment 19. The LNP of embodiment 18, further comprising one or more of a phospholipid, a sterol, a co-lipid, and a PEG-lipid, or a combination thereof.

[0305] Embodiment 20. The LNP of embodiment 18, wherein the phospholipid comprises dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC) or 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), or a combination thereof.

[0306] Embodiment 21. The LNP of embodiment 18 or 19, wherein the sterol comprises cholesterol, campesterol, sitosterol, or stigmasterol, or a combination thereof.

[0307] Embodiment 22. The LNP of any one of embodiments 18 to 21, wherein the co-lipid comprises cholesterol hemisuccinate (CHEMS) or a quaternary ammonium head group-containing lipid.

[0308] Embodiment 23. The LNP of embodiment 22, wherein the quaternary ammonium head group-containing lipid comprises 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA) or 3β-(N-(N′,N′-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or a combination thereof.

[0309] Embodiment 24. The LNP of any one of embodiments 18 to 23, wherein the PEG-lipid comprises a PEG moiety with a molecular weight (MW) of 1000 to 5000 Da.

[0310] Embodiment 25. The PEG-lipid is 14 ~C 18 25. The LNP of any one of embodiments 18 to 24, comprising a fatty acid having a fatty acid chain length of:

[0311] Embodiment 26. The PEG-lipid is selected from the group consisting of DMG-PEG2000 (1,2-dimyristoyl-glycero-3-methoxypolyethylene glycol-2000), DPG-PEG2000 (1,2-dipalmitoyl-glycero-3-methoxypolyethylene glycol-2000), DSG-PEG2000 (1,2-distearoyl-glycero-3-methoxypolyethylene glycol-2000), DOG-PEG2000 (1,2-dioleoyl-glycero-3-methoxypolyethylene glycol-2000), DMPE-PEG200 (1,2-dimyristoyl-glycero-3-phosphoethanolamine-3 26. The LNP of any one of embodiments 18 to 25, comprising 1,2-dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), DPPE-PEG2000 (1,2-dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), DSPE-PEG2000 (1,2-distearoyl-glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), DOPE-PEG2000 (1,2-dioleoyl-glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), or a combination thereof.

[0312] Embodiment 27. The LNP of any one of embodiments 18 to 26, wherein the PEG-lipid comprises an optically pure glycerol moiety.

[0313] Embodiment 28. The LNP of any one of embodiments 18 to 27, further comprising a functionalized PEG-lipid.

[0314] Embodiment 29. The LNP of embodiment 28, wherein the functionalized PEG-lipid is conjugated to a binding moiety.

[0315]

[0023] Embodiment 30. The LNP of embodiment 29, wherein the binding moiety comprises an antigen-binding domain of an antibody.

[0316] Embodiment 31. The functionalized PEG-lipid is 16 ~C 18The LNP of any one of embodiments 28 to 30, comprising a fatty acid having a fatty acid chain length of:

[0317] Embodiment 32. The LNP of embodiment 31, wherein the functionalized PEG-lipid comprises a dipalmitoyl lipid or a distearoyl lipid.

[0318] Embodiment 33. The LNP of any one of embodiments 18 to 32, comprising 40-60 mol % ionizable cationic lipid.

[0319] Embodiment 34. The LNP of any one of embodiments 19 to 33, comprising 7 to 30 mol % phospholipid.

[0320] Embodiment 35. The LNP of any one of embodiments 19 to 34, comprising 20-45 mol % sterol.

[0321] Embodiment 36. The LNP of any one of embodiments 19 to 35, comprising 1 to 30 mol % of a co-lipid.

[0322] Embodiment 37. The LNP of any one of embodiments 19 to 36, comprising 0-5 mol % PEG-lipid.

[0323] Embodiment 38. The LNP of any one of embodiments 19 to 37, comprising 0.1-5 mol % of functionalized PEG-lipid.

[0324] Embodiment 39. The LNP of any one of embodiments 18 to 38, further comprising a nucleic acid.

[0325] Embodiment 40. The LNP of embodiment 39, wherein the weight ratio of total lipid to nucleic acid is between 10:1 and 50:1.

[0326] Embodiment 41. The LNP of embodiment 39 or 40, which comprises mRNA.

[0327] Embodiment 42. A method of delivering a nucleic acid into a cell, comprising contacting said cell with the LNP of any one of embodiments 39 to 41.

Claims

1. Formula 1 【Chemistry 59】 An ionizable cationic lipid having the structure: In the formula, Y is O, NH, N-CH ３ , or C.H. ２ and n is an integer from 0 to 4, X is, 【Chemistry 60】 and m is an integer from 1 to 3, o is an integer from 1 to 4; p is an integer from 1 to 4; When p=1, each R is independently C ６ ~C １６ Straight chain alkyl; C ６ ~C １６ Branched alkyl; C ６ ~C １６ Straight chain alkenyl; C ６ ~C １６ Branched alkenyl; C ９ ~C １６ Cycloalkyl-alkyl, where the cycloalkyl is a C alkyl group located at either end or within the alkyl chain. ３ ~C ８ cycloalkyl); or C ８ ~C １８ aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=2, each R is independently C ６ ~C １４ Straight chain alkyl; C ６ ~C １４ Straight chain alkenyl; C ６ ~C １４ Branched alkyl; C ６ ~C １４ Branched alkenyl; C ９ ~C １４ Cycloalkyl-alkyl, where the cycloalkyl is a C alkyl group located at either end or within the alkyl chain. ３ ~C ８ cycloalkyl); C ８ ~C １６ aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=3, each R is independently C ６ ~C １２ Straight chain alkyl; C ６ ~C １２ Straight chain alkenyl; C ６ ~C １２ Branched alkyl; C ６ ~C １２ Branched alkenyl; C ９ ~C １２ Cycloalkyl-alkyl, where the cycloalkyl is a C alkyl group located at either end or within the alkyl chain. ３ ~C ８ cycloalkyl); C ８ ~C １４ aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=4, each R is independently C ６ ~C １０ Straight chain alkyl; C ６ ~C １０ Straight chain alkenyl; C ６ ~C １０ Branched alkyl; C ６ ~C １０ Branched alkenyl; C ９ ~C １０ Cycloalkyl-alkyl, wherein the cycloalkyl is a C alkyl group located at either end or within the alkyl group. ３ ~C ８ cycloalkyl); C ８ ~C １２ aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; Ionizable cationic lipids.

2. Formula 2 【Chemistry 61】 An ionizable cationic lipid having the structure: In the formula, Y is O, NH, N-CH ３ , or C.H. ２ and n is an integer from 0 to 4, X is, 【Chemistry 62】 and m is an integer from 1 to 3; o is an integer from 1 to 4; p is an integer from 1 to 4; When p=1, each R is independently C ６ ~C １６ Straight chain alkyl; C ６ ~C １６ Straight chain alkenyl; C ６ ~C １６ Branched alkyl; C ６ ~C １６ Branched alkenyl; C ９ ~C １６ Cycloalkyl-alkyl, where the cycloalkyl is a C alkyl group located at either end or within the alkyl chain. ３ ~C ８ cycloalkyl); or C ８ ~C １８ aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=2, each R is independently C ６ ~C １４ Straight chain alkyl; C ６ ~C １４ Straight chain alkenyl; C ６ ~C １４ Branched alkyl; C ６ ~C １４ Branched alkenyl; C ９ ~C １４ Cycloalkyl-alkyl, where the cycloalkyl is a C alkyl group located at either end or within the alkyl chain. ３ ~C ８ cycloalkyl); or C ８ ~C １６ aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=3, each R is independently C ６ ~C １２ Straight chain alkyl; C ６ ~C １２ Straight chain alkenyl; C ６ ~C １２ Branched alkyl; Branched C ６ ~C １２ Alkenyl; C ９ ~C １２ Cycloalkyl-alkyl, where the cycloalkyl is a C alkyl group located at either end or within the alkyl chain. ３ ~C ８ cycloalkyl); or C ８ ~C １４ aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=4, each R is independently C ６ ~C １０ Straight chain alkyl; straight chain C ６ ~C １０ Alkenyl; C ６ ~C １０ Branched alkyl; C ６ ~C １０ Branched alkenyl; C ９ ~C １０ Cycloalkyl-alkyl, wherein the cycloalkyl is a C alkyl group located at either end or within the alkyl group. ３ ~C ８ cycloalkyl); or C ８ ~C １２ aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; Ionizable cationic lipids.

3. Formula 3 【Chemistry 63】 An ionizable cationic lipid having the structure: In the formula, W is C=O or CH ２ and n is an integer from 0 to 4, X is, 【Chemistry 64】 and m is an integer from 1 to 3; o is an integer from 1 to 4; p is an integer from 1 to 4; When p=1, each R ｃ is independently ８ ~C １８ Straight chain alkyl; C ８ ~C １８ Straight chain alkenyl; C ８ ~C １８ Branched alkyl; C ８ ~C １８ Branched alkenyl; C １１ ~C １８ Cycloalkyl-alkyl, where the cycloalkyl is a C alkyl group located at either end or within the alkyl chain. ３ ~C ８ cycloalkyl); or C １０ ~C ２０ aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=2, each R ｃ is independently ８ ~C １６ Straight chain alkyl; C ８ ~C １６ Straight chain alkenyl; C ８ ~C １６ Branched alkyl; C ８ ~C １６ Branched alkenyl; C １１ ~C １６ Cycloalkyl-alkyl, where the cycloalkyl is a C alkyl group located at either end or within the alkyl chain. ３ ~C ８ cycloalkyl); or C １０ ~C １８ aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=3, each R ｃ is independently ８ ~C １４ Straight chain alkyl; C ８ ~C １４ Straight chain alkenyl; C ８ ~C １４ Branched alkyl; C ８ ~C １４ Branched alkenyl; C １１ ~C １４ Cycloalkyl-alkyl, where the cycloalkyl is a C alkyl group located at either end or within the alkyl chain. ３ ~C ８ cycloalkyl); or C １０ ~C １６ aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; When p=4, each R ｃ is independently ８ ~C １２ Straight chain alkyl; C ８ ~C １２ Straight chain alkenyl; C ８ ~C １２ Branched alkyl; C ８ ~C １２ Branched alkenyl; C １１ ~C １２ Cycloalkyl-alkyl, wherein the cycloalkyl is a C alkyl group located at either end or within the alkyl group. ３ ~C ８ cycloalkyl); or C １０ ~C １４ aryl-alkyl, where the aryl is phenyl or naphthalenyl and is located at either terminus or within the alkyl chain; Ionizable cationic lipids.

4. 3. The ionizable cationic lipid of claim 1 or 2, wherein Y is O.

5. 3. The ionizable cationic lipid of claim 1 or 2, wherein Y is NH.

6. Y is N-CH ３ 3. The ionizable cationic lipid of claim 1 or 2, wherein

7. Y is CH ２ 3. The ionizable cationic lipid of claim 1 or 2, wherein

8. X is 【Chemistry 65】 3. The ionizable cationic lipid of claim 1 or 2, wherein

9. 4. The ionizable cationic lipid of claim 3, wherein W is C=O.

10. R or R is a straight chain alkyl ｃ 10. The ionizable cationic lipid of claim 1 , comprising:

11. R or R is a straight chain alkenyl ｃ 10. The ionizable cationic lipid of claim 1 , comprising:

12. R or R is a branched alkyl ｃ 10. The ionizable cationic lipid of claim 1 , comprising:

13. 10. The ionizable cationic lipid of claim 1, comprising R that is branched alkenyl.

14. R or R is cycloalkyl-alkyl ｃ 10. The ionizable cationic lipid of claim 1 , comprising:

15. R or R is aryl-alkyl ｃ 10. The ionizable cationic lipid of claim 1 , comprising:

16. Each R or R ｃ 16. The ionizable cationic lipid of any one of claims 1 to 15, wherein the groups are the same.

17. R or R arising from the first branch point ｃ Both of the groups are the same, and the R or R arising from the second branch point ｃ Both of the groups are the same, but the R or R emanating from the first branch point ｃ The group is an R or R ｃ 16. The ionizable cationic lipid of claim 1 , wherein the ionizable cationic lipid is different from the group:

18. A lipid nanoparticle (LNP) comprising an ionizable cationic lipid according to any one of claims 1 to 17.

19. 20. The LNP of claim 18, further comprising one or more of a phospholipid, a sterol, a co-lipid, and a PEG-lipid, or a combination thereof.

20. 19. The LNP of claim 18, wherein the phospholipid comprises dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC) or 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), or a combination thereof.

21. 20. The LNP of claim 18 or 19, wherein the sterol comprises cholesterol, campesterol, sitosterol or stigmasterol, or a combination thereof.

22. 22. The LNP of any one of claims 18 to 21, wherein the co-lipid comprises cholesterol hemisuccinate (CHEMS) or a quaternary ammonium head group-containing lipid.

23. 23. The LNP of claim 22, wherein the quaternary ammonium head group-containing lipid comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA) or 3β-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or a combination thereof.

24. The LNP of any one of claims 18 to 23, wherein the PEG-lipid comprises a PEG moiety with a molecular weight (MW) of 1000 to 5000 Da.

25. The PEG-lipid is １４ ~C １８ 25. The LNP of any one of claims 18 to 24, comprising a fatty acid having a fatty acid chain length of:

26. The PEG-lipid may be selected from the group consisting of DMG-PEG2000 (1,2-dimyristoyl-glycero-3-methoxypolyethylene glycol-2000), DPG-PEG2000 (1,2-dipalmitoyl-glycero-3-methoxypolyethylene glycol-2000), DSG-PEG2000 (1,2-distearoyl-glycero-3-methoxypolyethylene glycol-2000), DOG-PEG2000 (1,2-dioleoyl-glycero-3-methoxypolyethylene glycol-2000), DMPE-PEG200 (1,2-dimyristoyl-glycero-3-phosphoethanolamine- 26. The LNP of any one of claims 18 to 25, comprising DPPE-PEG2000 (1,2-dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), DSPE-PEG2000 (1,2-distearoyl-glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), DOPE-PEG2000 (1,2-dioleoyl-glycero-3-phosphoethanolamine-3-methoxypolyethyleneglycol-2000), or a combination thereof.

27. The LNP of any one of claims 18 to 26, wherein the PEG-lipid comprises an optically pure glycerol moiety.

28. The LNP of any one of claims 18 to 27, further comprising a functionalized PEG-lipid.

29. The LNP of claim 28, wherein the functionalized PEG-lipid is conjugated to a binding moiety.

30. The LNP of claim 29 , wherein the binding portion comprises an antigen-binding domain of an antibody.

31. The functionalized PEG-lipid is １６ ~C １８ The LNP of any one of claims 28 to 30, comprising a fatty acid having a fatty acid chain length of:

32. 32. The LNP of claim 31 , wherein the functionalized PEG-lipid comprises a dipalmitoyl lipid or a distearoyl lipid.

33. The LNP of any one of claims 18 to 32, comprising 40-60 mol% ionizable cationic lipid.

34. The LNP of any one of claims 19 to 33, comprising 7 to 30 mol% phospholipid.

35. The LNP of any one of claims 19 to 34, comprising 20 to 45 mol% sterol.

36. The LNP of any one of claims 19 to 35, comprising 1 to 30 mol% co-lipid.

37. The LNP of any one of claims 19 to 36, comprising 0-5 mol% PEG-lipid.

38. The LNP of any one of claims 19 to 37, comprising 0.1 to 5 mol% functionalized PEG-lipid.

39. 39. The LNP of any one of claims 18 to 38, further comprising a nucleic acid.

40. The LNP of claim 39, wherein the weight ratio of total lipid to nucleic acid is 10:1 to 50:

1.

41. The LNP of claim 39 or 40, comprising mRNA.

42. 42. A method for delivering a nucleic acid into a cell, comprising contacting the cell with an LNP described in any one of claims 39 to 41.

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