Lipid Nanoparticles for Delivering Nucleic Acids and Methods of Using the Same

Abstract

The present disclosure provides an improved composition of ionizable lipid nanoparticles for delivering therapeutic nucleic acids to cells. Anionic phospholipids, including phosphatidylserine and phosphatidylglycerol, are included in lipid nanoparticles to increase transfection efficiency within human dendritic cells. Further incorporation of monounsaturated alkyl chain analogs in ionizable lipids of dimethylaminopropyl-dioxolane or heterocyclic ketal in the formulation demonstrated high levels of transfection within human dendritic cells and good stability against oxidative damage compared to other ionizable lipids of the same family. Finally, the use of the ammonium salt of phosphatidylserine enables the effective generation of PS-targeted LNPs.

Description

Technical Field

[0001] Related Applications This patent application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 345,823, filed May 25, 2022, U.S. Provisional Patent Application No. 63 / 346,197, filed May 26, 2022, and U.S. Provisional Patent Application No. 18 / 324,097, filed May 25, 2023, the entire contents of each of which are incorporated herein by reference in their entirety.

[0002] Reference to Sequence Listing This specification includes a sequence listing submitted herewith, including a file named 191016-010503.xml having a size of 17,585 bytes, created on May 24, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Field The present disclosure relates to cationic ionizable lipids and lipid nanoparticles (LNPs). In some embodiments, LNPs comprising one or more cationic ionizable lipids are useful for targeting dendritic cells or for delivering nucleic acid compounds for methods of using these LNP compositions as vaccines. In some embodiments, the LNP may comprise a bio-reducible ionizable cationic lipid or a non-conjugated polyolefinic ionizable cationic lipid.

Background Art

[0004] Lipid nanoparticles (LNPs) are used to deliver therapeutic nucleic acids to cells. For example, LNP pharmaceutical compositions are used in vaccines to deliver mRNA therapeutics. LNP formulations typically include ionizable cationic lipids (ICLs). However, it is known in the art that certain ICL compounds are undesirably sensitive to oxidation during storage. Therefore, there is a need for improved ICL compounds that have improved stability against oxidative degradation while in storage and that also provide desirable transfection activity or efficacy intracellularly when incorporated into LNPs together with therapeutic agents such as nucleic acids.

[0005] LNP compositions, including stable nucleic acid lipid particle (SNALP) compositions, are useful for the delivery of nucleic acid therapeutics for various infectious diseases. Infectious diseases such as tuberculosis, HIV / AIDS, malaria, and COVID-19 pose serious problems to human health. For example, Mycobacterium is a genus of bacteria involved in tuberculosis (TB). According to the World Health Organization, globally, TB is one of the top ten causes of death and the leading cause of death among single pathogens. Despite current maximal efforts, significant challenges have been posed to the development of vaccines effective in preventing many infectious diseases. New efforts in the identification of individual antigenic peptides or combinations of antigenic peptides have helped improve vaccine efficiency. Nevertheless, there remains great potential in the engineering of adjuvants to help efficiently deliver and present these antigenic sequences to specialized antigen-presenting cells such as dendritic cells. The mRNA coding of antigenic peptides or proteins in combination with ionizable cationic lipid nanoparticles represents a particularly promising strategy in vaccine development. There is a need for safe and effective treatments, including LNP pharmaceutical compositions (including vaccine compositions) for delivering mRNA for the treatment and prevention of various diseases. SUMMARY OF THE INVENTION

[0006] Aspects of the present disclosure are (a) a nucleic acid, (b) an ionizable cationic lipid having an N / P ratio of 3 to 8 with respect to the nucleic acid, wherein the ionizable lipid has a chemical structure [Chemical formula] [wherein, R1 is a C 15 ~C 19 alkyl group containing one or two olefins] having, present in the LNP vaccine composition in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition, and optionally selected from the group consisting of DLin-KC3-DMA, KC3-01, KC3-OA, KC3-PA, KC3-C17(8:1), KC3-C15(C8:1), Compound 3 (Table 1A), Compound 8 (Table 1A), an ionizable cationic lipid, (c) a sterol in a total amount of 25 to 45 mol% of the total lipid content of the LNP composition, (d) one or more phospholipids in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition, and (e) a complex lipid in a total amount of 0.5 to 2.5 mol% of the total lipid content of the LNP composition. It relates to an LNP vaccine composition.

[0007] In some embodiments, the one or more phospholipids include phosphatidylserine (PS) lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition. In some embodiments, the PS lipid is DSPS or DPPS.

[0008] In some embodiments, the second phospholipid is selected from the group consisting of DSPC, HSPC, DPPC, and sphingomyelin.

[0009] In some embodiments, the ionizable cationic lipid includes either a monounsaturated alkyl chain or a polyunsaturated alkyl chain, and the olefins are separated by at least two methylene groups.

[0010] In some embodiments, the ionizable cationic lipid is selected from the group consisting of KC3-01, KC3-OA, KC3-PA, KC3-C17(8:1), KC3-C15(C8:1), and Compound 8 (Table 1A).

[0011] In some embodiments, the composition has an N / P ratio of 5 to 6 with respect to the nucleic acid.

[0012] In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is chemically modified RNA. In some embodiments, the nucleic acid is modified with N-methylpseudouridine.

[0013] Another aspect of the present disclosure is (a) a nucleic acid, (b) an ionizable cationic lipid having an N / P ratio of 3 to 8 with respect to the nucleic acid, and having a chemical structure

Chemical formula

[0014] In some embodiments, the second phospholipid is selected from the group consisting of DSPC, HSPC, and sphingomyelin.

[0015] In some embodiments, the ionizable cationic lipid has an N / P ratio of 5 to 6 with respect to the nucleic acid.

[0016] In some embodiments, the ionizable cationic lipid contains either two monounsaturated alkyl chains or two polyunsaturated alkyl chains, and the olefin is separated by at least two methylene groups.

[0017] In some embodiments, the ionizable cationic lipid is selected from the group consisting of KC3-01, KC3-OA, KC3-PA, KC3-C17(8:1), KC3-C15(C8:1), and Compound 8 (Table 1A). In some embodiments, the ionizable cationic lipid is 3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine (KC3-OA).

[0018] In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA is chemically modified with N-methylpseudouridine.

[0019] Aspects of the present disclosure include (a) a nucleic acid, (b) an ionizable cationic lipid having an N / P ratio of 3 to 8 with respect to the nucleic acid, in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition, (c) a sterol in a total amount of 25 to 45 mol% of the total lipid content of the LNP composition, (d) one or more phospholipids in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition, and (e) a complex lipid in a total amount of 0.5 to 2.5 mol% of the total lipid content of the LNP composition, the complex lipid having a poly(ethylene glycol) chain terminally linked to a linking moiety and two hydrocarbon chains terminally linked to the same linking moiety, wherein the hydrocarbon chains are independently selected from a saturated C 12Relates to a nucleic acid-lipid nanoparticle (LNP) composition comprising a complex lipid that is a lock. In some embodiments, the linking moiety is a glyceryl group, an N-oxycarbonylglycerophosphorylethanolamino-carbonyl group, an oxycarbonylamide group, or an oxyacetamide group. In some embodiments, the poly(ethylene glycol) chain is methoxy-poly(ethylene glycol) with an average molecular weight of 2000. In some embodiments, the PEG-lipid is mPEG-1,2-dilauroylglycerol (PEG-DLG), mPEG-1,2-dilauroylglycerol (PEG-DLG), PEG-1,2-dilauroylglycerol, PEG-DLPE, PEG-oxycarbonyl-N,N-didodecylamide, or mPEG-N,N-didodecylacetamide.

[0020] In some embodiments, the LNP has a z-average particle size of 60-150 nm and is freeze-stable.

[0021] In some embodiments, one or more phospholipids comprise phosphatidylserine (PS) lipid in an amount of 2.5-10 mol% of the total lipid content of the LNP composition, and the LNP composition comprises phosphatidylserine (PS) lipid in an amount of 2.5-10 mol% of the total lipid content of the LNP composition.

[0022] Aspects of the present disclosure relate to a nucleic acid-lipid nanoparticle (LNP) human vaccine composition comprising (a) a nucleic acid, (b) an ionizable cationic lipid having an N / P ratio of 3-8 with respect to the nucleic acid in an amount of 40-65 mol% of the total lipid content of the LNP composition, (c) a sterol in an amount of 25-45 mol% of the total lipid content of the LNP composition, (d) one or more phospholipids in an amount of 5-25 mol% of the total lipid content of the LNP composition, the one or more phospholipids comprising phosphatidylglycerol (PG) in an amount of 1.0-10 mol% of the total lipid content of the LNP composition, and (e) a complex lipid in an amount of 0.5-2.5 mol% of the total lipid content of the LNP composition.

[0023] In some embodiments, the ionizable cationic lipid is selected from the group consisting of KC3-01, KC3-OA, KC3-PA, KC3-C17(8:1), KC3-C15(C8:1), and Compound 8 (Table 1A). In some embodiments, the sterol is cholesterol, and the phosphatidylglycerol (PG) is an anionic phospholipid selected from the group consisting of distearoyl phosphatidylglycerol (DSPG) and dipalmitoyl phosphatidylglycerol (DPPG).

[0024] In some embodiments, the nucleic acid is mRNA, and the ionizable cationic lipid is present at an N / P ratio of 4 to 7 with respect to the nucleic acid. In some embodiments, the complex lipid is selected from PEG-DMG, PEG-DLG, and PEG-DLPE.

[0025] In some embodiments, the one or more phospholipids include phospholipids selected from the group consisting of distearoyl phosphatidylcholine (DSPC) and hydrogenated soy phosphatidylcholine (HSPC).

[0026] Aspects of the present disclosure relate to a method of making a nucleic acid delivery composition comprising a lipid, wherein the lipid comprises phosphatidylserine, and the method comprises dissolving the phosphatidylserine in ethanol, wherein the phosphatidylserine is in the form of an ammonium salt of phosphatidylserine.

[0027] In some embodiments, phosphatidylserine is DPPS. In some embodiments, phosphatidylserine is dissolved in ethanol to a concentration greater than 0.2 mM. In some embodiments, the ammonium salt is a salt containing ammonium selected from the group consisting of ammonium, alkylammonium, dialkylammonium, trialkylammonium, and tetraalkylammonium. In some embodiments, ammonium is formed by being selected from the group consisting of ammonia, dimethylamine, diethylamine, triethylamine, trimethylamine, 2-(dimethylamino)ethanol, diethanolamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methyl-glucamine, imidazole, histidine, lysine, arginine, 4-(2-hydroxyethyl)-morpholine, piperazine, 1-(2-hydroxyethyl)-pyrrolidine, triethanolamine, or tromethamine (tris(hydroxymethyl)aminomethane).

[0028] Aspects of the present disclosure relate to a method for delivering nucleic acids into cells, comprising the step of contacting lipid nanoparticles (LNP) with the cells, wherein the cells are human dendritic cells and the LNP composition is obtained by the process of the aspects of the present disclosure.

[0029] In one embodiment, the LNP comprises the ionizable cationic lipid of the present disclosure.

[0030] In some embodiments, the cells are cells of a human or animal subject. In some embodiments, the LNP is administered intramuscularly, subcutaneously, intradermally, or topically to the subject.

[0031] Aspects of the present disclosure relate to an LNP composition comprising: (a) an mRNA, a nucleic acid; (b) cholesterol sterol in an amount of 25 to 40 mol% of the total lipid content of the LNP composition; (c) an ionizable cationic lipid having an N / P ratio of 3 to 8 with respect to the nucleic acid in an amount of 40 to 65 mol% of the total lipid content of the LNP composition; (d) one or more phospholipids selected from the group consisting of (i) an ammonium salt of dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS) lipid in an amount of 2.5 to 10 mol% of the total lipid content of the LNP composition and (ii) distearoylphosphatidylcholine (DSPC) phospholipid in an amount of 5 to 25 mol% of the total lipid content of the LNP composition, in an amount of 5 to 25 mol% of the total lipid content of the LNP composition; and (e) a PEG-containing complex lipid in an amount of 0.5 to 2.5 mol% of the total lipid content of the LNP composition.

[0032] Aspects of the present disclosure relate to a method for administering a nucleic acid to a subject in need thereof, the method comprising administering a nucleic acid-lipid nanoparticle (LNP) composition to a human subject, the nucleic acid LNP composition comprising: (a) a nucleic acid; (b) an ionizable cationic lipid having an N / P ratio of 3 to 8 with respect to the nucleic acid in an amount of 40 to 65 mol% of the total lipid content of the LNP composition; (c) a sterol in an amount of 25 to 45 mol% of the total lipid content of the LNP composition; (d) one or more phospholipids in an amount of 5 to 25 mol% of the total lipid content of the LNP composition, the one or more phospholipids comprising phosphatidylglycerol (PG) in an amount of 1.0 to 10 mol% of the total lipid content of the LNP composition; and (e) a complex lipid in an amount of 0.5 to 2.5 mol% of the total lipid content of the LNP composition.

[0033] Aspects of the present disclosure relate to nucleic acid-lipid nanoparticle (LNP) compositions comprising: (a) a nucleic acid; (b) an ionizable cationic lipid having an N / P ratio of 3 to 8 relative to the nucleic acid, in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition; (c) a sterol in a total amount of 25 to 45 mol% of the total lipid content of the LNP composition; (d) one or more phospholipids in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition, the one or more phospholipids comprising phosphatidylserine (PS) lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition; and (e) a complex lipid in a total amount of 0.5 to 2.5 mol% of the total lipid content of the LNP composition. In some embodiments, the nucleic acid is mRNA, the ionizable cationic lipid is present in the LNP composition at an N / P ratio of 4 to 7 relative to the nucleic acid, the sterol is cholesterol, and the complex lipid is a PEG-containing complex lipid.

[0034] In some embodiments, the one or more phospholipids comprise at least two phospholipids having incompatible acyl chain lengths.

[0035] In some embodiments, the phosphatidylserine (PS) lipid is dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS).

[0036] In some embodiments, the one or more phospholipids comprise a phospholipid selected from the group consisting of distearoylphosphatidylcholine (DSPC) and hydrogenated soy phosphatidylcholine (HSPC). In some embodiments, the one or more phospholipids consist of distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS). In some embodiments, the PEG-containing complex lipid is PEG(2000)-dimyristoyl glycerol (PEG-DMG).

[0037] Aspects of the present disclosure are directed to an ionizable lipid having a chemical structure [wherein, R1 is [wherein, R1 is [Chemical] where a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each independently (C1-C4) alkyl optionally substituted with hydroxyl, n is an integer equal to 2, 3 or 4. Relates to an LNP composition having

[0038] In some embodiments, R2 and R3 are each methyl and n is 3 or 4.

[0039] In some embodiments, the ionizable cationic lipid is one or more compounds selected from the group consisting of KC3-OA, KC3-PA, KC3-C17(8:1), and KC3-C15(C8:1). In some embodiments, the ionizable cationic lipid is KC3-PA. In some embodiments, the ionizable cationic lipid is KC3-OA. In some embodiments, the ionizable cationic lipid is KC3-C17(C8:1). Aspects of the present disclosure relate to ionizable cationic lipids selected from the group consisting of KC3-PA, KC3-C17(8:1), and KC3-C15(C8:1).

[0040] Aspects of the present disclosure relate to a nucleic acid lipid nanoparticle (LNP) composition comprising (a) a nucleic acid, (b) an ionizable cationic lipid having an N / P ratio of 3 to 8 with respect to the nucleic acid, in an amount of 40 to 65 mol% of the total lipid content of the LNP composition, (c) a sterol in an amount of 25 to 45 mol% of the total lipid content of the LNP composition, (d) one or more phospholipids in an amount of 5 to 25 mol% of the total lipid content of the LNP composition, the one or more phospholipids comprising phosphatidylserine (PS) lipid in an amount of 2.5 to 10 mol% of the total lipid content of the LNP composition, and (d) a complex lipid in an amount of 0.5 to 2.5 mol% of the total lipid content of the LNP composition.

[0041] In some embodiments, the sterol is cholesterol. In some embodiments, the one or more phospholipids include phospholipids having incompatible acyl chain lengths. In some embodiments, the PS lipid is dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS). In some embodiments, the one or more phospholipids include phospholipids selected from the group consisting of distearoylphosphatidylcholine (DSPC) and hydrogenated soy phosphatidylcholine (HSPC). In some embodiments, the one or more phospholipids include distearoylphosphatidylcholine (DSPC) and phosphatidylserine (PS). In some embodiments, the ionizable cationic lipid is 3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine (KC3-OA). In some embodiments, the ionizable cationic lipid is 3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine (KC3-OA). In some embodiments, the one or more phospholipids consist of distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS), and the phosphatidylserine (PS) is (L-serine) DPPS.

[0042] In some embodiments, the complex lipid is a PEG-containing complex lipid, and the PEG-containing complex lipid is selected from the group consisting of PEG(2000)-dimyristoyl glycerol (PEG-DMG), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-DLPE), and PEG(2000)-dilauroyl glycerol (PEG-DLG).

[0043] In some embodiments, the nucleic acid is mRNA, the one or more phospholipids include phosphatidylserine (PS) and one or more phospholipids selected from the group consisting of distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), and dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS), and the complex lipid includes polyethylene glycol (PEG). In some embodiments, the complex lipid is PEG(2000)-dimyristoyl glycerol (PEG-DMG). In some embodiments, the ionizable cationic lipid is 3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine (KC3-OA). In some embodiments, the one or more phospholipids consist of distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS).

[0044] In some embodiments, dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS) is the ammonium salt of (L-serine) DPPS.

[0045] In some embodiments, (a) the nucleic acid is mRNA, (b) the sterol is cholesterol sterol in a total amount of 25-45 mol% of the total lipid content of the LNP composition, (c) the ionizable cationic lipid is in a total amount of 40-65 mol% of the total lipid content of the LNP composition and has an N / P ratio of 3-8 with respect to the nucleic acid, (d) the one or more phospholipids are in a total amount of 5-25 mol% of the total lipid content of the LNP composition and consist of (i) dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS) lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition, and (ii) distearoylphosphatidylcholine (DSPC) phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition, and (e) the complex lipid is a PEG-containing complex lipid in a total amount of 0.5-2.5 mol% of the total lipid content of the LNP composition. In some embodiments, the PEG-containing complex lipid is selected from the group consisting of PEG(2000)-dimyristoyl glycerol (PEG-DMG), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-DLPE), and PEG(2000)-dilauroyl glycerol (PEG-DLG).

[0046] Aspects of the present disclosure relate to a nucleic acid-lipid nanoparticle (LNP) human vaccine composition comprising (a) a nucleic acid, (b) an ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition and having an N / P ratio of 3-8 with respect to the nucleic acid, (c) a sterol in a total amount of 25-45 mol% of the total lipid content of the LNP composition, (d) one or more phospholipids in a total amount of 5-25 mol% of the total lipid content of the LNP composition, the one or more phospholipids comprising phosphatidylglycerol (PG) in a total amount of 1.0-10 mol% of the total lipid content of the LNP composition, and (e) a complex lipid in a total amount of 0.5-2.5 mol% of the total lipid content of the LNP composition.

[0047] In some embodiments, the sterol is cholesterol, and the phosphatidylglycerol (PG) is an anionic phospholipid selected from the group consisting of distearoyl phosphatidylglycerol (DSPG) and dipalmitoyl phosphatidylglycerol (DPPG).

[0048] In some embodiments, the nucleic acid is mRNA, and the ionizable cationic lipid is present at an N / P ratio of 4 to 7 with respect to the nucleic acid.

[0049] Aspects of the present disclosure relate to a nucleic acid-lipid nanoparticle (LNP) composition comprising an ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition, wherein the ionizable cationic lipid is selected from the group consisting of KC3-PA, KC3-C17(8:1), and KC3-C15(C8:1), KC3-OA, and KC3-01.

[0050] [Chemical formula]

[0051] Aspects of the present disclosure are (a) a nucleic acid, (b) an ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition and having an N / P ratio of 3 to 8 with respect to the nucleic acid, (c) a sterol in a total amount of 25 to 45 mol% of the total lipid content of the LNP composition, (d) one or more phospholipids in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition, and (d) a complex lipid in a total amount of 0.5 to 2.5 mol% of the total lipid content of the LNP composition, the complex lipid comprising a PEG-lipid having a poly(ethylene glycol) chain terminally linked to a linking moiety and two hydrocarbon chains terminally linked to the same linking moiety, the two hydrocarbon chains being independently saturated C 12 chains selected from an n-dodecyl (lauryl) group and an n-dodecanoyl (lauroyl) group, and relates to a nucleic acid-lipid nanoparticle (LNP) composition comprising the complex lipid.

[0052] In some embodiments, the linking moiety is a glyceryl group, an N-oxycarbonylglycerophosphorylethanolamino-carbonyl group, an oxycarbonylamide group, or an oxyacetamide group. In some embodiments, the poly(ethylene glycol) chain is methoxy-poly(ethylene glycol) with an average molecular weight of 2000. In some embodiments, the PEG-lipid is mPEG-1,2-dilauroylglycerol (PEG-DLG), mPEG-1,2-dilauroylglycerol (PEG-DLG), PEG-1,2-dilauroylglycerol, PEG-DLPE, PEG-oxycarbonyl-N,N-didodecylamide, or mPEG-N,N-didodecylacetamide.

Brief Description of the Drawings

[0053]

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Mode for Carrying Out the Invention

[0054] It should be understood that both the foregoing summary and the following detailed description are exemplary and for illustrative purposes only and are not intended to limit the compositions and methods of the present disclosure.

[0055] Liposome nanoparticle (LNP) compositions can include ionizable lipids, sterols, and one or more phospholipids. In some embodiments, the LNP composition further includes a nucleic acid such as mRNA for administration in a pharmaceutical composition such as a vaccine. In some embodiments, the LNP composition optionally further includes a complex lipid.

[0056] Lipid nanoparticle (LNP) compositions containing mRNA include stabilized nucleic acid lipid particles (SNALP) used as vehicles for systemic delivery of mRNA or other nucleic acid therapeutics. The SNALP composition includes a cationic lipid such as MC3 or KC2, which includes a protonatable tertiary amine head group attached to a pair of straight-chain 18-carbon aliphatic chains (e.g., linoleic acid) containing a pair of carbon-carbon double bonds separated by a single methylene group. However, the structure of these hydrocarbon chains, each containing a pair of double bonds separated by a single methylene group, confers the desired biological properties of the SNALP composition, but this chemical substructure also presents an undesirable problem of increased susceptibility of the compound to oxidative degradation. For example, FIG. 1 is a diagram of the oxidative degradation mechanism of a lipid ester of linoleic acid containing multiple conjugated unsaturates that are particularly sensitive to oxidation. There is a need for new cationic lipids that are suitable for use in SNALP compositions but have enhanced resistance to oxidative degradation.

[0057] Disclosed herein are compounds, compositions, and methods related to the treatment of bacterial infections. As used herein, the terms "compound," "drug," and "active agent" are used interchangeably. Some aspects of the present disclosure relate to novel ionizable lipids or bio-reductive ionizable lipids. These lipids are cationic at acidic pH (i.e., have a positive charge) and are encountered intracellularly, for example, after endocytosis or phagocytosis by cells. The same lipids and compositions containing them have a nearly neutral charge when present at pH 7.4. These lipids may also have a single olefin present in their alkyl or acyl groups.

[0058] Some aspects of the present disclosure relate to processes for the synthesis of novel ionizable lipids.

[0059] Other aspects relate to compositions comprising lipid nanoparticles comprising an ionizable cationic lipid, wherein the lipid nanoparticles contain nucleic acids. In some embodiments, the nucleic acid is encapsulated within the lipid nanoparticles.

[0060] Aspects of the present disclosure provide improved compositions of ionizable lipid nanoparticles for delivering therapeutic nucleic acids to cells. Anionic phospholipids, including phosphatidylserine and phosphatidylglycerol, are included in the lipid nanoparticles to increase transfection efficiency within dendritic cells. Further incorporation of ionizable lipids into LNP formulations using gem-disubstitution of a monounsaturated alkyl chain (single olefin) at the 2-position of 1,3-dioxolane or ketal demonstrated high levels of transfection within human dendritic cells and good stability against oxidative damage compared to other ionizable lipids of the same family.

[0061] Definitions For convenience, certain terms used herein, in the examples, and in the appended claims are summarized here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0062] As used herein, the following terms and phrases are intended to have the following meanings.

[0063] The articles "a" and "an" are used to refer to one or more (i.e., at least one) grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0064] As used herein, the term "comprising" or "comprises" is used in connection with compositions, methods, and respective components thereof, which cover the inclusion of elements that are present in a given embodiment but not specified.

[0065] As used herein, the term "consisting essentially of" refers to the elements necessary for a given embodiment. This term allows for the presence of additional elements that do not substantially affect the basis and novel or functional characteristics of that embodiment of the present disclosure.

[0066] The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding all elements not recited during the description of the embodiment.

[0067] The term "comprising" as used herein includes "consisting of" and "consisting essentially of".

[0068] When reference is made in the description to "as described above" or "above-mentioned", "the foregoing", it refers to any of the disclosures made in the specification on any of the previous pages.

[0069] When reference is made in the description to "as described herein", "described in this specification", "provided in this specification", or "as described in this text", or "specified in this specification", it refers to any of the disclosures made in the specification on any of the previous or subsequent pages.

[0070] As used herein, the term "about" means an acceptable variation within 20%, within 10%, and within 5% of a specified value. In certain embodiments, "about" can mean a variation of + / -1%, 2%, 3%, 4%, 5%, 10%, or 20%.

[0071] As used herein in connection with a compound or composition, the term "effective amount" means the amount of an active compound (also referred to herein as an active agent or active drug) sufficient to produce a bactericidal or bacteriostatic effect. In some embodiments, the effective amount is a "therapeutically effective amount," meaning the amount of an active compound sufficient to reduce the symptoms of a bacterial infection being treated.

[0072] As used herein, the term "subject (alternatively, "patient")" refers to an animal, preferably a mammal, most preferably a human, that is to receive either a prophylactic or therapeutic treatment.

[0073] As used herein, the term "administration" or "administering" means all means of introducing a compound or pharmaceutical composition to a subject in need thereof, including but not limited to oral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, inhalation, buccal, ophthalmic, sublingual, intravaginal, and rectal. Administration of the compound or composition is preferably parenteral. For example, the compound or composition can preferably be administered intravenously, but can also be administered intraperitoneally or via inhalation, as is currently used clinically for liposomal amikacin in the treatment of Mycobacterium avium (see Shirley et al., Amikacin Liposome Inhalation Suspension: A Review in Mycobacterium avium Complex Lung Disease. Drugs. 2019 Apr;79(5):555-562).

[0074] As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures such as those described herein.

[0075] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic inorganic or organic acid addition salt of a compound of the disclosure that has the desired pharmacological activity.

[0076] The term "alkyl" means a saturated carbon chain having 1 to 20 carbon atoms, which may be linear, branched, or a combination thereof, unless otherwise specified in the carbon chain. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, and octyl. Unless otherwise specified herein, alkyl groups are optionally substituted.

[0077] The term "phosphatidylserine" refers to the L-isomer of serine in the head group, together with any of its acyl chain compositions, unless otherwise specified in a particular embodiment.

[0078] The term "lipid complex" refers to a complex lipid that inhibits the aggregation of lipid particles. Such lipid complexes include, but are not limited to, polysarcosine (see, e.g., WO2021191265A, which is incorporated herein by reference for all purposes), polyamide oligomers (e.g., ATTA-lipid complex), PEG-lipid complexes (PEG coupled to dialkyloxypropyl, PEG coupled to diacylglycerol, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, PEG conjugated to ceramide, etc.) (see, e.g., U.S. Patent No. 5,885,613, which is incorporated herein by reference for all purposes), cationic PEG lipids, and mixtures thereof. The PEG can be conjugated directly to the lipid or can be attached to the lipid via a linker moiety. Any linker moiety suitable for coupling PEG to a lipid (e.g., including non-ester-containing linker moieties and ester-containing linker moieties) can be used. In a preferred embodiment, a non-ester-containing linker moiety is used.

[0079] The abbreviations of ionizable cationic lipids can be truncated from those used in the table in the examples. For example, AKG-UO-1 or AKG-KC2-01 may be referred to as UO1 or KC2-01.

[0080] The abbreviation UT used in various studies refers to an untreated sample.

[0081] The term "lipid nanoparticle" or "LNP" refers to particles having a diameter of about 5 to 500 nm. In some embodiments, the lipid nanoparticles contain one or more active agents. In some embodiments, the lipid nanoparticles contain nucleic acids. In some embodiments, the nucleic acids are condensed inside the nanoparticles having a cationic lipid, a polymer, or a multivalent small molecule and an external lipid coat that interacts with the biological environment. Due to the repulsion between phosphate groups, nucleic acids are necessarily rigid polymers and preferably have an elongated shape. Inside cells, to cope with volume constraints, DNA can pack itself with the help of ions and other molecules under appropriate solution conditions. Usually, DNA condensation is defined as the collapse of an extended DNA strand into compact, systematic particles containing only one or a few molecules. By binding to phosphate groups, cationic lipids can condense DNA by neutralizing the phosphate charge and packing it densely.

[0082] In some embodiments, the active agent is encapsulated in the LNP. In some embodiments, the active agent may be an anionic compound, for example, DNA, RNA, natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNAs, and small interfering RNAs), nuclear proteins, peptides, nucleic acids, ribozymes, DNA-containing nuclear proteins, such as intact or partially deproteinized virus particles (virions), oligomers and polymeric anionic compounds other than DNA (for example, acidic polysaccharides and glycoproteins), but is not limited thereto. In some embodiments, the active agent may be mixed with an adjuvant.

[0083] In LNP vaccine products, the active agent is generally contained inside the LNP. In some embodiments, the active agent comprises nucleic acid. Typically, water-soluble nucleic acids are condensed with cationic lipids or polycationic polymers inside the particles, and the surface of the particles is rich in neutral lipids or PEG-lipid derivatives. Additional ionizable cationic lipids may also be present on the surface, promoting endosomal escape in response to acidification in the environment by carrying a positive charge.

[0084] Ionizable lipids can have different properties or functions from LNPs. Due to the pKa of the amino group, lipid molecules can become positively charged under acidic conditions. Under these conditions, lipid molecules can electrostatically bind to the phosphate groups of nucleic acids, enabling the formation of LNPs and encapsulation of nucleic acids. In some embodiments, the pKa may be low enough to substantially neutralize the surface charge of the LNP in a biological fluid such as blood at physiological pH values. High LNP surface charge is associated with toxicity, adherence, rapid removal from circulation by free macrophages, and hemolytic toxicity (including immune activation) (Filion et al Biochim Biophys Acta.1997 Oct 23;1329(2):345-56).

[0085] In some embodiments, the pKa may be high enough for the ionizable cationic lipid to be able to take a positively charged form at acidic endosomal pH values. In this way, the cationic lipid can combine with endogenous endosomal anionic lipids to promote a membrane-lytic non-bilayer structure such as the hexagonal HII phase, resulting in more efficient intracellular transport. In some embodiments, the pKa ranges from 6.2 to 6.5. For example, the pKa may be about 6.2, about 6.3, about 6.4, about 6.5. Unsaturated tails also contribute to the ability of lipids to adopt non-bilayer structures (Jayaraman et al.,Angew Chem Int Ed Engl.2012 Aug 20;51(34):8529-33).

[0086] Among other properties such as liposome removal and circulating half-life, the release of nucleic acids from LNP formulations can be modified by the presence of polyethylene glycol and / or sterols (e.g., cholesterol) or other potential additives in the LNP, as well as the overall chemical structure (including the pKa of any ionizable cationic lipid included as part of the formulation).

[0087] The term "bioreductive" refers to compounds that undergo accelerated degradation due to cleavage of disulfide bonds in a reducing environment. Unlike other nucleic acid therapeutics such as siRNA, the success of mRNA-based therapeutics depends on the utility of a safe and efficient delivery vehicle to encapsulate the mRNA. mRNA is fragile and requires a protective coating to maintain its active state until it reaches the target site. mRNA-containing LNPs are a promising vaccine option for Covid-19 immunity (Jackson et al., Preliminary Report. N Engl J Med. 2020 Nov 12;383(20):1920-1931). The efficiency and tolerability of LNPs are due to amino lipids, and unlike many biomaterial applications that require weeks or months of service, functional LNP-mediated delivery of mRNA occurs within hours, eliminating the need for persistent lipids. Indeed, this is particularly important in applications that require long-term administration. LNPs have been demonstrated to enter cells via endocytosis and accumulate in the endosomal compartment. Ionizable cationic lipids (ICLs) are susceptible to enzymatic hydrolysis by lipases or hydrolysis induced by the reducing environment of lysosomes in late endosomes / lysosomes and are completely biodegradable, but can efficiently deliver mRNA to the cytosol after endocytosis. The extracellular space is a relatively oxidative environment, while the intracellular space is a reducing environment, allowing disulfide bond molecules to remain intact in the extracellular space but be rapidly reduced upon internalization (Huang et al., Mol Ther. 2005 Mar;11(3):409-17, 2005). Some embodiments provide bioreductive disulfide bond ICL molecules (see Compounds 29-36 (Table 2)) that are stable in LNP formulations in circulation but undergo cleavage in the reducing environment of lysosomes. Such compounds and compositions can promote rapid biological destruction of lipids and prevent potential toxic accumulation of ICL lipids (as observed in rats with DLin-MC3-DMA (Sabins et al., Mol Ther. 2018 Jun 6;26(6):1509-1519)).

[0088] As used herein, the terms "encapsulation" and "encapsulated" refer to the incorporation of mRNA, DNA, siRNA or other nucleic acid pharmaceuticals into lipid nanoparticles or their association with lipid nanoparticles. As used herein, the term "encapsulated" refers to either complete encapsulation or partial encapsulation. siRNA can selectively knockdown or downregulate the expression of a target gene. For example, siRNA can be selected to silence a gene associated with a particular disease, disorder, or condition when a nanoparticle composition containing the siRNA is administered to a subject in need thereof. siRNA may contain a sequence complementary to the mRNA sequence encoding the target gene or protein.

[0089] The term "mol %" related to cholesterol refers to the molar amount of cholesterol relative to the total molar amount of cholesterol and non-PEGylated phospholipids expressed in percentage points. For example, "55 mol % cholesterol" in a liposome containing cholesterol and HSPC refers to a composition having 55 parts by mole of cholesterol per 45 parts by mole of HSPC.

[0090] The term "mol %" related to PEG-lipid refers to the ratio of the molar amounts of PEG-lipid and non-PEGylated phospholipid expressed in percentage points. For example, "5 mol % PEG-DSPE" in an LNP containing HSPC and PEG-DSPE refers to a composition having 5 parts by mole of PEG-DSPE per 100 parts by mole of HSPC.

[0091] In some embodiments, the "mol %" related to cholesterol refers to the molar amount of cholesterol relative to the total molar amount of total lipids expressed in percentage points. For example, "40.5 mol % cholesterol" in an LNP composition containing ICL, DSPC, PS, cholesterol, and PEG-DMG refers to a composition having 40.5 parts by mole of cholesterol relative to 59.5 parts by mole of the combined components of ICL, DSPC, PS, and PEG-DMG.

[0092] In some embodiments, the “mol%” related to the complex lipid refers to the ratio of the molar amount of the complex lipid expressed in percentage points. For example, “1.5 mol% of PEG-DMG” in ICL, DSPC, PS, cholesterol, and PEG-DMG, which are cholesterol-containing LNPs, refers to a composition of 1.5 mol parts of PEG-DMG relative to a combined component of 98.5 mol parts of ICL, DSPC, PS, and cholesterol.

[0093] In some embodiments, the “mol%” related to the PS lipid refers to the ratio of the molar amount of the PS lipid expressed in percentage points. For example, “5 mol% of DPPS” in ICL, DSPC, DPPS, cholesterol, and PEG-DMG, which are cholesterol-containing LNPs, refers to a composition of 5 mol parts of DPPS relative to a combined component of 95 mol parts of ICL, DSPC, PEG-DMG, and cholesterol.

[0094] In some embodiments, the “mol%” related to ICL refers to the ratio of the molar amount of ICL expressed in percentage points. For example, “48 mol% of ICL” in KC3-OA (for example, an example of ICL), DSPC, PS, cholesterol, and PEG-DMG, which are cholesterol-containing LNPs, refers to a composition of 48 mol parts of ICL relative to a combined component of 52 mol parts of PEG-DMG, DSPC, PS, and cholesterol.

[0095] As used herein, the term “pharmaceutically acceptable carrier, diluent or excipient” includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent approved by the US Food and Drug Administration as acceptable for use in humans or livestock.

[0096] Various aspects and embodiments are described in further detail in the following subsections.

[0097] Ionizable cationic lipid Provided herein are compounds useful for the preparation of lipid nanoparticle (LNP) compositions.

[0098] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I).

[0099] [Chemical formula] [wherein, R1 is [Chemical formula] wherein a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each independently (C1-C4) alkyl optionally substituted with hydroxyl, and n is an integer equal to 2, 3 or 4]

[0100] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), and the total length of the R1 hydrocarbon chain is C 15 ~C 18 In some embodiments, the total length of the R1 hydrocarbon chain is C 16 ~C 18 In some embodiments, the total length of the R1 hydrocarbon chain is C 16 or C 18

[0101] ​In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein a is 0 and b is 1, 2, 3, or 4. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein a is 0 and b is 1 or 3. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein a is 0 and b is 1. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein a is 0 and b is 3.

[0102] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein a is 1 and b is 1, 2, 3, or 4. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein a is 1 and b is 1 or 3. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein a is 1 and b is 1. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein a is 1 and b is 3.

[0103] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein R 10 and R 12 are the same. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein R 10 and R 12 are each independently (C1-C4) alkyl optionally substituted with hydroxyl. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein R10 and R 12 are each (C1-C4) alkyl. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein R 10 and R 12 are each methyl. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein R 10 and R 12 are each ethyl. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein R 10 and R 12 are each independently selected from methyl or ethyl. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein R 10 and R 12 are each independently selected from methyl, ethyl, -(CH2)(CH2)OH, and -(CH2)2(CH2)OH.

[0104] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each methyl, and n is an integer equal to 2, 3 or 4. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each methyl, and n is an integer equal to 2 or 3. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each methyl, and n is an integer equal to 2. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each methyl, and n is an integer equal to 3.

[0105] In some embodiments, the ionizable cationic lipid has the chemical structure of formula (II)

Chemical formula

Chemical formula

[0106] In some embodiments, R in formula (II) 22 is a polyene hydrocarbon chain of formula A.

[0107] In some embodiments, R in formula (II) 10 and R 12 are each independently selected from methyl, ethyl, propyl, -(CH2)(CH2)OH, and -(CH2)2(CH2)OH. In some embodiments, in formula (II), R 10 and R 12 are each independently methyl. In some embodiments, in formula (II), R 10 and R 12 are each independently ethyl. In some embodiments, in formula (II), at least one of R 10 and R 12 is n-propyl optionally substituted with hydroxyl. In some embodiments, in formula (II), R 10 is methyl and R 12 is selected from methyl, ethyl, -(CH2)(CH2)OH, and -(CH2)2(CH2)OH. In some embodiments, in formula (II), R 10 is methyl and R 12 is selected from -(CH2)(CH2)OH and -(CH2)2(CH2)OH. In some embodiments, in a compound containing the chemical structure of formula (II), R 10 is methyl and R 12 is selected from -(CH2)(CH2)OH and -(CH2)2(CH2)OH. In some embodiments, in formula (II), R 10 and R 12 are independently selected from methyl or ethyl optionally substituted with one or more hydroxyls. In some embodiments, in formula (II), R 10 and R 12One or both of them are -(CH2)(CH2)OH or -(CH2)2(CH2)OH in formula (II). In some embodiments, in formula (II), R 10 is methyl, and R 12 is methyl or ethyl substituted with hydroxyl. In some embodiments, one or both of R 10 in formula (II) are methyl, and R 12 is -(CH2)(CH2)OH in formula (II). In some embodiments, one or both of R 10 in formula (II) are methyl, and R 12 is -(CH2)2(CH2)OH in formula (II).

[0108] In some embodiments, the compound has the structure of the compounds listed in the following table. Table 1A and Table 1B show examples of cationic lipids. Table 2 shows examples of bioreducible cationic lipids.

[0109] JPEG2025522311000011.jpg172170

[0110] JPEG2025522311000012.jpg160170

[0111] JPEG2025522311000013.jpg150170

[0112] JPEG2025522311000014.jpg161170

[0113] JPEG2025522311000015.jpg182170

[0114] JPEG2025522311000016.jpg140170

[0115] JPEG2025522311000017.jpg109170

[0116] In some embodiments, the ionizable lipid encapsulates the nucleic acid. In some embodiments, the ionizable lipid encapsulates the nucleic acid in an LNP formulation. In some embodiments, the nucleic acid is an siRNA molecule. In some embodiments, the nucleic acid is an mRNA molecule. In some embodiments, the nucleic acid is a DNA molecule.

[0117] In some embodiments, provided are compositions further comprising a ligand, such as an antibody conjugate to a cell surface receptor, to target the lipid nanoparticles to dendritic cells in a highly specific manner. In some embodiments, the composition further comprises a targeting ligand, which is oriented on the outside of the nanoparticles. In some embodiments, the targeting ligand is an antibody.

[0118] In some embodiments, the lipid nanoparticles are in an aqueous medium.

[0119] In some embodiments, the nucleic acid is encapsulated in the lipid nanoparticles together with a compound disclosed herein, including the compounds of Formula I, II, III, IV-B, V-A-1 or combinations thereof, and the nucleic acid is either RNA or DNA. In some embodiments, the nucleic acid is encapsulated in the lipid nanoparticles together with a compound disclosed herein, including the compounds disclosed herein or combinations thereof, and the nucleic acid is either RNA or DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid is DNA.

[0120] In some embodiments, the lipid nanoparticles comprise a membrane comprising phosphatidylcholine and cholesterol. In some embodiments, the cholesterol is cholesterol. In some embodiments, the lipid nanoparticles comprise a membrane comprising phosphatidylcholine and ionizable cationic lipid (ICL). In some embodiments, the ICL has the structure of Formula I, II, III, IV-B, V-A-1, and cholesterol, and the membrane separates the inside of the lipid nanoparticles from the aqueous medium. In some embodiments, the ICL has the structures shown in Table 1A and Table 2. In some embodiments, the ICL has the structures shown in Table 1B. In some embodiments, the phosphatidylcholine is distearoyl phosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC). In some embodiments, the molar ratio of the ionizable cationic lipid to cholesterol is about 65:35 to 40:60. In some embodiments, the molar ratio of the ICL to cholesterol is about 60:40 to about 45:55.

[0121] In some embodiments, the molar ratio of phosphatidylcholine to cholesterol is about 1:5 to about 1:2.

[0122] In some embodiments, the membrane further comprises a polymer-conjugated lipid.

[0123] In some embodiments, the lipid nanoparticles comprise ICL, DSPC, cholesterol, and polymer-conjugated lipid in a molar ratio of about 49.5:10.3:39.6:2.5.

[0124] In some embodiments, the polymer-conjugated lipid is PEG(2000)-dimyristoyl glycerol (PEG-DMG) or PEG (molecular weight 2,000)-dimyristoyl phosphatidylethanolamine (PEG-DMPE).

[0125] In some embodiments, the percentage of oxidative degradation products of the ionizable lipid is less than 50% of the DLin-KC2-DMA or DLin-MC3-DMA control formulation.

[0126] In some embodiments, the composition is a liquid pharmaceutical formulation for parenteral administration.

[0127] In some embodiments, the composition is a liquid pharmaceutical formulation for subcutaneous, intramuscular, or intradermal administration.

[0128] In some embodiments, the composition is in the form of a lyophilized powder and is reconstituted with an aqueous medium subsequent to administration.

[0129] Another aspect of the present disclosure relates to a method of preventing a bacterial or viral infection, the method comprising administering to a subject in need thereof an effective amount of the composition provided herein to elicit an immune response. Some embodiments provide a method of vaccinating a subject in need thereof, the method comprising administering a composition comprising a nucleic acid encoding an antigen protein.

[0130] In some embodiments, the composition is administered subcutaneously, intramuscularly, or intradermally.

[0131] In some embodiments, the bacterial infection is a Mycobacterium tuberculosis infection. In some embodiments, the bacterial infection is in the form of nontuberculosis mycobacterium.

[0132] In some embodiments, the viral infection is a coronavirus. In some embodiments, the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2.

[0133] In some embodiments, the viral infection is HIV / AIDS.

[0134] In some embodiments, the lipid nanoparticles are administered parenterally.

[0135] In some embodiments, the lipid nanoparticle composition is administered as part of a single dose.

[0136] The present disclosure features lipid nanoparticles that include a nucleic acid, such as DNA, mRNA, siRNA, an antisense oligonucleotide, CRISPR components, such as guide RNA (gRNA or sgRNA) and a CRISPR-associated endonuclease (Cas protein), and a lipid. Exemplary lipids include ionizable cationic lipids (ICLs), phospholipids, sterol lipids, alkylene glycol lipids (e.g., polyethylene glycol lipids), sphingolipids, glycerolipids, glycerophospholipids, prenol lipids, glycolipids, fatty acids, and polyketides. In some embodiments, the LNP includes a single type of lipid. In some embodiments, the LNP includes multiple (e.g., two or more) lipids. The LNP can include one or more of an ionizable cationic lipid, a phospholipid, a sterol, or an alkylene glycol lipid (e.g., polyethylene glycol lipid).

[0137] In one embodiment, the LNP comprises an ionizable cationic lipid. As used herein, "ionizable cationic lipid," "ionizable lipid," and "ICL" are used interchangeably. An ICL is a lipid that contains an ionizable moiety that can carry a charge (e.g., a positive charge, e.g., a cationic lipid) under certain conditions (e.g., under physiological conditions, e.g., within a certain pH range). The ionizable moiety may include an amine, preferably a substituted amine. The ionizable lipid may be a cationic lipid or an anionic lipid. In addition to the ionizable moiety, the ionizable lipid may contain, for example, an alkyl or alkenyl group having a length of more than 6 carbon atoms (e.g., a length of more than about 8 carbons, 10 carbons, 12 carbons, 14 carbons, 16 carbons, 18 carbons, 20 or more carbons). Additional ionizable lipids that may be included in the LNP described herein are disclosed in Jayaraman et al. (Angew. Chem. Int. Ed. 51:8529-8533 (2012)), Semple et al. Nature Biotechnol. 28:172-176 (2010)), and U.S. Pat. Nos. 8,710,200 and 8,754,062, each of which is incorporated herein by reference in its entirety.

[0138] In some embodiments, the LNP further comprises an ionizable lipid having the structure of formula (IV-A) or a pharmaceutically acceptable salt thereof.

[0139]

Chemical formula

Chemical formula

Chemical formula

[0140] In some embodiments, v of the compound of formula (III) is equal to 0. In some embodiments, v of the compound of formula (III) is equal to 1. In some embodiments, v of the compound of formula (III) is equal to 1 and q1 is equal to 1. In some embodiments, v of the compound of formula (III) is equal to 1 and q1 is equal to 2.

[0141] In some embodiments, R of the compound of formula (III) 22 wherein the sum of a and c is 6, 7, 8 or 9. In some embodiments, R of the compound of formula (III) 22 wherein the sum of a and c is 6. In some embodiments, R of the compound of formula (III) 22 wherein the sum of a and c is 7. In some embodiments, R of the compound of formula (III) 22 wherein the sum of a and c is 9.

[0142] In some embodiments, R of the compound of formula (III) 22 wherein v is equal to 0 and the sum of a and c is 6, 7, 8 or 9. In some embodiments, R of the compound of formula (IV-B) 22 wherein v is equal to 0 and the sum of a and c is 6. In some embodiments, R of the compound of formula (III) 22 wherein v is equal to 0 and the sum of a and c is 7. In some embodiments, R of the compound of formula (III) 22 wherein v is equal to 0 and the sum of a and c is 9.

[0143] In some embodiments, in the compound of formula (III), R 10 and R 12 are independently selected from methyl, ethyl, -(CH2)(CH2)OH, and -(CH2)2(CH2)OH. In some embodiments, R of the compound of formula (III) 22 wherein, R10 and R 12 are each methyl, and the sum of a and c is 6, 7, 8 or 9. In some embodiments, for the compound of formula (III), R 22 in, R 10 and R 12 are each methyl, v is 0, and the sum of a and c is 6, 7, 8 or 9.

[0144] In some embodiments, for the compound of formula (IV-B), v is equal to 0, and R 22 is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0145] The LNP may contain ionizable lipids at a concentration of more than about 0.1 mol% of the total lipid content of the LNP, for example. In one embodiment, the LNP contains ionizable lipids at a concentration of, for example, about 1 mol%, about 2 mol%, about 4 mol%, about 8 mol%, about 20 mol%, about 40 mol%, about 50 mol%, about 60 mol%, more than about 80 mol% of the total lipid content of the LNP. In one embodiment, the LNP contains ionizable lipids at a concentration of more than about 20 mol%, about 40 mol%, or about 50 mol%. In one embodiment, the LNP contains ionizable lipids at a concentration of, for example, about 1 mol% to about 95 mol% of the total lipid content of the LNP. In one embodiment, the LNP contains ionizable lipids at a concentration of, for example, about 2 mol% to about 90 mol%, about 4 mol% to about 80 mol%, about 10 mol% to about 70 mol%, about 20 mol% to about 60 mol%, about 40 mol% to about 55 mol% of the total lipid content of the LNP. In one embodiment, the LNP contains ionizable lipids at a concentration of about 20 mol% to about 60 mol%. In one embodiment, the LNP contains ionizable lipids at a concentration of about 40 mol% to about 55 mol%.

[0146] In one embodiment, the LNP contains a phospholipid. The phospholipid is a lipid containing a phosphate group and at least one alkyl, alkenyl, or heteroalkyl chain. The phospholipid may be natural or non-natural (e.g., synthetic phospholipid). The phospholipid may contain an amine, amide, ester, carboxyl, choline, hydroxyl, acetal, ether, carbohydrate, sterol, or glycerol. In some embodiments, the phospholipid may contain phosphocholine, phosphosphingolipid, or plasmalogen. Exemplary phospholipids include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DAPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), bis(monoacylglycerol) phosphate (BMP), L-α-phosphatidylcholine, 1,2-diheptadecanoyl-sn-glycero-3-phosphorylcholine (DHDPC), and 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC).Additional phospholipids that may be included in the LNPs described herein are disclosed in Li, J. et al. (Asian J. Pharm. Sci. 10:81-98 (2015)), which is hereby incorporated by reference in its entirety.

[0147] In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, the phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In some embodiments, the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0148] Uptake of phosphatidylserine LNP (e.g., as described herein) comprises the following components: (i) an ionizable cationic lipid (ICL) containing a C16 alkyl or C16 alkenyl group or a C18 alkyl or C18 alkenyl group at a concentration of about 1 mol% to about 95 mol% (or any value therebetween, e.g., about 20 mol% to about 80 mol%); (ii) a phospholipid containing a C16 or C18 alkyl or alkenyl group at a concentration of 0.1 mol% to about 20 mol% (or any value therebetween, e.g., about 2.5 mol% to about 10 mol%); (iii) cholesterol at a concentration of about 1 mol% to about 95 mol% (or any value therebetween, e.g., about 20 mol% to about 80 mol%); (iv) phosphatidylserine (PS) or phosphatidylglycerol (PG) added to the LNP lipid formulation at a concentration of about 0.5 mol% to about 20 mol%, about 2.5 mol% to about 10 mol%, about 4 mol% to about 8 mol%, or any value therebetween of the total lipid content of the LNP; (v) one or more polyethylene glycol (PEG)-2000-containing lipids (e.g., DPG-PEG2000, DPPE-PEG2000, DMPE-PEG2000, DMG-PEG2000) at a concentration of about 0.1 mol% to about 5 mol% (any value therebetween, e.g., about 1 mol% to about 2.5 mol%). In some embodiments, the LNP comprises two of (i)-(v). In some embodiments, the LNP comprises three of (i)-(v). In some embodiments, the LNP comprises four of (i)-(v). In some embodiments, the LNP comprises each of (i)-(v). In some embodiments, the LNP comprises (i) and (ii). In some embodiments, the LNP comprises (i) and (iii). In some embodiments, the LNP comprises (i) and (v). In some embodiments, the LNP comprises (ii) and (iii). In some embodiments, the LNP comprises (ii) and (v). In some embodiments, the LNP comprises (iii) and (iv). In some embodiments, the LNP comprises (iii) and (v). In some embodiments, the LNP comprises (i), (ii) and (iii).In some embodiments, the LNP comprises (i), (ii), and (v). In some embodiments, the LNP comprises (ii), (iii), and (v). In some embodiments, the LNP comprises (ii), (iii), (iv), and (v). In one embodiment, the LNP consists of, or consists essentially of, four out of (i)-(v). In one embodiment, the LNP consists of, or consists essentially of, each of (i)-(v). In some embodiments, the LNP consists of, or consists essentially of, (i) and (ii). In some embodiments, the LNP consists of, or consists essentially of, (i) and (iii). In some embodiments, the LNP consists of, or consists essentially of, (i) and (v). In some embodiments, the LNP consists of, or consists essentially of, (ii) and (iii). In some embodiments, the LNP comprises (ii) and (v). In some embodiments, the LNP consists of, or consists essentially of, (iii) and (iv). In some embodiments, the LNP consists of, or consists essentially of, (iii) and (v). In some embodiments, the LNP consists of, or consists essentially of, (i), (ii), and (iii). In some embodiments, the LNP consists of, or consists essentially of, (i), (ii), and (v). In some embodiments, the LNP comprises (ii), (iii), and (v). In some embodiments, the LNP consists of, or consists essentially of, (ii), (iii), (iv), and (v).

[0149] The LNP may contain phospholipids, for example, at a concentration of more than about 0.1 mol% of the total lipid content of the LNP. In one embodiment, the LNP contains phospholipids, for example, at a concentration of about 0.5 mol%, about 1 mol%, about 1.5 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 8 mol%, about 10 mol%, about 12 mol%, about 15 mol%, about 20 mol%, more than about 50 mol% of the total lipid content of the LNP. In one embodiment, the LNP contains phospholipids at a concentration of more than about 1 mol%, about 5 mol%, or about 10 mol%. In one embodiment, the LNP contains phospholipids, for example, at a concentration of about 0.1 mol% to about 50 mol% of the total lipid content of the LNP. In one embodiment, the LNP contains phospholipids, for example, at a concentration of about 0.5 mol% to about 40 mol%, about 1 mol% to about 30 mol%, about 5 mol% to about 25 mol%, about 10 mol% to about 20 mol%, about 10 mol% to about 15 mol%, or about 15 mol% to about 20 mol% of the total lipid content of the LNP. In one embodiment, the LNP contains phospholipids at a concentration of about 5 mol% to about 25 mol%. In one embodiment, the LNP contains phospholipids at a concentration of about 10 mol% to 20 mol%.

[0150] In one embodiment, the LNP comprises a sterol or an ionized sterol molecule. A sterol is a lipid that contains a polycyclic structure and an optional hydroxyl or ether substituent, and can be a natural or non-natural (e.g., synthetic sterol) lipid. A sterol may or may not contain a double bond, may contain a single double bond, or may contain multiple double bonds. A sterol may further contain an alkyl, alkenyl, halo, ester, ketone, hydroxyl, amine, polyether, carbohydrate, or cyclic moiety. Exemplary lists of sterols include cholesterol, dehydroergosterol, ergosterol, campesterol, β-sitosterol, stigmasterol, lanosterol, dihydrolanosterol, desmosterol, brassicasterol, lasasterol, thymosterol, 7-dehydrodesmosterol, avenasterol, campestanol, lupeol, and cycloartenol. In some embodiments, the sterol comprises cholesterol, dehydroergosterol, ergosterol, campesterol, β-sitosterol, or stigmasterol. Additional sterols that can be included in the LNP described herein are disclosed in Fahy, E. et al. (J. Lipid Res. 46:839-862 (2005)).

[0151] Ionized sterol In some embodiments, the LNP comprises a sterol. In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is dehydroergosterol. In some embodiments, the sterol is ergosterol. In some embodiments, the sterol is campesterol. In some embodiments, the sterol is β-sitosterol. In some embodiments, the sterol is stigmasterol. In some embodiments, the sterol is a corticosteroid (e.g., corticosterone, hydrocortisone, cortisone, or aldosterone).

[0152] In some embodiments, the ionizable lipid may be a branched ionizable lipid selected from ALC-0315 and SM-102.

[0153]

Chemical Formula

[0154] The LNP may contain, for example, sterols at a concentration greater than about 0.1 mol% of the total lipid content of the LNP. In one embodiment, the LNP contains, for example, sterols at a concentration of about 0.5 mol%, about 1 mol%, about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, or greater than about 70 mol% of the total lipid content of the LNP. In one embodiment, the LNP contains sterols at a concentration greater than about 10 mol%, about 15 mol%, about 20 mol%, or about 25 mol%. In one embodiment, the LNP contains, for example, sterols at a concentration of about 1 mol% to about 95 mol% of the total lipid content of the LNP. In one embodiment, the LNP contains, for example, sterols at a concentration of about 5 mol% to about 90 mol%, about 10 mol% to about 85 mol%, about 20 mol% to about 80 mol%, about 20 mol% to about 60 mol%, about 20 mol% to about 50 mol%, or about 20 mol% to 40 mol% of the total lipid content of the LNP. In one embodiment, the LNP contains sterols at a concentration of about 20 mol% to about 50 mol%. In one embodiment, the LNP contains sterols at a concentration of about 30 mol% to about 60 mol%.

[0155] In some embodiments, the LNP comprises an alkylene glycol-containing lipid. The alkylene glycol-containing lipid is a lipid that contains at least one alkylene glycol moiety, such as a methylene glycol or ethylene glycol moiety. In some embodiments, the alkylene glycol-containing lipid comprises polyethylene glycol (PEG). The alkylene glycol-containing lipid may be a PEG-containing lipid. The polymer conjugate lipid may include poly(ethylene glycol) conjugated (pegylated) phospholipids (PEG-lipids), such as PEG (molecular weight 2,000) methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glycerol (PEG-DSG), PEG (molecular weight 2,000) methoxy-poly(ethylene glycol)-1,2-palmitoyl-sn-glycerol (PEG-DPG), PEG (molecular weight 2,000) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-DSPE) or N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]} (PEG-ceramide). The molecular weight of the PEG moiety in the PEG-lipid component can also vary from 500 to 10,000 g / mol, 1,500 to 6,000 g / mol, but is preferably about 2,000 MW. Other polymers used for conjugation to the lipid anchor may include poly(2-methyl-2-oxazoline) (PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), poly-N-vinylpyrrolidone (PVP), polyglycerol, poly(hydroxyethyl L-asparagine) (PHEA), and poly(hydroxyethyl L-glutamine) (PHEG).

[0156] The PEG-containing lipid may further contain an amine, amide, ester, carboxyl, phosphate, choline, hydroxyl, acetal, ether, heterocycle, or carbohydrate. The PEG-containing lipid may contain, for example, in addition to the PEG moiety, at least one alkyl or alkenyl group having a length of more than 6 carbon atoms (e.g., a length of more than about 8 carbons, 10 carbons, 12 carbons, 14 carbons, 16 carbons, 18 carbons, 20 or more carbons). In one embodiment, the PEG-containing lipid contains a PEG moiety having at least 20 PEG monomers, for example, at least 30 PEG monomers, 40 PEG monomers, 45 PEG monomers, 50 PEG monomers, 100 PEG monomers, 200 PEG monomers, 300 PEG monomers, 500 PEG monomers, 1000 PEG monomers, or 2000 PEG monomers. Exemplary PEG-containing lipids include PEG-DMG (e.g., DMG-PEG2k), PEG-c-DMG, PEG-DSG, PEG-DPG, PEG-DSPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, and PEG-DLPE. In some embodiments, the PEG-lipids include PEG-DMG (e.g., DMG-PEG2k), PEG-c-DMG, PEG-DSG, and PEG-DPG. Additional PEG-lipids that can be included in the LNPs described herein are disclosed in Fahy, E. et al. (J. Lipid Res. 46:839-862 (2005)), which is hereby incorporated by reference in its entirety.

[0157] In some embodiments, the PEG-lipid is PEG-DMG (e.g., DMG-PEG2k). In some embodiments, the PEG-lipid is α-(3’-{[1,2-di(myristyloxy)propanoxy]carbonylamino}propyl)-ω-methoxy, polyoxyethylene (PEG-c-DMG). In some embodiments, the PEG-lipid is PEG-DSG. In some embodiments, the PEG-lipid is PEG-DPG.

[0158] The LNP may comprise an alkylene glycol-containing lipid at a concentration of more than about 0.1 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration of, for example, about 0.5 mol%, about 1 mol%, about 1.5 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 8 mol%, about 10 mol%, about 12 mol%, about 15 mol%, about 20 mol%, more than about 50 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration of more than about 1 mol%, about 4 mol%, or about 6 mol%. In one embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration of, for example, from about 0.1 mol% to about 50 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration of, for example, from about 0.5 mol% to about 40 mol%, from about 1 mol% to about 35 mol%, from about 1.5 mol% to about 30 mol%, from about 2 mol% to about 25 mol%, from about 2.5 mol% to about 20 mol%, from about 3 mol% to about 15 mol%, from about 3.5 mol% to about 10 mol%, or from about 4 mol% to about 9 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration of from about 4 mol% to 9 mol%.

[0159] In some embodiments, the LNP comprises at least two types of lipids. In one embodiment, the LNP comprises two of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP comprises at least three types of lipids. In one embodiment, the LNP comprises three of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP comprises at least four types of lipids. In one embodiment, the LNP comprises each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid.

[0160] LNP (e.g., as described herein) may comprise one or more of the following components: (i) an ionizable cationic lipid at a concentration of from about 1 mol% to about 95 mol% (e.g., from about 20 mol% to about 80 mol%); (ii) a phospholipid at a concentration of from 0.1 mol% to about 50 mol% (e.g., from about 2.5 mol% to about 20 mol%); (iii) a sterol at a concentration of from about 1 mol% to about 95 mol% (e.g., from about 20 mol% to about 80 mol%); (iv) a PEG-containing lipid at a concentration of from about 0.1 mol% to about 50 mol% (e.g., from about 2.5 mol% to about 20 mol%). In some embodiments, the LNP comprises one of (i)-(iv). In some embodiments, the LNP comprises two of (i)-(iv). In some embodiments, the LNP comprises three of (i)-(iv). In some embodiments, the LNP comprises each of (i)-(iv). In some embodiments, the LNP comprises (i) and (ii). In some embodiments, the LNP comprises (i) and (iii). In some embodiments, the LNP comprises (i) and (iv). In some embodiments, the LNP comprises (ii) and (iii). In some embodiments, the LNP comprises (ii) and (iv). In some embodiments, the LNP comprises (iii) and (iv). In some embodiments, the LNP comprises (i), (ii) and (iii). In some embodiments, the LNP comprises (i), (ii) and (iv). In some embodiments, the LNP comprises (ii), (iii) and (iv).

[0161] LNP (e.g., as described herein) may include one or more of the following components: (i) an ionizable cationic lipid at a concentration of about 1 mol% to about 95 mol% (e.g., about 20 mol% to about 80 mol%); (ii) DSPC at a concentration of 0.1 mol% to about 50 mol% (e.g., about 2.5 mol% to about 20 mol%); (iii) cholesterol at a concentration of about 1 mol% to about 95 mol% (e.g., about 20 mol% to about 80 mol%); (iv) DMG-PEG2k at a concentration of about 0.1 mol% to about 50 mol% (e.g., about 2.5 mol% to about 20 mol%). In some embodiments, the LNP includes two of (i)-(iv). In some embodiments, the LNP includes three of (i)-(iv). In some embodiments, the LNP includes each of (i)-(iv). In some embodiments, the LNP includes (i) and (ii). In some embodiments, the LNP includes (i) and (iii). In some embodiments, the LNP includes (i) and (iv). In some embodiments, the LNP includes (ii) and (iii). In some embodiments, the LNP includes (ii) and (iv). In some embodiments, the LNP includes (iii) and (iv). In some embodiments, the LNP includes (i), (ii) and (iii). In some embodiments, the LNP includes (i), (ii) and (iv). In some embodiments, the LNP includes (ii), (iii) and (iv).

[0162] In one embodiment, the LNP has a ratio of ionizable lipid to phospholipid of about 50:1 to about 1:1 (e.g., 40:1, 32:3, 6:1, 7:1, 5:1, 24:5, 26:5, 10:3, 15:2, 16:7, 18:1, 3:1, 3:2, or 1:1). In one embodiment, the LNP has a ratio of ionizable lipid to phospholipid of about 15:2. In one embodiment, the LNP has a ratio of ionizable lipid to phospholipid of about 5:1. In one embodiment, the LNP has a ratio of ionizable lipid to sterol of about 10:1 to about 1:10 (e.g., 9:1, 8:1, 8:7, 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3:1, 2:1, 1:1, 1:2, 1:3, 3:4, 1:4, 3:5, 1:5, 4:5, 1:6, 5:6, 7:6, 7:8, or 8:9). In one embodiment, the LNP has a ratio of ionizable lipid to alkylene-containing lipid of about 1:10 to about 10:1 (e.g., 1:9, 1:8, 7:8, 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3:1, 2:1, 1:1, 1:2, 1:3, 3:4, 1:4, 3:5, 1:5, 4:5, 1:6, 5:6, 7:6, 7:8, or 8:9). In one embodiment, the LNP has a ratio of phospholipid to alkylene-containing lipid of about 10:1 to about 1:10 (e.g., 9:1, 8:1, 8:7, 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3:1, 2:1, 1:1, 1:2, 1:3, 3:4, 1:4, 3:5, 1:5, 4:5, 1:6, 5:6, 7:6, 7:8, or 8:9). In one embodiment, the LNP has a ratio of sterol to alkylene-containing lipid of about 50:1 to about 1:1 (e.g., 40:1, 32:3, 6:1, 7:1, 5:1, 24:1, 22:1, 20:1, 22:5, 24:5, 26:5, 10:3, 15:2, 16:7, 18:1, 3:1, 3:2, or 1:1).

[0163] In some embodiments, the LNP (e.g., as described herein) comprises two of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid (e.g., a PEG-containing lipid). In another embodiment, the LNP (e.g., as described herein) comprises three of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid (e.g., a PEG-containing lipid). In some embodiments, the LNP (e.g., as described herein) comprises each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid (e.g., a PEG-containing lipid).

[0164] In some embodiments, the LNP described herein has a diameter of 5 to 500 nm, such as 10 to 400 nm, 20 to 350 nm, 25 to 325 nm, 30 to 300 nm, 50 to 250 nm, 60 to 200 nm, 75 to 190 nm, 80 to 180 nm, 100 to 200 nm, 200 to 300 nm, and 150 to 250 nm. The diameter of the LNP can be determined by any method known in the art, such as dynamic light scattering, transmission electron microscopy (TEM) or scanning electron microscopy (SEM). In some embodiments, the LNP has a diameter of 50 to 100 nm, 70 to 100 nm, and 80 to 100 nm. In one embodiment, the LNP has a diameter of about 90 nm. In some embodiments, the LNP described herein has a diameter greater than about 30 nm. In some embodiments, the LNP has a diameter of greater than about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about 225 nm, about 250 nm, about 275 nm or greater than about 300 nm. In one embodiment, the LNP has a diameter greater than about 70 nm. In one embodiment, the LNP has a diameter greater than about 90 nm. In one embodiment, the LNP has a diameter greater than about 180 nm.

[0165] In some embodiments, the plurality of LNPs described herein have an average diameter in the range of about 40 nm to about 180 nm. In some embodiments, the plurality of LNPs described herein have an average diameter of about 50 nm to about 150 nm. In some embodiments, the plurality of LNPs described herein have an average diameter of about 50 nm to about 120 nm. In some embodiments, the plurality of LNPs described herein have an average diameter of about 60 nm to about 120 nm. In some embodiments, the plurality of LNPs have an average diameter of about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm.

[0166] In some embodiments, one nanoparticle or a plurality of nanoparticles described herein have an average surface charge that is negative from neutral, less than -100 mV, for example, less than -90 mV, -80 mV, -70 mV, -60 mV, -50 mV, -40 mV, -30 mV, and -20 mV. In some embodiments, one nanoparticle or a plurality of nanoparticles have a surface charge that is negative from neutral from -100 mV to 100 mV, from -75 mV to 0, or from -50 mV to -10 mV.

[0167] In some embodiments, at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the plurality of nanoparticles have an average surface charge that is negative from neutral, less than -100 mV. In some embodiments, one nanoparticle or a plurality of nanoparticles have an average surface charge of from -20 mV to +20, from -10 mV to +10 mV, or from -5 mV to +5 mV at pH 7.4. Neutral-charged LNPs have improved pharmacokinetics and biological performance compared to cationic LNPs.

[0168] Generation of Lipid Nanoparticles (LNPs) The method for generating LNP can include mixing a first solution and a second solution. The mixing can be achieved using standard solution mixing techniques such as propeller mixing, vortex of the solution or preferably microfluidic mixing or high-efficiency T mixing. In some embodiments, the first solution contains one lipid or a plurality of lipids and nucleic acids, and all components are solubilized in a water / solvent system. The solvent can be any water-miscible solvent (e.g., ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane or tetrahydrofuran). In some embodiments, the first solution contains a small proportion of water or pH-buffered water. The first solution contains water up to at least 60% by volume, for example, at least about 0.05% by volume, 0.1% by volume, 0.5% by volume, 1% by volume, 2% by volume, 3% by volume, 4% by volume, 5% by volume, 10% by volume, 15% by volume, 20% by volume, 25% by volume, 30% by volume, 35% by volume, 40% by volume, 45% by volume, 50% by volume, 55% by volume or 60% by volume of water. In one embodiment, the first solution contains from about 0.05% to 60% by volume of water, for example, from about 0.05% to 50% by volume, from about 0.05% to 40% by volume, or from about 5% to 20% by volume of water.

[0169] In some embodiments, the first solution comprises a single type of lipid, such as an ionizable lipid, a phospholipid, a sterol, or a PEG-containing lipid. In some embodiments, the first solution comprises a plurality of lipids. In some embodiments, the plurality comprises an ionizable lipid, a phospholipid, a sterol, or a PEG-containing lipid. In some embodiments, the plurality of lipids comprises cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DMG-PEG2k) or α-(3’-{[1,2-di(myristyloxy)propanoxy]carbonylamino}propyl)-ω-methoxy, polyoxyethylene (PEG2000-C-DMG), and an ionizable lipid. The plurality of lipids can be present in any ratio. In one embodiment, the plurality of lipids comprises an ionizable lipid or a sterol, a phospholipid, a sterol, a PEG-containing lipid of the foregoing lipids or a combination thereof in a specific ratio (e.g., the ratios described herein).

[0170] In some embodiments, the second solution is water. In some embodiments, the second solution is an aqueous buffer having a pH of 3-6 (e.g., a pH of about 3, about 4, about 5, or about 6). The second solution may contain a payload component, such as a nucleic acid (e.g., mRNA). The second solution may contain a small proportion of a water-miscible organic solvent. The second solution may contain at least one water-miscible organic solvent up to at least 60% by volume, e.g., at least about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or any volume % therebetween of at least one organic solvent (e.g., a water-miscible organic solvent). In one embodiment, the second solution contains from about 0.05% to 60% by volume of an organic solvent, e.g., from about 0.05% to 50% by volume, from about 0.05% to 40% by volume, or from about 5% to 20% by volume of an organic solvent (e.g., a water-miscible organic solvent). The aqueous buffer solution may be an aqueous solution of a citrate buffer. In some embodiments, the aqueous buffer solution is a citrate buffer having a pH of 4-6 (e.g., a pH of about 4, about 5, or about 6). In one embodiment, the aqueous buffer solution is a citrate buffer having a pH of about 6.

[0171] In some embodiments, a solution containing a mixture of the first and second solutions containing the LNP suspension may be diluted. In some embodiments, the pH of a solution containing a mixture of the first and second solutions containing the LNP suspension can be adjusted. Dilution or pH adjustment of the LNP suspension can be achieved by adding water, an acid, a base, or an aqueous buffer. In some embodiments, no dilution or pH adjustment of the LNP suspension is performed. In some embodiments, both dilution and pH adjustment of the LNP suspension are performed.

[0172] In some embodiments, tangential flow filtration (TFF) (e.g., hemodiafiltration) can be used to remove excess reagents, solvents, and unencapsulated nucleic acids from the LNP suspension. Organic solvents (e.g., ethanol) and buffers can also be removed from the LNP suspension by TFF. In some embodiments, the LNP suspension is dialyzed and not subjected to TFF. In some embodiments, the LNP suspension is subjected to TFF and not dialyzed. In some embodiments, the LNP suspension is subjected to both dialysis and TFF.

[0173] In one aspect, the present disclosure features a method comprising treating an LNP sample containing a nucleic acid with a fluid containing a detergent (e.g., Triton X-100, or an anionic detergent such as, but not limited to, sodium dodecyl sulfate (SDS), or a nonionic detergent such as, but not limited to, β-octyl glucoside, or an amphoteric detergent 3 - 14) for a period suitable for releasing the encapsulated and / or enclosed nucleic acid by degrading the lipid layer. In one embodiment, the method further comprises analyzing the sample for the presence, absence, and / or amount of the released nucleic acid.

[0174] LNP containing a ligand Some aspects of the present disclosure relate to an LNP containing a ligand (also referred to herein as a target ligand) having binding specificity for a cell surface antigen, wherein binding of the ligand to the antigen induces internalization of the ligand. Some embodiments relate to a composition comprising an LNP containing a ligand described herein.

[0175] Targeting of LNPs can also be achieved by adding lipids to the formulation. For example, phosphatidylserine is known to redistribute to the outer surface of the plasma membrane during apoptosis and is a molecular stimulus for the cellular attraction of phagocytosis (Fadok et al. Curr Biol. 2003 Aug 19;13(16):R655-7). Phosphatidylserine (PS) and phosphatidylglycerol (PG) can be recognized by dendritic cells and can induce the uptake and activation of dendritic cells. Targeting of LNPs can also be achieved by adding specific anionic phospholipids to the formulation (Table 3A). For example, phosphatidylserine is known to redistribute to the outer surface of the plasma membrane during apoptosis and is a molecular stimulus for the cellular attraction of phagocytosis (Fadok et al. Curr Biol. 2003 Aug 19;13(16):R655-7). Phosphatidylserine (PS) and phosphatidylglycerol (PG) can be recognized by dendritic cells and can induce the uptake and activation of dendritic cells (Caronni et al., Nat Comm. 2021 April 14;12:2237-2253; Ischihashi et al., PLOS One 2013). Anionic phospholipids have been used previously in the context of liposomes, but inclusion in lipid nanoparticles containing condensed nucleic acids can be unexpected because the anionic headgroups can compete for the binding sites of ionizable cationic lipids with the phosphate backbone of mRNA, can inhibit intracellular escape by changing the surface charge, or can lead to aggregation of LNPs during formation or storage.

[0176] JPEG2025522311000027.jpg193170

[0177] JPEG2025522311000028.jpg140170

[0178] In one embodiment, the anionic target ligand is selected from the group consisting of phosphatidylserine (PS), phosphatidylglycerol (PG), N-glutaryl-phosphatidylethanolamine (N-glu-PE), or N-succinyl-phosphatidylethanolamine (N-Suc-PE). In one embodiment, the anionic phospholipid used is phosphatidylserine. In another embodiment, the phosphatidylserine contains the L-isomer of serine. In another embodiment, the acyl chains of phosphatidylserine, such as in the case of dimyristoylphosphatidyl-L-serine (DMPS), dipalmitoylphosphatidyl-L-serine (DPPS), or distearoylphosphatidyl-L-serine (DSPS), are fully saturated. In a preferred embodiment, the PS used is the L-isomer of either DPPS or DSPS. Phosphatidylserine may also contain an asymmetric acyl chain composition, for example, where one acyl chain is stearic acid and the other is palmitic acid.

[0179] In some embodiments, the anionic phospholipid is selected from groups other than phosphatidylserine. In some embodiments, these non-PS anionic phospholipids include phosphatidylglycerol (PG), phosphatidic acid (PA), N-glutaryl-phosphatidylethanolamine (N-Glu-PE), N-succinyl-phosphatidylethanolamine (N-Suc-PE), and cardiolipin. In one embodiment, these anionic phospholipids include saturated acyl chains of 16 or 18 carbons, such as distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), N-succinyl-distearoylphosphatidylethanolamine (N-Suc-DSPE), N-glutaryl-distearoylphosphatidylethanolamine (N-Glu-DSPE), distearoylphosphatidic acid (DSPA), and cardiolipin.

[0180] JPEG2025522311000029.jpg171170

[0181] In some embodiments, the anionic phospholipid used is phosphatidylglycerol. In another embodiment, for cases such as dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), or distearoyl phosphatidylglycerol (DSPG), the acyl chains of phosphatidylglycerol are fully saturated. In a preferred embodiment, the PG used is either DPPS or DSPS. Phosphatidylglycerol may also contain, for example, an asymmetric acyl chain composition such that one acyl chain is stearic acid and the other is palmitic acid.

[0182] In some embodiments, the salt form of phosphatidylglycerol or phosphatidylserine is highly soluble in ethanol. In some embodiments, the salt form of phosphatidylserine is highly soluble in ethanol. In some embodiments, it is soluble at greater than 0.5 mg / ml, greater than 1 mg / ml, greater than 5 mg / ml, greater than 10 mg / ml, or greater than 20 mg / ml. In some embodiments, the salt form of phosphatidylglycerol or phosphatidylserine is soluble at at least 0.3 mM, at least 0.4 mM, at least 0.5 mM, at least 0.6 mM, or at least 0.8 mM as determined by the shake flask method in ethanol at 22 degrees or less at a standard strength of 200. In some embodiments, the salt is an ammonium salt. In one embodiment, phosphatidylserine is added to the LNP lipid in the form of an ammonium or substituted ammonium salt. The substituted ammonium salt can be a mono-, di-, tri-, or tetraalkylammonium having an alkyl group of 1 to 6, 1 to 4, 1 to 3, 1, 2, or 3 carbon atoms, respectively. One or more of the alkyl groups can be an n-alkyl or a branched alkyl group (e.g., isopropyl group, etc.) or can form a ring (e.g., cyclohexyl group, etc.). The alkyl group and the nitrogen ammonium atom can form a heterocycle. The substituted ammonium salt can also be formed by an alkylenediamine. Tris(hydroxymethyl)aminomethane and triethanolamine can also be used as amine bases to form the PS salt. In some embodiments, the amine is selected from ammonia, dimethylamine, diethylamine, triethylamine, trimethylamine, 2-(dimethylamino)ethanol, diethanolamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methyl-glucamine, imidazole, histidine, lysine, arginine, 4-(2-hydroxyethyl)-morpholine, piperazine, 1-(2-hydroxyethyl)-pyrrolidine, triethanolamine, and tromethamine (tris(hydroxymethyl)aminomethane). In some embodiments, this targeted lipid is the ammonium salt of DPPS.

[0183] JPEG2025522311000030.jpg141170

[0184] Any method known in the art can be used to obtain phosphatidylserine in the form of ammonium or substituted ammonium salts. In one embodiment, the sodium salt of phosphatidylserine (PS) is dissolved in a one-phase system of chloroform, methanol, and water (Bligh-Dyer one-phase) containing the chloride salt of ammonium or substituted ammonium, and the system is brought to a two-phase state by adding excess methanol and / or water containing ammonium chloride or substituted ammonium chloride. The chloroform-rich phase containing PS is separated and the process is repeated. Finally, the chloroform-rich phase is washed with water to remove excess chloride, and the ammonium (substituted ammonium) salt of PS is obtained by evaporation of the chloroform-rich phase. Optionally, the obtained ammonium or substituted ammonium salt of PS is vacuum dried or dissolved in cyclohexane and lyophilized. In another embodiment, PS as a sodium or potassium salt is dissolved in a water-miscible organic solvent such as chloroform or a chloroform-methanol mixture, washed with a dilute aqueous solution of an acid such as HCl to obtain the free acid form of PS, and then neutralized with ammonium hydroxide or a substituted amine in the free base form. In yet another embodiment, the organic solution of PS as a sodium or potassium salt is treated with a cation exchange resin in ammonium in the form of substituted ammonium. In yet another embodiment, PS is prepared in the form of a calcium or magnesium salt and treated with an ammonium or substituted ammonium salt of a chelating agent such as EDTA or ammonium phosphate or substituted ammonium phosphate in the presence of an organic solvent, causing substitution of calcium or magnesium ions in the form of a chelate or less soluble phosphate, and separated, for example, by filtration, while the ammonium or substituted ammonium salt of PS remains in an organic (e.g., ethanol) solution.

[0185] In one embodiment, PS or PG is added to the LNP lipid formulation at a concentration of about 0.1 mol% to about 20 mol%, about 0.1 mol% to about 10 mol%, about 0.1 mol% to about 5 mol%, about 0.5 mol% to about 20 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 5 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 10 mol%, or about 1 mol% to about 5 mol% of the total lipid content of the LNP. In one embodiment, PS is added to the LNP lipid formulation at a concentration of about 1 mol% to about 20 mol%, about 2.5 mol% to about 10 mol%, about 3 mol% to about 9 mol%, or about 4 mol% to about 8 mol% of the total lipid content of the LNP.

[0186] In one embodiment, the PS or PG lipid is included in an LNP composition containing an ionizable cationic lipid known in the art, including DODAP, AKG-OA-DM2, O-11769, DLin-MC3-DMA, DLin-KC2-DMA, DLin-KC3-DMA, ALC-0315, and SM-102.

[0187] In another embodiment, the PS lipid is included in an LNP composition containing ICLs of formula I, II, III, IV-B, V-A-1, combinations thereof, or pharmaceutically acceptable salts thereof. In another embodiment, the PS lipid is included in the LNP composition using an N / P ratio of 3 to 8, 4 to 7, or 5 to 6.

[0188] In some embodiments, provided is a method of delivering a nucleic acid to a cell, the method comprising contacting the cell with a composition comprising a lipid nanoparticle (LNP) that includes a ligand having binding specificity for a cell surface antigen (also referred to herein as a target ligand), wherein binding of the ligand to the antigen induces internalization of the ligand. In some embodiments, the target ligand may be, but is not limited to, an internalizing antibody or fragment thereof, a small molecule conjugate, or a glycoconjugate. In some embodiments, binding of the target ligand to a specific cell surface antigen induces internalization of the LNP, and when contacted with the cell and incubated with the cell under internalization conditions, the target ligand conjugated to the cell expresses at least 100,000 or at least 1,000,000 antigen molecules.

[0189] JPEG2025522311000031.jpg188170

[0190] JPEG2025522311000032.jpg80170

[0191] Composition In some embodiments, the lipid nanoparticle composition comprises a lipid and a nucleic acid, and the lipid nanoparticle comprises a compound of Formula I, II, III, IV-B, V-A-1, a combination thereof, or a pharmaceutically acceptable salt thereof.

[0192] Other aspects of the disclosure relate to the use of these ionizable lipids or lipid nanoparticle compositions containing ionizable lipids in vaccines for the prevention of infectious diseases or cancer. In some embodiments, the infectious disease may be a bacterial or viral infection. In some embodiments, the compositions described herein can be used to prevent infectious diseases associated with tuberculosis, HIV / AIDS, malaria, or coronavirus-related infectious diseases such as COVID-19. In other embodiments, the infectious disease is influenza, hepatitis B, hepatitis C, dengue fever, human papillomavirus (HPV), norovirus, mumps, measles, meningococcal disease, pneumococcal disease, polio, rotavirus, respiratory syncytial virus (RSV), rubella, herpes zoster / herpes virus, tetanus, or whooping cough.

[0193] In some embodiments, the compounds and compositions described herein can promote efficient uptake and transfection of target cells, including tissue macrophages and dendritic cells. As a result, efficient delivery of a nucleic acid encoding an antigen specific for an infectious virus or bacterium and subsequent presentation of the antigen, which elicits the desired immune response to protect against the corresponding infectious disease, occurs. In some embodiments, the nucleic acid may be a synthetic nucleic acid (e.g., codon-optimized mRNA) encoding an epitope of a coronavirus such as SARS-CoV, MERS-CoV, or SARS-CoV-2. In some embodiments, the nucleic acid may be a synthetic nucleic acid (e.g., codon-optimized mRNA) encoding the S-protein (spike protein) of a coronavirus such as SARS-CoV, MERS-CoV, or SARS-CoV-2 or a fragment thereof.

[0194] In some embodiments, the vaccine is used for the prevention of mycobacterium infection. In some embodiments, the vaccine is tuberculosis, nontuberculous mycobacteria (NTM), nontuberculous lung disease, leprosy, Mycobacterium avium-intracellulare, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium abscessus, and other infectious diseases, such as those caused by coronaviruses (SARS-CoV-2, Covid-19; SARS-CoV, SARS; MERS-CoV; HCoV-229E; HCoV-NL63; HCoV-OC43; HCoV-HKU1), chikungunya, dengue, diphtheria, Ebola, EV-D68, influenza (flu), hepatitis viruses (including HAV, HBV, HCV, HDV, HEV, and GB virus C), Haemophilus influenzae type B (Hib), Hendra virus, HIV / AIDS, human metapneumovirus (hMPV), human papillomavirus (HPV), Lassa, Lyme, malaria, Marburg, measles, meningococcal disease, mumps, Nipah virus, norovirus, parainfluenza virus (PIV), plague, pneumococcal disease, polio, respiratory syncytial virus (RSV), Rocky Mountain spotted fever, rotavirus, rubella (German measles), varicella-zoster virus (chickenpox, shingles), smallpox, tetanus (lockjaw), West Nile, whooping cough (pertussis), and Zika for prevention.

[0195] In some embodiments, the composition further comprises a pharmaceutical excipient.

[0196] In some embodiments, the lipid nanoparticles are in an aqueous medium.

[0197] In some embodiments, the nucleic acid is encapsulated in the lipid nanoparticles together with the compound ionizable cationic lipid compounds presented herein or combinations thereof, and the nucleic acid is either RNA or DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid is DNA.

[0198] In some embodiments, the lipid nanoparticles comprise a membrane containing phosphatidylcholine and sterol. In some embodiments, the sterol is cholesterol. In some embodiments, the lipid nanoparticles comprise a membrane containing phosphatidylcholine and ionizable cationic lipid (ICL). In some embodiments, the ICL has the structure of Formula I and cholesterol, and the membrane separates the interior of the lipid nanoparticles from the aqueous medium. In some embodiments, the ICL has the structures shown in Table 1A and Table 2. In some embodiments, the ICL has the structure shown in Table 1B. In some embodiments, the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC). In some embodiments, the molar ratio of the ionizable cationic lipid to cholesterol is about 65:35 to 40:60. In some embodiments, the molar ratio of the ICL to cholesterol is about 60:40 to about 45:55.

[0199] In some embodiments, the molar ratio of phosphatidylcholine to cholesterol is about 1:5 to about 1:2.

[0200] In some embodiments, the membrane further comprises a polymer composite lipid.

[0201] In some embodiments, the lipid nanoparticles comprise ICL, DSPC, cholesterol and a polymeric complex lipid in a molar ratio of about 49.5:10.3:39.6:2.5.

[0202] In some embodiments, the polymeric complex lipid is PEG(2000)-dimyristoyl glycerol (PEG-DMG) or PEG (molecular weight 2000)-dimyristoyl phosphatidylethanolamine (PEG-DMPE).

[0203] The compositions of the present disclosure can be administered for systemic delivery by various routes, for example, via intravenous, parenteral, intraperitoneal, or topical routes. The compositions can be administered to a subject intravenously, subcutaneously, or intraperitoneally. In some embodiments, the present disclosure provides a method for performing in vivo delivery of nucleic acids to a subject.

[0204] In some embodiments, the composition is a liquid pharmaceutical formulation for oral administration.

[0205] In some embodiments, the composition is a liquid pharmaceutical formulation for subcutaneous, intramuscular, or intradermal administration.

[0206] In some embodiments, the composition is in the form of a lyophilized powder, which is subsequently reconstituted with an aqueous medium prior to administration.

[0207] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I).

[0208]

Chemical Formula

Chemical Formula

[0209] In some embodiments, an ionizable cationic lipid composition is provided. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I-A).

[0210] [Chemical formula] [wherein R1 is [Chemical formula] where a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each independently (C1-C4) alkyl optionally substituted with hydroxyl, n is an integer equal to 2, 3 or 4]

[0211] In some embodiments, a and b of the two R1 hydrocarbon chains are the same.

[0212] In some embodiments, a and b of the two R1 hydrocarbon chains are different.

[0213] In some embodiments, one of the two R1 hydrocarbon chains is saturated C 12 ~C 18 alkyl.

[0214] In some embodiments, an ionizable cationic lipid composition is provided. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I-A).

[0215] [Chemical formula] [wherein, R1 is [Chemical formula] where a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each methyl, and n is an integer equal to 3]

[0216] In some embodiments, a and b of the two R1 hydrocarbon chains are the same.

[0217] In some embodiments, a and b of the two R1 hydrocarbon chains are different.

[0218] In some embodiments, one of the two R1 hydrocarbon chains is saturated C 12 ~C 18 alkyl.

[0219] In some embodiments, an ionizable cationic lipid composition is provided. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I-A).

[0220] [Chemical formula] [wherein, R1 is a saturated C 15 ~C 18 hydrocarbon chain, R2 and R3 are each methyl, and n is an integer equal to 3]

[0221] In some embodiments, a and b of the two R1 hydrocarbon chains are the same.

[0222] In some embodiments, a and b of the two R1 hydrocarbon chains are different.

[0223] In some embodiments, one of the two R1 hydrocarbon chains is a saturated C 12 ~C 18 alkyl.

[0224] Method of Use Targeting of Dendritic Cells Dendritic cells (DCs) are specialized antigen-presenting cells that play a central role in initiating and regulating adaptive immunity. Due to their potent antigen (Ag)-presenting ability and unique capacity to generate T cell responses, the efficient and specific delivery of Ag to DCs is the basis for generating Ag-specific effectors and memory cells against tumors or pathogens.

[0225] Dendritic cells can be generated from human blood monocytes by adding granulocyte macrophage colony-stimulating factor (GM-CSF), IL-4, and IFN-gamma and differentiating monocyte-derived DCs in vitro. Cells under culture show both dendritic and veiled shapes, with the former being adherent and the latter being suspended. Phenotypically, they are CD1a- / dim, CD11a+, CD11b++, CD11c+, CD14dim / -, CD16a- / dim, CD18+, CD32dim / -, CD33+, CD40+, CD45R0+, CD50+, CD54+, CD64- / dim, CD68+, CD71+, CD80dim, CD86+ / ++, MHC class I++, HLA-DR++, HLA-DP+, and HLA-DQ (Geiseler et al. Dev Immunol. 1998;6(1-2):25-39).

[0226] Alternatively, human primary blood dendritic cell lines have been developed and are commercially available from Creative Biolabs.

[0227] CD8+ T cells are capable of producing the cytokines IL2, IFN-γ, and TNF, which are known to have important functions during Mycobacterium tuberculosis infection. Importantly, CD8+ T cells have a cytolytic function to kill Mycobacterium tuberculosis-infected cells via a granule-mediated mechanism (via perforin, granzyme, and granulysin) or a Fas-Fas ligand interaction to induce apoptosis. In humans, CD8+ T cells are capable of producing granulysin, which can directly kill Mycobacterium tuberculosis. Thus, antigen-producing mRNA LNPs delivered to DCs are expected to stimulate CD8+ T cell responses to fight Mycobacterium tuberculosis infection.

[0228] CD8+ T cells can recognize Mycobacterium tuberculosis-specific antigens (as peptides) presented by classical and non-classical MHC molecules. Classically restricted CD8+ T cells that recognize antigens presented by antigen-presenting cells in the context of classical MHC Ia (HLA-A, B, C) molecules have been identified. Non-classically restricted CD8+ T cells include CD8+ T cells that can recognize Mg antigens in the context of MHC I-related molecules (MR1) such as HLA-E molecules (non-MHC Ia), glycolipids associated with group 1 CD1 molecules, and mucosal-associated invariant T cells (MAIT). Finally, γδ T cells represent a distinct population of CD8 (and CD4) T cells that have both innate and adaptive functions in response to Mycobacterium tuberculosis infection. Although CD8+ T cells have been shown to function directly in response to Mycobacterium tuberculosis infection, they also play an important role in modulating many different functions (e.g., interactions to provide optimal CD4 T cell function) in the overall host immune response.

[0229] In one embodiment, the LNP may be added to cultured human dendritic cells at an appropriate concentration (e.g., 1 - 5 μg / mL mRNA). After allowing some time for cell uptake and antigen expression, human T cells (HemaCare) can be added, and the cell culture medium can be sampled at various time points for INF-γ by Elisa (R&D Systems, DIF50C). Alternatively, the cells can be analyzed by flow cytometry for the CD8+ marker or intracellular INFγ production (PE anti-human IFN-γ antibody, Biolegend).

[0230] In one embodiment, the LNP can be administered to a subject at a dose of 0.01 - 5 mg / kg mRNA by any of the administration routes summarized above. According to some embodiments, a proportion of the LNP is taken up by DC cells, while most accumulates in the liver and spleen. The DC cells can express antigen peptides, process them for MHC I presentation, and migrate to lymph nodes to present them to naive T cells, which induces the education of memory T cells against the antigen.

[0231] In one embodiment, the LNP modified with a targeting ligand such as anti-DEC205-PEG-DSPE can be administered to a subject at a dose of 0.01 - 5 mg / kg mRNA. According to some embodiments, a higher proportion of the LNP can be taken up by DC cells, increasing the production of antigen peptides compared to non-targeted LNP and enabling more effective vaccination against pathogens. Additional targeting ligands for dendritic cells include, but are not limited to, CLEC9A, CLEC4A, XCR1, CD141, and HLD-DR. For example, the evaluation of the CD8+ reactivity against antigens generated in vivo can be achieved by measuring the concentration of INFγ in plasma by species-specific IFN-gamma Quantikine ELISA Kits (R&D Systems).

[0232] In some embodiments, the LNP composition provides desired pharmacokinetic properties such as an extended plasma half-life and encapsulation of mRNA stability. The plasma half-life can be measured as the percentage of the injected volume (ID) remaining in the blood 6 or 24 hours after intravenous injection into immunocompetent mice. The stability of mRNA encapsulation over 24 hours in plasma can be determined by the change in the ratio of mRNA to lipid (mRNA / L ratio) after intravenous administration to mice. In some embodiments, the percentage of encapsulated mRNA remaining in the blood is greater than 20% of the injected dose at 6 hours, preferably greater than 30%, and most preferably greater than 40%. The percentage retained in the blood after 24 hours is preferably greater than 10%, more preferably greater than 20% of the injected dose.

[0233] Disclosed herein are methods for preventing mycobacterial infections such as Mycobacterium tuberculosis, or Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Additional mycobacteria and Gram-positive bacteria include, but are not limited to, Mycobacterium avium complex, Mycobacterium leprae, Mycobacterium gordonae, Mycobacterium abscessus, Mycobacterium abscessus, Mycobacterium mucogenicum, streptococci, vancomycin-resistant enterococci (VRE), Staphylococcus pneumoniae, Enterococcus faecium, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes (group A streptococcus), viridans group streptococci, Listeria monocytogenes, Nocardia, and Corynebacterium.

[0234] Administration of a vaccine to induce a second immune response can result in MHC class II-presented epitopes that can induce a CD4+ helper T cell response against cells expressing an antigen that induces an MHC-presented epitope. Alternatively, or in addition, administration of a vaccine to induce a second immune response can result in MHC class I-presented epitopes that can induce a CD8+ T cell response against cells expressing an antigen that induces an MHC-presented epitope. Further, administration of a vaccine to induce a second immune response can result in one or more neo-epitopes (including known neoepitopes) and one or more epitopes that are not contained in cancer-specific somatic mutations but are expressed by cancer cells and preferably induce an immune response against cancer cells, preferably a cancer-specific immune response. In one embodiment, administration of a vaccine to induce a second immune response results in a neo-epitope that is an MHC class II-presented epitope and / or can induce a CD4+ helper T cell response against cells expressing an antigen that induces an MHC-presented epitope, and an epitope that is an MHC class I-presented epitope and / or does not contain cancer-specific somatic mutations that can induce a CD8+ T cell response against cells expressing an antigen that induces an MHC-presented epitope. In one embodiment, the epitope does not contain cancer-specific somatic mutations.

[0235] "Cellular immune response", "cellular response", "cellular response to an antigen", or synonyms thereof, means including a cellular response directed at cells characterized by the presentation of an antigen having MHC class I or class II. The cellular response relates to cells called T cells or T-lymphocytes that act as either "helper cells" or "killer cells". Helper T cells (also referred to as CD4+ T cells) play a central role by regulating the immune response, and killer cells (also referred to as cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill abnormal cells such as cancer cells and prevent the production of more abnormal cells. In a preferred embodiment, the present disclosure requires stimulation of an anti-tuberculosis CTL response against Mycobacterium that expresses one or more expressed antigens and preferably presents the expressed antigen having MHC class I.

[0236] The "antigen" according to the present disclosure includes any substance that induces an immune response. In particular, the "antigen" relates to any substance that specifically reacts with an antibody or a T-lymphocyte (T cell), preferably a peptide or a protein. As used herein, the term "antigen" includes any molecule that contains at least one epitope. Preferably, the antigen in the context of the present disclosure is optionally, after processing, preferably a molecule that induces an immune reaction specific to the antigen (including cells expressing the antigen). According to the present disclosure, any suitable antigen that is a candidate for an immune reaction (preferably a cellular immune reaction) may be used. In the context of the embodiments of the present disclosure, the antigen is preferably presented by cells, preferably antigen-presenting cells including abnormal cells, particularly cancer cells, and an immune reaction against the antigen occurs in the context of MHC molecules. The antigen is preferably a product corresponding to or derived from a native antigen. Such native antigens may include tumor antigens.

[0237] As used herein, an "antigenic peptide" relates to an antigen or a portion or fragment of an antigen that can stimulate an immune response, preferably a cellular response, against an antigen characterized by the expression of the antigen, preferably the presentation of an antigen by abnormal cells, particularly cancer cells. Preferably, the antigenic peptide can stimulate a cellular response against cells characterized by the presentation of an antigen having class I MHC, preferably can stimulate antigen-responsive cytotoxic T lymphocytes (CTLs). Preferably, the antigenic peptide according to the present disclosure is a MHC class I and / or class II presenting peptide, or can be processed to generate a MHC class I and / or class II presenting peptide. Preferably, the antigenic peptide contains an amino acid sequence substantially corresponding to the amino acid sequence of a fragment of the antigen. Preferably, the fragment of the antigen is a MHC class I and / or class II presenting peptide. Preferably, the antigenic peptide according to the present disclosure contains an amino acid sequence substantially corresponding to the amino acid sequence of the fragment, and is processed to generate the fragment, i.e., a MHC class I and / or class II presenting peptide derived from the antigen. When a peptide is presented directly, i.e., without processing, particularly without cleavage, it has a length suitable for binding to MHC molecules, particularly class I MHC molecules, preferably a length of 7 to 20 amino acids, more preferably a length of 7 to 12 amino acids, still more preferably a length of 8 to 11 amino acids, particularly a length of 9 or 10 amino acids.

[0238] The main types of professional antigen-presenting cells are dendritic cells, which have the broadest range of antigen presentation, and probably the most important antigen-presenting cells, macrophages, B cells, and certain activated epithelial cells. Dendritic cells (DCs) are a population of leukocytes that present antigens captured in peripheral tissues to T cells via the MHC class II and I antigen presentation pathways. Dendritic cells are powerful inducers of immune responses, and it is well known that the activation of these cells is an important step for the induction of antitumor immunity. Dendritic cells can be classified, for convenience, into "immature" cells and "mature" cells, and can be used as a simple way to distinguish two well-characterized phenotypes.

[0239] However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as antigen-presenting cells with a high capacity for antigen uptake and processing, which correlates with high expression of Fcγ receptors and mannose receptors. The mature phenotype is typically characterized by low expression of these markers, but high expression of cell surface molecules involved in T cell activation, such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB). Dendritic cell maturation is a state of dendritic cell activation in which presentation by immature dendritic cells leads to tolerance, whereas the antigen-presenting dendritic cells lead to T cell priming. Dendritic cell maturation is triggered mainly by innate receptors (such as bacterial DNA, viral RNA, endotoxin), inflammatory cytokines (TNF, IL-1, IFN), ligation of CD40 on the dendritic cell surface by CD40L, and biomolecules with microbial features released from cells undergoing stressed cell death. Dendritic cells can be induced in vitro by culturing bone marrow cells with cytokines such as granulocyte macrophage colony-stimulating factor (GM CSF) and tumor necrosis factor alpha. Non-professional antigen-presenting cells do not constitutively express MHC class II proteins required for interaction with naive T cells, and these are expressed only upon stimulation of non-professional antigen-presenting cells by certain cytokines such as IFNγ. An "antigen-presenting cell" can be loaded with MHC class I-presented peptides by transducing the cell with a nucleic acid encoding a peptide or polypeptide containing the presented peptide, preferably mRNA, such as a nucleic acid encoding an antigen.

[0240] In some embodiments, a pharmaceutical composition comprising a gene delivery vehicle that targets dendritic cells or other antigen-presenting cells can be administered to a patient to effect transfection that occurs in vivo. As used herein, "nucleic acid" is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, most preferably RNA transcribed in vitro (IVT RNA) or synthetic RNA. Nucleic acids include genomic DNA, cDNA, mRNA, and molecules produced by recombinant techniques and chemically synthesized molecules according to the present disclosure. According to the present disclosure, nucleic acids can exist as single-stranded or double-stranded linear or covalently closed circular molecules. Nucleic acids can be isolated according to the present disclosure. The term "isolated nucleic acid" means, according to the present disclosure, that the nucleic acid has been (i) amplified in vitro, for example via polymerase chain reaction (PCR), (ii) produced by recombinant techniques by cloning, (iii) purified by cleavage and separation, for example by gel electrophoresis, or (iv) synthesized, for example by chemical synthesis. Nucleic acids can be used, in particular, for introduction into cells, i.e., transfection of cells, in the form of RNA prepared by in vitro transcription from a DNA template. Moreover, the RNA can be modified prior to application by sequence stabilization, capping, and polyadenylation.

[0241] As used herein, the term "RNA" relates to molecules that contain ribonucleotide residues and are preferably composed entirely or substantially of ribonucleotide residues. "Ribonucleotide" relates to a nucleotide having a hydroxyl group at the 2'-position of the B-D-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA (such as partially or fully purified RNA), substantially pure RNA, synthetic RNA, and RNA produced by recombinant techniques (such as modified RNA that is different from natural RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides). Such modifications can include, for example, the addition of non-nucleotide material at the ends or within the RNA, such as at one or more nucleotides of the RNA. Nucleotides in the RNA molecule can also include non-standard nucleotides such as unnatural nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be referred to as analogs or analogs of natural RNA.

[0242] As used herein, the term "RNA" includes "mRNA" and preferably relates to "mRNA". The term "mRNA" means "messenger RNA" and relates to a "transcript" generated using a DNA template and encoding a peptide or polypeptide. Typically, mRNA includes a 5'-UTR, a protein coding region, and a 3'-UTR. mRNA has only a limited half-life in cells and in vitro. In the context of the present disclosure, mRNA can be generated by in vitro transcription from a DNA template. The term "modification" in the context of RNA as used in the present disclosure includes any modification of the RNA that does not naturally occur in said RNA. In one embodiment of the present disclosure, the RNA used according to the present disclosure does not have an uncapped 5'-triphosphate. Removal of such uncapped 5'-triphosphate can be achieved by treating the RNA with a phosphatase. The RNA according to the present disclosure can have modified ribonucleotides for improving its stability and / or reducing its cytotoxicity. For example, in one embodiment, in the RNA used according to the present disclosure, in the case of cytidine, 5-methylcytidine is partially or completely replaced, preferably completely replaced. Alternatively, or in addition, in one embodiment, in the RNA used according to the present disclosure, in the case of uridine, pseudouridine is partially or completely replaced, preferably completely replaced.

[0243] In one embodiment, the term "capping" relates to generating an RNA having a 5-cap or 5'-cap analog. The term "5-cap" refers to the cap structure present at the 5'-end of an mRNA molecule and generally consists of a guanosine nucleotide that is connected to the mRNA via a 5'-5 triphosphate bond that is not normally found. In one embodiment, this guanosine is methylated at the 7-position. The term "conventional 5'-cap" refers to a native RNA 5'-cap, preferably a 7-methylguanosine cap (m'G). As used herein, the term "5'-cap" includes 5'-cap analogs that are similar to the RNA cap structure and are preferably modified to have the ability to stabilize RNA and / or enhance the translation of the RNA (when bound to the RNA) in vivo and / or intracellularly.

[0244] According to the present disclosure, the stability and translation efficiency of RNA may be modified as needed. For example, RNA can be stabilized and its translation increased by one or more modifications having a stabilizing effect on the RNA and / or an improved translation efficiency. Such modifications are described, for example, in PCT / EP2006 / 009448, which is hereby incorporated by reference in its entirety. To increase the expression of RNA used in accordance with the present disclosure, preferably, without modifying the sequence of the expressed peptide or protein, the coding region, i.e., the sequence encoding the expressed peptide or protein, is modified to increase the GC content to improve mRNA stability and perform codon optimization, thus enhancing translation in the cell.

[0245] Aspect of the present disclosure relate to a method of preventing a bacterial or viral infection, the method comprising administering to a subject in need thereof an effective amount of a composition produced herein to induce an immune response.

[0246] Aspects of the present disclosure provide a method of vaccinating a subject, comprising administering to the subject a single dose of a composition described herein that comprises a nucleic acid (e.g., mRNA) encoding an effective amount of a polypeptide for vaccination of the subject. In some embodiments, the nucleic acid is formulated in cationic lipid nanoparticles. In some embodiments, the lipid nanoparticle composition is administered by a single injection method.

[0247] In some embodiments, the bacterial infection is a Mycobacterium tuberculosis infection.

[0248] In some embodiments, the viral infection is a coronavirus. In some embodiments, the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2.

[0249] In some embodiments, the viral infection is HIV / AIDS.

[0250] In some embodiments, the lipid nanoparticles are administered parenterally.

[0251] Generally, administration to patients by intradermal injection is possible. However, the injection may also be made into lymph nodes via the muscle (Maloy et al. (2001), Proc Natl Acad Sci USA 98:3299-3033). The resulting cells provide the target complex and are recognized by autologous cytotoxic T lymphocytes, which then proliferate.

[0252] In some embodiments, the composition is administered by inhalation. In some embodiments, the composition is formulated as a nasal spray and / or aerosol.

[0253] The actual dosage level of the active agent in the pharmaceutical compositions disclosed herein may vary to obtain an amount of the active agent that is effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration, without undue toxicity to the patient.

[0254] As used herein in the context of administration, "parenteral" means a route of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, epidural and intrasternal injection and infusion.

[0255] As used herein, the phrases "parenteral administration" and "administered parenterally" typically refer to a route of administration other than enteral (i.e., via the gastrointestinal tract) and topical administration by injection or infusion, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, inhalation, subcapsular, subdural, respiratory mucosa, intraspinal, epidural and intrasternal injection and infusion. Intravenous injection and infusion are often (but not exclusively) used for the administration of liposomal drugs.

[0256] The dosing regimen can be adjusted so as to obtain the optimal desired response (e.g., a therapeutic response). For example, more than one dose may be administered over time, or the dose may be proportionally decreased or increased as indicated by the requirements of the therapeutic situation.

[0257] In some embodiments, the dose comprises 0.01 - 5 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 - 5 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 - 3 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 - 3 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 - 1 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 - 1 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 - 0.5 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 - 0.5 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 - 1 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 - 0.1 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 - 0.05 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 - 0.1 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 - 0.05 mg / kg of mRNA.

[0258] The dosage of the compound and / or its pharmaceutically acceptable salt or the LNP comprising the compound and / or its pharmaceutically acceptable salt can vary within a wide range and, in each particular case, should necessarily be adjusted according to the individual conditions and the pathogen to be controlled.

[0259] In some embodiments, an ionizable cationic lipid (ICL) is provided. The cationic lipid is engineered to improve stability against oxidative degradation during storage while retaining high transfection activity or efficacy intracellularly. Aspects of the present disclosure are based in part on the discovery that an LNP composition comprising mRNA and a particular ionizable cationic lipid (ICL) enhanced mRNA expression in human dendritic cells.

[0260] In some embodiments, the LNP composition comprises a targeting ligand to a cell surface receptor for targeting lipid nanoparticles in a highly specific manner, including to dendritic cells. In some embodiments, the LNP composition comprises a phosphatidyl-L-serine compound as a targeting ligand, such as dipalmitoylphosphatidyl-L-serine (DPPS), or distearoylphosphatidyl-L-serine (DSPS). In some embodiments, the LNP composition comprises a phosphatidyl-L-serine compound and an anionic phospholipid as targeting ligands. In some embodiments, the LNP composition comprises a phosphatidylglycerol-containing compound as a targeting ligand, such as distearoylphosphatidylglycerol (DSPG) or dipalmitoylphosphatidylglycerol (DPPG), to enhance expression in human dendritic cells. In some embodiments, the LNP composition comprises both a phosphatidyl-L-serine compound as a targeting ligand and distearoylphosphatidylcholine (DSPC) as a second phospholipid. In some embodiments, the LNP composition does not comprise dipalmitoylphosphatidylcholine (DPPC) and comprises both a phosphatidyl-L-serine compound as a targeting ligand and distearoylphosphatidylcholine (DSPC) as a second phospholipid.

[0261] Aspects of the present disclosure are based in part on the discovery that the selection of certain cationic ionizable lipids can enhance the transfection of human dendritic cells. For example, the KC3 cationic lipid was more active than either KC2 or the diacyl ionizable lipid (UO series) in transfecting human dendritic cells with the LNP composition. Among the LNP compositions containing the KC3 ionizable cationic lipid, those with an ionizable cationic lipid having a monounsaturated alkyl chain were, unexpectedly, both more active and more stable against oxidative degradation than those containing one having a dilinoleyl alkyl chain. In addition, a decrease in transfection in the activity of human dendritic cells in LNP compositions containing monounsaturated lipids, such as the diacyl ionizable lipid (UO series), was observed.

[0262] In some embodiments, certain salts of phosphatidylserine-targeted lipids are provided. For example, in some embodiments, the phosphatidylserine-targeted lipid can be provided as an ammonium salt of DPPS having improved biophysical properties and higher solubility in the presence of ethanol, which is a preferred solvent for the preparation of LNPs. DSPS or the sodium salt of DSPS is insoluble in ethanol and requires both the presence of methanol and heating to enable its formation, and the same was true for the ammonium salt of DSPS. It is envisioned that other ammonium salts of phosphatidylserine will provide the same advantages in terms of solubility and biophysical properties.

[0263] In some embodiments, an ionizable cationic lipid composition useful for the preparation of liposome nanoparticle (LNP) compositions is provided. In some embodiments, each polyene hydrocarbon chain, each covalently bonded to a head group containing a dialkylaminoalkyl group, contains a single unsaturated alkenyl double bond within (a) a pair of linear C 16 or C 18 A liposome composition comprising an ionizable cationic lipid having a hydrocarbon chain is provided. In some embodiments, the head group of the ionizable cationic lipid has a dialkylamino group having a pKa of about 6.3 - 7.5. In some embodiments, the head group of the ionizable cationic lipid contains a heterocyclyl or alkyl moiety covalently bonded to the dialkylamino group. In some embodiments, the head group of the ionizable cationic lipid further optionally contains a phosphate group. In some embodiments, each lipid tail of the ionizable cationic lipid compound is identical, and each lipid tail has a total of one olefin with a full length of 15, 16, 17, or 18 carbons.

[0264] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I).

[0265] [Chemical formula] [In the formula, R1 is [Chemical formula] where a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each independently (C1-C4) alkyl optionally substituted with hydroxyl, and n is an integer equal to 2, 3 or 4]

[0266] In some embodiments, an ionizable cationic lipid composition is provided. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I), wherein a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each methyl, and n is an integer equal to 3.

[0267] In some embodiments, an ionizable cationic lipid composition is provided. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of formula (I-A).

[0268] [Chemical formula] [In the formula, R1 is [Chemical formula] where a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each methyl, and n is an integer equal to 3]

[0269] In some embodiments, the LNP composition comprises a pair of identical lipid hydrocarbon tails having a total of 15, 16, 17, or 18 carbons, and an ionizable cationic lipid comprising a single olefin group or a pair of olefin groups. In some embodiments, the LNP composition [Chemical formula] comprises an ionizable cationic lipid selected from the group consisting of

[0270] In some embodiments, the ionizable cationic lipid is KC3-PA. In some embodiments, the ionizable cationic lipid is KC3-C15(C8:1). In some embodiments, the ionizable cationic lipid is KC3-C16(C8:1). In some embodiments, the ionizable cationic lipid is KC3-C17(C8:1). In some embodiments, the ionizable cationic lipid is KC3-C18(C8:1).

[0271] In some embodiments, the ionizable cationic lipid is KC3-15. In some embodiments, the ionizable cationic lipid is KC3-16. In some embodiments, the ionizable cationic lipid is KC3-17. In some embodiments, the ionizable cationic lipid is KC3-18.

[0272] The salt form of the targeted lipid may affect its solubility in the alcohol-containing solvent used in the preparation of lipid nanoparticles. In some embodiments, an ionizable cationic lipid composition is provided. In some embodiments, the lipid nanoparticle (LNP) composition comprises a nucleic acid, an ionizable lipid disclosed herein, a sterol, and one or more phospholipids including phosphatidylserine (PS) lipids, and optionally further comprises a complex lipid. In some embodiments, the lipid nanoparticle (LNP) composition comprises an mRNA nucleic acid, an ionizable lipid disclosed herein, cholesterol, one or more phospholipids selected from the group consisting of DSPC, DPPC, and DOPC, and a PS lipid selected from the group consisting of DPPS, DSPS, and DOPS, and optionally further comprises a complex lipid comprising PEG.

[0273]

Chemical formula

[0274]

Chemical formula

[0275]

Chemical formula

[0276]

Chemical formula

[0277]

Chemical formula

[0278] [Chem.] In some embodiments, the ionizable lipid is AKG-UO-5.

[0279] [Chem.]

[0280] In some embodiments, the ionizable lipid is AKG-UO-6, AKG-UO-7, AKG-UO-7, AKG-UO-8, AKG-UO-9, or AKG-UO-10.

[0281] [Chem.]

[0282] In some embodiments, the LNP composition can include an anionic phospholipid. In some embodiments, the LNP composition is prepared using a sodium or ammonium salt of the anionic phospholipid. In some embodiments, the anionic phospholipid salt is a compound of formula (V-A-1) having a chemical structure.

[0283] [Chem.] [wherein, X + is an ammonium (NH4 + ) or sodium (Na + ) cation, and a is 14, 15, or 16]

[0284] In some embodiments, the anionic phospholipid salt is [Chem.] selected from the group consisting of. In some embodiments, the anionic lipid salt is the DSPS (L-isomer) sodium salt. In some embodiments, the anionic lipid salt is the DSPS (L-isomer) ammonium salt. In some embodiments, the anionic lipid salt is the DPPS (L-isomer) sodium salt. In some embodiments, the anionic lipid salt is the DPPS (L-isomer) ammonium salt. In some embodiments, the targeted lipid is the sodium or ammonium salt of dipalmitoylphosphatidyl-L-serine (DPPS) or distearoylphosphatidyl-L-serine (DSPS). In some embodiments, the targeted lipid is the sodium or ammonium salt of dipalmitoylphosphatidyl-L-serine (DPPS) or distearoylphosphatidyl-L-serine (DSPS).

[0285] In some embodiments, the LNP composition

Chemical formula

[0286] In some embodiments, the salt form of phosphatidylserine is highly soluble in ethanol. In some embodiments, it is soluble at greater than 0.5 mg / ml, greater than 1 mg / ml, greater than 5 mg / ml, greater than 10 mg / ml, or greater than 20 mg / ml. In some embodiments, the salt is an ammonium salt. In some embodiments, the salt is the ammonium atom itself, an alkylammonium, dialkylammonium, or trialkylammonium salt. In some embodiments, the amine is selected from ammonia, dimethylamine, diethylamine, triethylamine, trimethylamine, 2-(dimethylamino)ethanol, diethanolamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methyl-glucamine, imidazole, histidine, lysine, arginine, 4-(2-hydroxyethyl)-morpholine, piperazine, 1-(2-hydroxyethyl)-pyrrolidine, triethanolamine, and tromethamine (tris(hydroxymethyl)aminomethane). In some embodiments, this targeted lipid is the ammonium salt of DPPS.

[0287] Anionic phospholipids isolated from phosphatidyl-L-serine were also considered as targeting lipids for LNPs. These include phosphatidylglycerol (PG), phosphatidic acid (PA), N-glutaryl-phosphatidylethanolamine (N-Glu-PE), N-succinyl-phosphatidylethanolamine (N-Suc-PE), and cardiolipin. In some embodiments, the LNP comprises an anionic phospholipid isolated from phosphatidyl-L-serine that is useful as a targeting lipid for the LNP. In some embodiments, the LNP comprises an anionic phospholipid selected from the group consisting of phosphatidylglycerol (PG), phosphatidic acid (PA), N-glutaryl-phosphatidylethanolamine (N-Glu-PE), N-succinyl-phosphatidylethanolamine (N-Suc-PE), and cardiolipin. Distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), N-succinyl-distearoylphosphatidylethanolamine (N-Suc-DSPE), N-glutaryl-distearoylphosphatidylethanolamine (N-glu-DSPE), distearoylphosphatidic acid (DSPA), and cardiolipin are also provided as anionic phospholipids.

[0288] In some embodiments, a lipid nanoparticle (LNP) composition is provided that comprises an ionizable cationic lipid composition. In some embodiments, a lipid nanoparticle (LNP) composition is provided that comprises an ionizable cationic lipid. In some embodiments, the LNP composition comprises an mRNA nucleic acid. In some embodiments, the lipid nanoparticle (LNP) composition further comprises PS lipid in an amount of 2.5 to 10 mol% of the total lipids in the LNP composition. In some embodiments, the lipid nanoparticle (LNP) composition further comprises a PS lipid selected from the group consisting of DSPS (L-isomer) and DPPS. In some embodiments, the lipid nanoparticle (LNP) composition comprises a total amount of a complex lipid in an amount of 0.5 to 2.0 mol% of the total lipid content of the LNP composition. In some embodiments, the lipid nanoparticle (LNP) composition comprises a total amount of a complex lipid in an amount less than 2 mol% of the total lipid content of the LNP composition, and the complex lipid is PEG-DMG.

[0289] In some embodiments, the lipid nanoparticle (LNP) composition comprises one or more phospholipids including a nucleic acid, an ionizable lipid disclosed herein, a sterol, and a phosphatidylserine (PS) lipid, and optionally further comprises a complex lipid. In some embodiments, the lipid nanoparticle (LNP) composition comprises an mRNA nucleic acid, an ionizable lipid disclosed herein, cholesterol, one or more phospholipids selected from the group consisting of SM, DSPC, HSPC, DPPC, and DOPC, and a PS lipid selected from the group consisting of DPPS and DSPS, and optionally further comprises a complex lipid comprising PEG. In some embodiments, the nucleic acid lipid nanoparticle (LNP) composition comprises a nucleic acid, an ionizable cationic lipid in an amount of 40 to 65 mol% of the total lipid content of the LNP composition, a sterol in an amount of 25 to 45 mol% of the total lipid content of the LNP composition, and one or more phospholipids in an amount of 5 to 25 mol% of the total amount of phospholipids in the LNP composition, the one or more phospholipids comprising phosphatidylserine (PS) in an amount of 2.5 to 10 mol% of the total lipid content of the LNP composition, and optionally further comprises a complex lipid in an amount of 0.5 to 2.5 mol% of the total lipid content of the LNP composition.

[0290] In some embodiments, the LNP composition further comprises an anionic lipid selected from the group consisting of DSPS (L-isomer), DPPS (L-isomer), DMPS (L-isomer), DOPS (L-isomer), and DSPS (D-isomer).

[0291] Aspects of the present disclosure relate to lipid nanoparticle (LNP) compositions comprising an ionizable lipid having a chemical structure.

[0292] [Chemical formula] [wherein, R1 is [Chemical formula] where a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each independently (C1-C4) alkyl optionally substituted with hydroxyl, and n is an integer equal to 2, 3 or 4]

[0293] In some embodiments, n is 2 or 3. In some embodiments, a is 0. In some embodiments, b is 1, 2 or 3. In some embodiments, a is 1. In some embodiments, b is 1, 2 or 3. In some embodiments, R2 and R3 are each methyl.

[0294] In some embodiments, R1 is [Chemical formula] where a is 0 or 1, b is 1 or 3, R2 and R3 are each methyl, and n is 2 or 3. In some embodiments, n is 3.

[0295] In some embodiments, the composition includes a nucleic acid, an ionizable lipid disclosed herein, a sterol, one or more phospholipids including phosphatidylserine (PS) lipids, and optionally a complex lipid.

[0296] In some embodiments, the nucleic acid is mRNA.

[0297] In some embodiments, the sterol is cholesterol.

[0298] In some embodiments, the one or more phospholipids consist of one or more phospholipids selected from the group consisting of SM, DSPC, HSPC, DPPC, and DOPC, and a PS lipid selected from the group consisting of DPPS and DSPS.

[0299] In some embodiments, the one or more phospholipids consist of DSPC and one or more PS lipids selected from the group consisting of (L-serine)DPPS and (L-serine)DSPS.

[0300] In some embodiments, the composition includes PS lipids in a total amount of 2.5 to 10 mol% of the total lipids in the composition.

[0301] In some embodiments, the complex lipid includes PEG.

[0302] Aspects of the present disclosure relate to a nucleic acid lipid nanoparticle (LNP) composition including a nucleic acid, an ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition, a sterol in a total amount of 25 to 45 mol% of the total lipid content of the LNP composition, and one or more phospholipids in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition, wherein the one or more phospholipids include phosphatidylserine (PS) in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition, and optionally a complex lipid in a total amount of 0.5 to 2.5 mol% of the total lipid content of the LNP composition.

[0303] In some embodiments, the nucleic acid is mRNA.

[0304] In some embodiments, the sterol is cholesterol.

[0305] In some embodiments, the one or more phospholipids consist of DSPC and L-serine PS.

[0306] In some embodiments, the composition comprises a total amount of PS that is 2.5 to 7.5 mol% of the total lipids in the composition.

[0307] In some embodiments, the composite lipid comprises PEG. In some embodiments, the composite lipid is PEG-DMG.

[0308] In some embodiments, the LNP comprises a total amount of composite lipid that is 0.5 to 2.0 mol% of the total lipid content of the LNP composition. In some embodiments, the total amount of composite lipid is less than 2 mol% of the total lipid content of the LNP composition.

[0309] In some embodiments, the nucleic acid is mRNA, the ionizable cationic lipid is in an amount that is 45 to 55 mol% of the total lipid content of the LNP composition, the sterol is cholesterol in an amount that is 35 to 45 mol% of the total lipid content of the LNP composition, the total amount of phospholipids is 7 to 15 mol% of the total lipid content of the LNP composition, the one or more phospholipids consist of DSPC, the PS lipid is one or more lipids selected from the group consisting of the L-serine forms of DPPS and DSPS, and the total amount of PS lipid is about 5 mol% of the total lipid content of the LNP composition.

[0310] In some embodiments, the composition comprises a total amount of PS lipid selected from 1.25 mol%, 2.5 mol%, 5 mol%, 7.5 mol%, and 10 mol% of the total lipid content of the LNP composition.

[0311] Aspects of the present disclosure relate to a nucleic acid lipid nanoparticle (LNP) composition comprising a nucleic acid that is mRNA, an ionizable cationic lipid in a total amount of 45 to 55 mol% of the total lipid content of the LNP composition, a sterol that is cholesterol in a total amount of 35 to 45 mol% of the total lipid content of the LNP composition, one or more phospholipids in a total amount of 10 mol% of the total lipid content of the LNP composition, the one or more phospholipids comprising phosphatidylserine (PS) in a total amount of 3 to 9 mol% of the total lipid content of the LNP composition, and a complex lipid in a total amount of 0.5 to 2.0 mol% of the total lipid content of the LNP composition.

[0312] In some embodiments, the one or more phospholipids are selected from the group consisting of DSPS (L-isomer), DPPS (L-isomer), DMPS (L-isomer), DOPS (L-isomer), and DSPS (D-isomer).

[0313] In some embodiments, the complex lipid is PEG-DMG, and the PS lipid is selected from the group consisting of DSPS (L-isomer) and DPPS.

[0314] In some embodiments, the ionizable cationic lipid is one or more compounds selected from the group consisting of KC3-OA, KC3-PA, KC3-C17(8:1), and KC3-C15(C8:1). In some embodiments, the ionizable cationic lipid is KC3-PA. In some embodiments, the ionizable cationic lipid is KC3-OA. In some embodiments, the ionizable cationic lipid is KC3-C17(C8:1).

[0315] In some embodiments, the LNP comprises nucleic acid, an ionizable cationic lipid in a total amount of 50 mol% of the total lipid content of the LNP composition, cholesterol in a total amount of 38.5 mol% of the total lipid content of the LNP composition, one or more phospholipids in a total amount of 7-15 mol% of the total lipid content of the LNP composition, wherein the one or more phospholipids comprise phosphatidylserine (PS) lipid in a total amount of 3-9 mol% of the total lipid content of the LNP composition, and a PEG-containing lipid in a total amount of 0.5-2.0 mol% of the total lipid content of the LNP composition.

[0316] In some embodiments, the phospholipid consists of one or more phospholipids selected from the group consisting of DSPC, DOPC, DPPC, HSPC, and SM.

[0317] In some embodiments, the PS lipid is one or more L-serine lipids selected from the group consisting of DPPS and DSPS.

[0318] In some embodiments, the one or more phospholipids comprise at least two (L-serine) PS lipids having incompatible acyl chain lengths.

[0319] In some embodiments, the phospholipids are DSPC and DPPS. In some embodiments, DSPC and DPPS are each present in the LNP in a total amount of 5 mol% relative to the total lipid content of the LNP composition.

[0320] Aspects of the present disclosure relate to a nucleic acid lipid nanoparticle (LNP) composition comprising nucleic acid, an ionizable cationic lipid KC3-PA or KC3-OA, and an (L-serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition.

[0321] In some embodiments, the nucleic acid is mRNA, the PS lipid is (L-serine) DSPS, (L-serine) DPPS, or a mixture thereof, and the LNP composition further comprises cholesterol and a second phospholipid selected from the group consisting of DSPC, DPPC, HSPC, and SM.

[0322] In some embodiments, the LNP composition further comprises 0.5 - 2.0 mol% of PEG-DMG or PEG-DSG relative to the total lipid content in the LNP composition.

[0323] In some embodiments, the ionizable cationic lipid is KC3-PA. In some embodiments, the ionizable cationic lipid is KC3-OA.

[0324] Aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) compositions comprising nucleic acid, KC3-C17(C8:1) ionizable cationic lipid, and (L-serine)PS lipid in a total amount of 2.5 - 10 mol% of the total lipid content of the LNP composition.

[0325] In some embodiments, the LNP composition has an N / P ratio of 4 - 7. In some embodiments, the composition has an N / P ratio of 5 - 6. In some embodiments, the composition has an N / P ratio of 5.3.

[0326] Aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) compositions comprising nucleic acid, the ionizable cationic lipid KC3-PA, and (L-serine)PS lipid in a total amount of 2.5 - 10 mol% of the total lipid content of the LNP composition.

[0327] In some embodiments, the nucleic acid is mRNA, the PS lipid is (L-serine)DSPS, (L-serine)DPPS, or a mixture thereof, and the LNP composition further comprises cholesterol and a second phospholipid selected from the group consisting of DSPC, DOPC, DPPC, HSPC, and SM.

[0328] In some embodiments, the LNP composition further comprises 0.5 - 2.0 mol% of PEG-DMG or PEG-DSG relative to the total lipid content in the LNP composition.

[0329] Aspects of the present disclosure relate to a nucleic acid lipid nanoparticle (LNP) composition comprising a nucleic acid, an ionizable cationic lipid selected from KC3-C17(C8:1), and an (L-serine)PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition.

[0330] In some embodiments, the N / P ratio is from 4 to 7. In some embodiments, the N / P ratio is from 5 to 6. In some embodiments, the N / P ratio is 3. In some embodiments, the N / P ratio is 7.

[0331] In some embodiments, the nucleic acid is an mRNA encoding a SARS-CoV-2 spike protein.

[0332] Aspects of the present disclosure relate to an mRNA nucleic acid with an N / P ratio of 4 to 7, a KC3-PA ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition, cholesterol in a total amount of 25 to 40 mol% of the total lipid content of the LNP composition, an (L-serine)PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition, DSPC phospholipid in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition, and PEG-DMG in a total amount of 0 to 2.5 mol% of the total lipid content of the LNP composition, a nucleic acid lipid nanoparticle (LNP) vaccine composition.

[0333] Aspects of the present disclosure relate to an mRNA nucleic acid with an N / P ratio of 3 to 8, a KC3-C17(C8:1) ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition, cholesterol in a total amount of 25 to 40 mol% of the total lipid content of the LNP composition, an (L-serine)PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition, DSPC phospholipid in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition, and PEG-DMG in a total amount of 0 to 2.5 mol% of the total lipid content of the LNP composition, a nucleic acid lipid nanoparticle (LNP) vaccine composition.

[0334] Aspects of the present disclosure relate to a nucleic acid lipid nanoparticle (LNP) vaccine composition comprising an mRNA nucleic acid having an N / P ratio of 4 to 7, a total amount of KC3-C15(C8:1) ionizable cationic lipid of 40 to 65 mol% of the total lipid content of the LNP composition, a total amount of cholesterol of 25 to 40 mol% of the total lipid content of the LNP composition, a total amount of (L-serine)PS lipid of 2.5 to 10 mol% of the total lipid content of the LNP composition, a total amount of DSPC phospholipid of 5 to 25 mol% of the total lipid content of the LNP composition, and a total amount of PEG-DMG of 0 to 2.5 mol% of the total lipid content of the LNP composition.

[0335] Aspects of the present disclosure relate to a nucleic acid lipid nanoparticle (LNP) vaccine composition comprising an mRNA nucleic acid having an N / P ratio of 3 to 8, a total amount of KC3-C18 ionizable cationic lipid of 40 to 65 mol% of the total lipid content of the LNP composition, a total amount of cholesterol of 25 to 40 mol% of the total lipid content of the LNP composition, a total amount of (L-serine)PS lipid of 2.5 to 10 mol% of the total lipid content of the LNP composition, a total amount of DSPC phospholipid of 5 to 25 mol% of the total lipid content of the LNP composition, and a total amount of PEG-DMG of 0 to 2.5 mol% of the total lipid content of the LNP composition.

[0336] In some embodiments, the nucleic acid is the mRNA of SEQ ID NO: 2.

[0337] Aspects of the present disclosure relate to the use of (L-serine)PS lipids in combination with the ionizable cationic lipids disclosed herein in LNPs for targeting of the LNPs to dendritic cells. In some embodiments, the LNP contains mRNA. In some embodiments, the LNP further contains cholesterol. In some embodiments, the total amount of (L-serine)PS lipid in the LNP is 2.5 to 10 mol% of the total lipid content of the LNP composition. In some embodiments, the LNP further contains one or more additional phospholipids including DSPC. In some embodiments, the LNP further contains a complex lipid. In some embodiments, the LNP contains an mRNA nucleic acid with an N / P ratio of 3 to 8, a total amount of KC3-PA or KC3-C17(C8:1) ionizable cationic lipid (ICL) of 40 to 65 mol% of the total lipid content of the LNP composition, a total amount of cholesterol of 25 to 40 mol% of the total lipid content of the LNP composition, a total amount of (L-serine)PS lipid of 2.5 to 10 mol% of the total lipid content of the LNP composition, a total amount of DSPC phospholipid of 5 to 25 mol% of the total lipid content of the LNP composition, and a total amount of complex lipid of 0 to 2.5 mol% of the total lipid content of the LNP composition. In some embodiments, the ICL is KC3-PA. In some embodiments, the ICL is KC3-C17(C8:1).

[0338] Some aspects of the present disclosure relate to lipid nanoparticle (LNP) compositions comprising an ionizable lipid having a chemical structure.

[0339] [Chemical formula] [wherein R1 is [Chemical formula] and here, a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each independently methyl, and n is an integer equal to 2 or 3]

[0340] In some embodiments, a is 0. In some embodiments, b is 1. In some embodiments, b is 3.

[0341] In some embodiments, a is 1. In some embodiments, b is 1. In some embodiments, b is 3.

[0342] In some embodiments, n is 2. In some embodiments, n is 3.

[0343] In some embodiments, the composition comprises an anionic lipid selected from the group consisting of phosphatidylglycerol (PG), phosphatidic acid (PA), N-glutaryl-phosphatidylethanolamine (N-Glu-PE), N-succinyl-phosphatidylethanolamine (N-Suc-PE), and cardiolipin. Distearoyl phosphatidylglycerol (DSPG), dipalmitoyl phosphatidylglycerol (DPPG), N-succinyl-distearoyl phosphatidylethanolamine (N-Suc-DSPE), N-glutaryl-distearoyl phosphatidylethanolamine (N-glu-DSPE), distearoyl phosphatidic acid (DSPA), and cardiolipin.

[0344] In some embodiments, the composition comprises an anionic targeted phospholipid other than phosphatidyl-L-serine.

[0345] In some embodiments, the composition comprises an anionic phospholipid selected from the group consisting of DSPG and DPPG.

[0346] In some embodiments, the composition comprises an anionic phospholipid selected from the group consisting of N-Glu-DSPE and N-Suc-DSPE.

[0347] In some embodiments, the composition comprises DSPA anionic phospholipid.

[0348] In some embodiments, the composition comprises cardiolipin anionic phospholipid.

[0349] In some embodiments, the ionized lipid has a chemical structure.

[0350] [Chemical formula]

[0351] In some embodiments, the ionized lipid has a chemical structure. [Chemical formula]

[0352] Aspects of the present disclosure relate to sodium or ammonium salts of compositions of anionic phospholipids of formula (V-A-1) having a chemical structure.

[0353] [Chemical formula] [wherein, X + is an ammonium cation or a sodium (Na + ) cation, and a is 14, 15, or 16]

[0354] In some embodiments, a is 14 or 16. In some embodiments, X + is an ammonium cation (NH4 + ).

[0355] In some embodiments, X + is a sodium cation (Na + ).

[0356] In some embodiments, X is ammonium (NH4 +) is an ammonium cation selected from the group consisting of alkylammonium, dialkylammonium, and trialkylammonium salts. In some embodiments, X is an ammonium cation selected from the group consisting of ammonium, dimethylamine, diethylamine, triethylamine, trimethylamine, 2-(dimethylamino)ethanol, diethanolamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methyl-glucamine, imidazole, histidine, lysine, arginine, 4-(2-hydroxyethyl)-morpholine, piperazine, 1-(2-hydroxyethyl)-pyrrolidine, triethanolamine, and tromethamine (tris(hydroxymethyl)aminomethane).

[0357] In some embodiments, the anionic phospholipid of formula (V-A-1) is the sodium salt of distearoylphosphatidyl-L-serine (DSPS L-isomer). In some embodiments, the anionic phospholipid of formula (V-A-1) is the ammonium salt of distearoylphosphatidyl-L-serine (DSPS L-isomer). In some embodiments, the anionic phospholipid of formula (V-A-1) is the sodium salt of DPPS (L-isomer). In some embodiments, the anionic phospholipid of formula (V-A-1) is the ammonium salt of DPPS (L-isomer). Some embodiments relate to the use of the salt form of the composition in the preparation of liposome nanoparticle (LNP) compositions. In some embodiments, the use comprises combining, during the preparation of the LNP composition, with one or more of the following LNP components: mRNA nucleic acid, ionizable cationic lipid (ICL), cholesterol, (L-serine)PS lipid, one or more phospholipids, and complex lipids. In some embodiments, the use comprises the step of combining an ammonium or salt form of a compound of formula (V-A-1) with one or more of the following LNP components: mRNA nucleic acid, ionizable cationic lipid (ICL), cholesterol, (L-serine)PS lipid, one or more phospholipids, and complex lipids during the preparation of the LNP composition.

[0358] In some embodiments, the LNP is a nucleic acid lipid nanoparticle (LNP) vaccine composition comprising an mRNA nucleic acid with an N / P ratio of 4 to 7, a total amount of Dlin-KC2-DMA ionizable cationic lipid of 40 to 65 mol% of the total lipid content of the LNP composition, a total amount of cholesterol of 25 to 40 mol% of the total lipid content of the LNP composition, a total amount of (L-serine)PS lipid of 2.5 to 10 mol% of the total lipid content of the LNP composition, a total amount of DSPC phospholipid of 5 to 25 mol% of the total lipid content of the LNP composition, and a total amount of PEG-DMG of 0 to 2.5 mol% of the total lipid content of the LNP composition.

[0359] In some embodiments, the nucleic acid is the mRNA of SEQ ID NO: 2.

[0360] In some embodiments, the LNP composition comprises a total amount of ionizable cationic lipid of 46 to 65 mol% of the total lipid content of the LNP composition. In some embodiments, the LNP composition comprises a total amount of PS of about 5 mol% of the total lipids in the composition. In some embodiments, the LNP composition comprises a total amount of complex lipid of about 1.5 mol% of the total lipid content of the LNP composition.

[0361] In some embodiments, the complex lipid is PEG-DMG and the PS lipid is selected from the group consisting of DSPS (L-isomer) and DPPS.

[0362] In some embodiments, the ionizable cationic lipid is one or more compounds selected from the group consisting of KC3-OA, KC3-PA, KC3-C17(C8:1), and KC3-C15(C8:1). In some embodiments, the ionizable cationic lipid is KC3-PA. In some embodiments, the ionizable cationic lipid is KC3-OA. In some embodiments, the ionizable cationic lipid is KC3-C17(C8:1).

[0363] Some embodiments relate to the use of (L-serine)PS lipids in combination with the ionizable cationic lipids disclosed herein in LNPs for the targeting of LNPs to dendritic cells.

[0364] In some embodiments, the LNP contains mRNA. In some embodiments, the LNP further contains cholesterol. In some embodiments, the total amount of (L-serine)PS lipid in the LNP is 2.5 to 10 mol% of the total lipid content of the LNP composition. In some embodiments, the LNP further contains one or more additional phospholipids including DSPC. In some embodiments, the LNP further contains a complex lipid.

[0365] In some embodiments, the LNP contains an mRNA nucleic acid with an N / P ratio of 3 to 8, a total amount of KC3-PA or KC3-C17(C8:1) ionizable cationic lipid (ICL) of 40 to 65 mol% of the total lipid content of the LNP composition, a total amount of cholesterol of 25 to 40 mol% of the total lipid content of the LNP composition, a total amount of (L-serine)PS lipid of 2.5 to 10 mol% of the total lipid content of the LNP composition, a total amount of DSPC phospholipid of 5 to 25 mol% of the total lipid content of the LNP composition, and a total amount of complex lipid of 0 to 2.5 mol% of the total lipid content of the LNP composition.

[0366] In some embodiments, the ICL is KC3-PA. In some embodiments, the ICL is KC3-C17(C8:1).

[0367] In some embodiments, the composition contains an anionic phospholipid selected from the group consisting of DSPG and DPPG in a total amount of 2.5 to 7.5 mol% of the total lipid content of the LNP composition. In some embodiments, the composition contains DSPG anionic phospholipid in a total amount of 2.5 to 7.5 mol% of the total lipid content of the LNP composition. In some embodiments, the composition contains DPPG anionic phospholipid in a total amount of 2.5 to 7.5 mol% of the total lipid content of the LNP composition.

[0368] In some embodiments, the LNP further comprises one or more additional phospholipids including DSPC.

[0369] Aspects of the present disclosure relate to nucleic acid-lipid nanoparticle (LNP) compositions comprising nucleic acids, a total amount of KC3 ionizable cationic lipid of 40 to 65 mol% of the total lipid content of the LNP composition, a total amount of cholesterol of 23.5 to 43.5 mol% of the total lipid content of the LNP composition, a total amount of (L-serine)PS lipid of 2.5 to 10 mol% of the total lipid content of the LNP composition, a total amount of DSPC or HSPC phospholipid of 5 to 25 mol% of the total lipid content of the LNP composition, and a total amount of PEG-containing complex lipid of 0.5 mol% to 2.5 mol% of the total lipid content of the LNP composition.

[0370] In some embodiments, the nucleic acid is mRNA. In some embodiments, the N / P ratio is 3 to 8.

[0371] In some embodiments, the KC3 ionizable cationic lipid is selected from the group consisting of KC3-OA, KC3-PA, KC3-C17(8:1), and KC3-C15(C8:1). In some embodiments, the KC3 ionizable cationic lipid is KC3-OA. In some embodiments, the KC3 ionizable cationic lipid is KC3-PA. In some embodiments, the KC3 ionizable cationic lipid is KC3-C17(C8:1). In some embodiments, the KC3 ionizable cationic lipid is KC3-C15(C8:1).

[0372] In some embodiments, the complex lipid is PEG-DMG or PEG-DSG.

[0373] In some embodiments, the composition comprises a total amount of PEG-containing complex lipid of 0.5 to 2.0 mol% of the total lipid content of the LNP composition.

[0374] In some embodiments, the composition comprises a total amount of KC3 ionizable cationic lipid of 48 mol% of the total lipid content of the LNP composition.

[0375] In some embodiments, the composition comprises DSPC and DSPS in a total amount of 10 mol% of the total lipid content of the LNP composition.

[0376] In some embodiments, the composition comprises 5% DSPC or HSPC in a total amount of 5 mol% of the total lipid content of the LNP composition.

[0377] In some embodiments, the composition comprises PEG-DMG in a total amount of 1.5 mol% of the total lipid content of the LNP composition.

[0378] In some embodiments, the composition comprises cholesterol in a total amount of 40.5 mol% of the total lipid content of the LNP composition.

[0379] In some embodiments, the composition comprises DSPC phospholipid in a total amount of 10 mol% of the total lipid content of the LNP composition.

[0380] In some embodiments, the PEG-containing complex lipid is PEG 2000 -DMG.

[0381] In some embodiments, the composition comprises cholesterol in an amount of 23.5 mol% of the total lipid content of the LNP composition. In some embodiments, the composition comprises cholesterol in an amount of 33.5 mol% of the total lipid content of the LNP composition. In some embodiments, the composition comprises cholesterol in an amount of 38.5 mol% of the total lipid content of the LNP composition. In some embodiments, the composition comprises cholesterol in an amount of 40.5 mol% of the total lipid content of the LNP composition. In some embodiments, the composition comprises cholesterol in an amount of 42.7 mol% of the total lipid content of the LNP composition. In some embodiments, the composition comprises cholesterol in an amount of 43.5 mol% of the total lipid content of the LNP composition. In some embodiments, the composition comprises cholesterol in an amount of 33.5 to 43.5 mol% of the total lipid content of the LNP composition. In some embodiments, the composition comprises KC3 ionizable cationic lipid in an amount of 45 to 55 mol% of the total lipid content of the LNP composition.

[0382] Aspects of the present disclosure relate to a nucleic acid lipid nanoparticle (LNP) composition selected from the group consisting of an mRNA nucleic acid, KC3-OA, KC3-PA, KC3-C17(8:1), and KC3-C15(C8:1), comprising KC3 ionizable cationic lipid in an amount of 45 to 55 mol% of the total lipid content of the LNP composition, cholesterol in an amount of 33.5 to 43.5 mol% of the total lipid content of the LNP composition, (L-serine)DPPS lipid in an amount of 5 mol% of the total lipid content of the LNP composition, DSPC or HSPC phospholipid in an amount of 5 mol% of the total lipid content of the LNP composition, and PEG-DMG complex lipid in an amount of 1.5 mol% of the total lipid content of the LNP composition.

[0383] Aspects of the present disclosure relate to a lipid nanoparticle (LNP) composition comprising a KC3 ionizable cationic lipid, a (L-serine)PS lipid, cholesterol, one or more phospholipids comprising at least one anionic phospholipid, and a complex lipid, wherein the LNP is obtained by a process comprising the step of dissolving a sodium or ammonium salt of the anionic phospholipid.

[0384] In some embodiments, the composition comprises a nucleic acid. In some embodiments, the nucleic acid is mRNA.

[0385] In some embodiments, the composition is a vaccine.

[0386] In some embodiments, the total amount of phospholipids in the composition is 5 to 25 mol% of the total lipid content of the LNP composition, the total amount of phosphatidylserine (PS) is 2.5 to 10 mol% of the total lipid content of the LNP composition, and the total amount of complex lipids in the composition is 0.5 to 2.5 mol% of the total lipid content of the LNP composition.

[0387] In some embodiments, the composition comprises 48 mol% of KC3 ionizable cationic lipid, 40.5 mol% of cholesterol, and 5 mol% of (L-serine) DPPS lipid, where each mol% refers to the mol% of the total lipid content of the LNP composition.

[0388] In some embodiments, the composition comprises 48 mol% of KC3 ionizable cationic lipid, 38.5 mol% of cholesterol, and 5 mol% of (L-serine) DPPS lipid, where each mol% refers to the mol% of the total lipid content of the LNP composition.

[0389] In some embodiments, the composition comprises 46 to 54 mol% of KC3 ionizable cationic lipid and 5 mol% of (L-serine) DPPS lipid, where each mol% refers to the mol% of the total lipid content of the LNP composition.

[0390] In some embodiments, the composition comprises 45 mol% of KC3 ionizable cationic lipid, 42.7 mol% of cholesterol, and 5 mol% of (L-serine) DPPS lipid, where each mol% refers to the mol% of the total lipid content of the LNP composition.

[0391] In some embodiments, the composition comprises 50 mol% of KC3 ionizable cationic lipid, 38.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, and a total phospholipid concentration of 10 mol%, where each mol% refers to the mol% of the total lipid content of the LNP composition.

[0392] In some embodiments, the composition comprises 48 mol% of KC3 ionizable cationic lipid, 40.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, and a total phospholipid concentration of 10 mol%, where each mol% refers to the mol% of the total lipid content of the LNP composition.

[0393] In some embodiments, the composition comprises 48 mol% of KC3 ionizable cationic lipid, 40.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, 5 mol% DSPC or DPPC, and a total phospholipid concentration of 10 mol%, where each mol% refers to the mol% of the total lipid content of the LNP composition.

[0394] In some embodiments, the composition comprises 46.5 mol% of KC3 ionizable cationic lipid, 42 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, where each mol% refers to the mol% of the total lipid content of the LNP composition.

[0395] In some embodiments, the composition comprises 5 mol% in total of DSPC or HSPC of the total lipid content of the LNP composition.

[0396] In some embodiments, the composition further comprises 1.5 mol% in total of PEG-DMG of the total lipid content of the LNP composition.

[0397] In some embodiments, the composition comprises 10 mol% in total of DSPC / DPPC phospholipid of the total lipid content of the LNP composition.

[0398] Aspects of the present disclosure relate to phosphatidylserine salts selected from the group consisting of DSPS sodium, DPPS sodium, DSPS ammonium, and DPPS ammonium.

[0399] Aspects of the present disclosure relate to the use of DSPS-Na salt or DPPS-NH4 in the preparation of LNPs comprising (L-serine)PS lipid, sterol, complex lipid, phospholipid for targeting dendritic cells. + Salt.

[0400] Aspects of the present disclosure relate to a solution comprising ethanol and DSPS or DPPS, the solution being obtained by a process comprising dissolving a phosphatidylserine salt in ethanol, the phosphatidylserine salt being selected from the group consisting of DSPS sodium, DPPS sodium, DSPS ammonium, and DPPS ammonium.

[0401] Embodiments Non-limiting embodiments are described below or are considered to be within the present disclosure.

[0402] Embodiment 1: A lipid nanoparticle (LNP) composition comprising an ionizable lipid having a chemical structure.

[0403] [Chemical formula] [Wherein, R1 is [Chemical formula] And where a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each independently (C1-C4) alkyl optionally substituted with hydroxyl, n is an integer equal to 2, 3 or 4]

[0404] Embodiment 2: The composition of Embodiment 1, wherein n is 2 or 3.

[0405] Embodiment 3: The composition according to any one of Embodiments 1 to 2, wherein a is 0.

[0406] Embodiment 4: The composition according to any one of Embodiments 1 to 3, wherein b is 1, 2 or 3.

[0407] Embodiment 5: The composition according to any one of Embodiments 1 to 2, wherein a is 1.

[0408] Embodiment 6: The composition according to any one of Embodiments 1 to 3, wherein b is 1, 2 or 3.

[0409] Embodiment 7: The composition according to any one of Embodiments 1 to 6, wherein R2 and R3 are each methyl.

[0410] Embodiment 8: R1 is

Chemical formula

[0411] Embodiment 9: The composition according to any one of Embodiments 1 to 8, wherein n is 3.

[0412] Embodiment 10: a. Nucleic acid, b. The ionizable lipid according to any one of Embodiments 1 to 9, c. Sterol, d. Containing one or more phospholipids including phosphatidylserine (PS) lipid, e. Optionally further containing complex lipid, The composition according to any one of Embodiments 1 to 9.

[0413] Embodiment 11: The composition according to Embodiment 10, wherein the nucleic acid is mRNA.

[0414] Embodiment 12: The composition of Embodiment 11, wherein the sterol is cholesterol.

[0415] Embodiment 13: One or more phospholipids are a. One or more phospholipids selected from the group consisting of SM, DSPC, HSPC, DPPC, and DOPC, and b. PS lipids selected from the group consisting of DPPS and DSPS The composition according to Embodiment 12, comprising.

[0416] Embodiment 14: One or more phospholipids are a. DSPC, and b. One or more PS lipids selected from the group consisting of (L-serine) DPPS and (L-serine) DSPS The composition according to Embodiment 13, comprising.

[0417] Embodiment 15: The composition according to Embodiment 13, comprising PS lipids in a total amount of 2.5 to 10 mol% of the total lipids in the composition.

[0418] Embodiment 16: The composition according to any one of Embodiments 10 to 15, wherein the complex lipid contains PEG.

[0419] Embodiment 17: a. Nucleic acid, b. Ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition, c. Sterol in a total amount of 25 to 45 mol% of the total lipid content of the LNP composition, and d. One or more phospholipids in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition, comprising phosphatidylserine (PS) in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition, e. Optionally further comprising complex lipid in a total amount of 0.5 to 2.5 mol% of the total lipid content of the LNP composition, Nucleic acid lipid nanoparticle (LNP) composition.

[0420] Embodiment 18: The composition according to Embodiment 17, wherein the nucleic acid is mRNA.

[0421] Embodiment 19: The composition according to Embodiment 18, wherein the sterol is cholesterol.

[0422] Embodiment 20: The composition according to Embodiment 19, wherein the total amount of one or more phospholipids consists of DSPC and L-serine PS.

[0423] Embodiment 21: The composition according to Embodiment 20, which contains PS in a total amount of 2.5 to 7.5 mol% of the total lipids in the composition.

[0424] Embodiment 22: The composition according to any one of Embodiments 17 to 21, wherein the complex lipid contains PEG.

[0425] Embodiment 23: The composition according to Embodiment 22, wherein the complex lipid is PEG-DMG.

[0426] Embodiment 24: The composition according to Embodiment 23, wherein the LNP contains the total amount of the complex lipid in an amount of 0.5 to 2.0 mol% of the total lipid content of the LNP composition.

[0427] Embodiment 25: The composition according to Embodiment 24, wherein the LNP contains the total amount of the complex lipid in an amount less than 2 mol% of the total lipid content of the LNP composition.

[0428] Embodiment 26: a. The nucleic acid is mRNA, b. The ionizable cationic lipid is in a total amount of 45 to 55 mol% of the total lipid content of the LNP composition, c. The sterol is cholesterol in a total amount of 35 to 45 mol% of the total lipid content of the LNP composition, d. The total amount of phospholipids is 7 to 15 mol% of the total lipid content of the LNP composition, e. One or more phospholipids consist of DSPC, and the PS lipid is one or more lipids selected from the group consisting of the L-serine type of DPPS and DSPS. f. The total amount of PS lipid is about 5 mol% of the total lipid content of the LNP composition, The composition according to claim 17.

[0429] Embodiment 27: The composition according to embodiment 26, comprising a total amount of PS lipid selected from 1.25 mol%, 2.5 mol%, 5 mol%, 7.5 mol%, and 10 mol% of the total lipid content of the LNP composition.

[0430] Embodiment 28: a. A nucleic acid that is mRNA, b. An ionizable cationic lipid in a total amount of 45-55 mol% of the total lipid content of the LNP composition, c. A sterol that is cholesterol in a total amount of 35-45 mol% of the total lipid content of the LNP composition, d. One or more phospholipids that are the total amount of phospholipids in a total amount of 10 mol% of the total lipid content of the LNP composition, and include phosphatidylserine (PS) in a total amount of 3-9 mol% of the total lipid content of the LNP composition, e. A total amount of complex lipid in a total amount of 0.5-2.0 mol% of the total lipid content of the LNP composition A nucleic acid-lipid nanoparticle (LNP) composition comprising.

[0431] Embodiment 29: The composition according to any one of embodiments 17-28, wherein one or more phospholipids are selected from the group consisting of DSPS (L-isomer), DPPS (L-isomer), DMPS (L-isomer), DOPS (L-isomer), and DSPS (D-isomer).

[0432] Embodiment 30: a. The complex lipid is PEG-DMG, b. The PS lipid is selected from the group consisting of DSPS (L-isomer) and DPPS, The composition according to claim 29.

[0433] Embodiment 31: The composition according to any one of Embodiments 17 to 28, wherein the ionizable cationic lipid is one or more compounds selected from the group consisting of KC3-OA, KC3-PA, KC3-C17(8:1), and KC3-C15(C8:1).

[0434] Embodiment 32: The composition according to any one of Embodiments 17 to 28, wherein the ionizable cationic lipid is KC3-PA.

[0435] Embodiment 33: The composition according to any one of Embodiments 17 to 28, wherein the ionizable cationic lipid is KC3-OA.

[0436] Embodiment 34: The composition according to any one of Embodiments 17 to 28, wherein the ionizable cationic lipid is KC3-C17(C8:1).

[0437] Embodiment 35: a. Nucleic acid, b. Ionizable cationic lipid in an amount of 50 mol% of the total lipid content of the LNP composition, c. Cholesterol in an amount of 38.5 mol% of the total lipid content of the LNP composition, d. One or more phospholipids in an amount of 7 to 15 mol% of the total lipid content of the LNP composition, including phosphatidylserine (PS) lipid in an amount of 3 to 9 mol% of the total lipid content of the LNP composition, and e. PEG-containing lipid in an amount of 0.5 to 2.0 mol% of the total lipid content of the LNP composition The nucleic acid lipid nanoparticle (LNP) composition according to claim 17, comprising:

[0438] Embodiment 36: The composition according to Embodiment 34, wherein the phospholipid consists of one or more phospholipids selected from the group consisting of DSPC, DOPC, DPPC, HSPC, and SM.

[0439] Embodiment 37: The composition according to Embodiment 36, wherein the PS lipid is one or more L-serine lipids selected from the group consisting of DPPS and DSPS.

[0440] Embodiment 38: The composition according to any one of Embodiments 17 to 28, wherein the one or more phospholipids comprise at least two (L-serine) PS lipids having incompatible acyl chain lengths.

[0441] Embodiment 39: The composition according to Embodiment 38, wherein the phospholipids are DSPC and DPPS.

[0442] Embodiment 40: The composition according to Embodiment 39, wherein DSPC and DPPS are each present in the LNP in a total amount of 5 mol% with respect to the total lipid content of the LNP composition.

[0443] Embodiment 41: A nucleic acid lipid nanoparticle (LNP) composition comprising a nucleic acid, an ionizable cationic lipid KC3-PA or KC3-OA, and a (L-serine) PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition.

[0444] Embodiment 42: The composition according to Embodiment 41, wherein the nucleic acid is mRNA, the PS lipid is (L-serine) DSPS, (L-serine) DPPS, or a mixture thereof, and the LNP composition further comprises cholesterol and a second phospholipid selected from the group consisting of DSPC, DPPC, HSPC, and SM.

[0445] Embodiment 43: The composition according to Embodiment 42, wherein the LNP composition further comprises 0.5 to 2.0 mol% of PEG-DMG or PEG-DSG with respect to the total lipid content of the LNP composition.

[0446] Embodiment 44: The composition according to any one of Embodiments 41 to 43, wherein the ionizable cationic lipid is KC3-PA.

[0447] Embodiment 45: The composition according to any one of Embodiments 41 to 43, wherein the ionizable cationic lipid is KC3-OA.

[0448] Embodiment 46: A nucleic acid lipid nanoparticle (LNP) composition comprising a nucleic acid, a KC3-C17(C8:1) ionizable cationic lipid, and an (L-serine)PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition.

[0449] Embodiment 47: The composition according to any one of Embodiments 41 to 46, wherein the LNP composition has an N / P ratio of 4 to 7.

[0450] Embodiment 48: The composition according to Embodiment 47, wherein the LNP composition has an N / P ratio of 5 to 6.

[0451] Embodiment 49: The composition according to Embodiment 48, wherein the LNP composition has an N / P ratio of 5.3.

[0452] Embodiment 50: A nucleic acid lipid nanoparticle (LNP) composition comprising a nucleic acid, an ionizable cationic lipid KC3-PA, and an (L-serine)PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition.

[0453] Embodiment 51: The composition according to Embodiment 50, wherein the nucleic acid is mRNA, the PS lipid is (L-serine)DSPS, (L-serine)DPPS, or a mixture thereof, and the LNP composition further comprises cholesterol and a second phospholipid selected from the group consisting of DSPC, DOPC, DPPC, HSPC, and SM.

[0454] Embodiment 52: The composition according to Embodiment 51, wherein the LNP composition further comprises 0.5 to 2.0 mol% of PEG-DMG or PEG-DSG based on the total lipid content of the LNP composition.

[0455] Embodiment 53: A nucleic acid lipid nanoparticle (LNP) composition comprising a nucleic acid, an ionizable cationic lipid selected from KC3-C17(C8:1), and an (L-serine)PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition.

[0456] Embodiment 54: The composition according to any one of Embodiments 17 to 21, 26 to 28, 35 to 44, or 53, wherein the N / P ratio is 4 to 7.

[0457] Embodiment 55: The composition according to Embodiment 54, wherein the N / P ratio is 5 to 6.

[0458] Embodiment 56: The composition according to Embodiment 55, wherein the N / P ratio is 3.

[0459] Embodiment 57: The composition according to Embodiment 55, wherein the N / P ratio is 7.

[0460] Embodiment 58: The composition according to any one of Claims 17 to 21, 26 to 28, 35 to 47, or 53, wherein the nucleic acid is mRNA encoding the SARS-CoV-2 spike protein.

[0461] Embodiment 59: a. An mRNA nucleic acid with an N / P ratio of 4 to 7, b. The total amount of KC3-PA ionizable cationic lipid, which is 40 to 65 mol% of the total lipid content of the LNP composition, c. The total amount of cholesterol, which is 25 to 40 mol% of the total lipid content of the LNP composition, d. The total amount of (L-serine)PS lipid, which is 2.5 to 10 mol% of the total lipid content of the LNP composition, e. The total amount of DSPC phospholipid, which is 5 to 25 mol% of the total lipid content of the LNP composition, and f. The total amount of PEG-DMG, which is 0 to 2.5 mol% of the total lipid content of the LNP composition A nucleic acid-lipid nanoparticle (LNP) vaccine composition comprising the same.

[0462] Embodiment 60: a. An mRNA nucleic acid with an N / P ratio of 3 to 8, b. The total amount of KC3-C17(C8:1) ionizable cationic lipid, which is 40 to 65 mol% of the total lipid content of the LNP composition, c. The total amount of cholesterol, which is 25 to 40 mol% of the total lipid content of the LNP composition, d. From 2.5 to 10 mol% of the total lipid content of the LNP composition of (L-serine)PS lipid, e. From 5 to 25 mol% of the total lipid content of the LNP composition of DSPC phospholipid, and f. From 0 to 2.5 mol% of the total lipid content of the LNP composition of PEG-DMG A nucleic acid-lipid nanoparticle (LNP) vaccine composition comprising.

[0463] Embodiment 61: a. mRNA nucleic acid with an N / P ratio of 4 to 7, b. From 40 to 65 mol% of the total lipid content of the LNP composition of KC3-C15(C8:1) ionizable cationic lipid, c. From 25 to 40 mol% of the total lipid content of the LNP composition of cholesterol, d. From 2.5 to 10 mol% of the total lipid content of the LNP composition of (L-serine)PS lipid, e. From 5 to 25 mol% of the total lipid content of the LNP composition of DSPC phospholipid, and f. From 0 to 2.5 mol% of the total lipid content of the LNP composition of PEG-DMG A nucleic acid-lipid nanoparticle (LNP) vaccine composition comprising.

[0464] Embodiment 62: a. mRNA nucleic acid with an N / P ratio of 3 to 8, b. From 40 to 65 mol% of the total lipid content of the LNP composition of KC3-C18 ionizable cationic lipid, c. From 25 to 40 mol% of the total lipid content of the LNP composition of cholesterol, d. From 2.5 to 10 mol% of the total lipid content of the LNP composition of (L-serine)PS lipid, e. From 5 to 25 mol% of the total lipid content of the LNP composition of DSPC phospholipid, and f. From 0 to 2.5 mol% of the total lipid content of the LNP composition of PEG-DMG A nucleic acid-lipid nanoparticle (LNP) vaccine composition comprising.

[0465] Embodiment 63: The composition according to any one of Embodiments 59 to 62, wherein the nucleic acid is the mRNA of SEQ ID NO: 2.

[0466] Embodiment 64: Use of (L-serine)PS lipid in combination with the ionizable cationic lipid according to any one of Embodiments 1 to 9 in an LNP for targeting to dendritic cells.

[0467] Embodiment 65: The use according to Embodiment 64, wherein the LNP contains mRNA.

[0468] Embodiment 66: The use according to any one of Embodiments 64 to 65, wherein the LNP further contains cholesterol.

[0469] Embodiment 67: The use according to Embodiment 66, wherein the total amount of (L-serine)PS lipid in the LNP is 2.5 to 10 mol% of the total lipid content of the LNP composition.

[0470] Embodiment 68: The use according to Embodiment 67, wherein the LNP further contains one or more additional phospholipids including DSPC.

[0471] Embodiment 69: The use according to Embodiment 68, wherein the LNP further contains a complex lipid.

[0472] Embodiment 70: The LNP is a. mRNA nucleic acid with an N / P ratio of 3 to 8, b. KC3-PA or KC3-C17(C8:1) ionizable cationic lipid (ICL) in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition, c. Cholesterol in a total amount of 25 to 40 mol% of the total lipid content of the LNP composition, d. (L-serine)PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition, e. DSPC phospholipid in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition, and f. Complex lipid in a total amount of 0 to 2.5 mol% of the total lipid content of the LNP composition and the use according to Embodiment 64.

[0473] Embodiment 71: Use according to Embodiment 70, wherein the ICL is KC3-PA.

[0474] Embodiment 72: Use according to Embodiment 70, wherein the ICL is KC3-C17(C8:1).

[0475] Embodiment 73: A lipid nanoparticle (LNP) composition comprising an ionizable lipid having a chemical structure.

[0476] [Chemical formula] [wherein, R1 is [Chemical formula] and here, a is 0 or 1, b is 1, 2, 3 or 4, provided that the sum of a + b is 1, 2, 3 or 4, R2 and R3 are each independently methyl, n is an integer equal to 2 or 3]

[0477] Embodiment 74: The composition according to Embodiment 73, wherein a is 0.

[0478] Embodiment 75: The composition according to Embodiment 74, wherein b is 1.

[0479] Embodiment 76: The composition according to Embodiment 74, wherein b is 3.

[0480] Embodiment 77: The composition according to Embodiment 73, wherein a is 1.

[0481] Embodiment 78: The composition according to Embodiment 77, wherein b is 1.

[0482] Embodiment 79: The composition according to Embodiment 77, wherein b is 3.

[0483] Embodiment 80: The composition according to any one of Embodiments 73 to 79, wherein n is 2.

[0484] Embodiment 81: The composition according to any one of Embodiments 73 to 79, wherein n is 3.

[0485] Embodiment 82: The composition according to any one of Embodiments 17 to 28, comprising an anionic lipid selected from the group consisting of phosphatidylglycerol (PG), phosphatidic acid (PA), N-glutaryl-phosphatidylethanolamine (N-Glu-PE), N-succinyl-phosphatidylethanolamine (N-Suc-PE), and cardiolipin. Distearoyl phosphatidylglycerol (DSPG), dipalmitoyl phosphatidylglycerol (DPPG), N-succinyl-distearoyl phosphatidylethanolamine (N-Suc-DSPE), N-glutaryl-distearoyl phosphatidylethanolamine (N-glu-DSPE), distearoyl phosphatidic acid (DSPA), and cardiolipin.

[0486] Embodiment 83: The composition according to any one of Embodiments 17 to 28 or 82, comprising an anionic targeted phospholipid other than phosphatidyl-L-serine.

[0487] Embodiment 84: The composition according to any one of Embodiments 17 to 28 or 82 to 83, comprising an anionic phospholipid selected from the group consisting of DSPG and DPPG.

[0488] Embodiment 85: The composition according to any one of Embodiments 17 to 28 or 82 to 83, comprising an anionic phospholipid selected from the group consisting of N-Glu-DSPE and N-Suc-DSPE.

[0489] Embodiment 86: The composition according to any one of Embodiments 17 to 28 or 82 to 83, comprising DSPA anionic phospholipid.

[0490] Embodiment 87: The composition according to any one of Embodiments 17 to 28 or 82 to 83, comprising cardiolipin anionic phospholipid.

[0491] Embodiment 88: The composition according to any one of Embodiments 1 to 63 or 73 to 87, wherein the ionizable lipid has a chemical structure.

[0492]

Chemical formula

[0493] Embodiment 89: Use of any one of Embodiments 64 to 72, wherein the ionizable lipid has a chemical structure.

[0494]

Chemical formula

[0495] Embodiment 90: Sodium or ammonium salts of the composition of the anionic phospholipid of formula (V-A-1) having a chemical structure.

[0496]

Chemical formula

[0497] Embodiment 91: The composition according to Embodiment 90, wherein a is 14 or 16.

[0498] Embodiment 92: The composition according to any one of Embodiments 90 or 91, wherein X + is an ammonium cation (NH4 + ).

[0499] Embodiment 93: The composition according to any one of Embodiments 90 or 91, wherein X + is a sodium cation (Na + ).

[0500] Embodiment 94: The composition according to any one of Embodiments 90 to 93, wherein the anionic phospholipid of formula (V-A-1) is the sodium salt of distearoyl phosphatidyl-L-serine (DSPS L-isomer).

[0501] Embodiment 95: The composition according to any one of Embodiments 90 to 93, wherein the anionic phospholipid of formula (V-A-1) is the ammonium salt of distearoyl phosphatidyl-L-serine (DSPS L-isomer).

[0502] Embodiment 96: The composition according to any one of Embodiments 90 to 93, wherein the anionic phospholipid of formula (V-A-1) is the sodium salt of DPPS (L-isomer).

[0503] Embodiment 97: The composition according to any one of Embodiments 90 to 93, wherein the anionic phospholipid of formula (V-A-1) is the ammonium salt of DPPS (L-isomer).

[0504] Embodiment 98: Use of the composition in the salt form according to any one of Embodiments 90 to 97 in the preparation of a liposome nanoparticle (LNP) composition.

[0505] Embodiment 99: During the preparation of the LNP composition, the following LNP components: mRNA nucleic acid, Ionized cationic lipid (ICL), Cholesterol, (L-serine)PS lipid, One or more phospholipids, and Complex lipid The use of Embodiment 98, in combination with one or more of the above.

[0506] Embodiment 100: The ammonium or salt form of the compound of formula (V-A-1) is used during the preparation of the LNP composition with the following LNP components: mRNA nucleic acid, The ionized cationic lipid (ICL) according to any one of Claims 1 to 9 or 73 to 88, Cholesterol, (L-Serine)PS lipid, one or more phospholipids, and complex lipids The use of embodiment 98, comprising the step of combining with one or more of them.

[0507] Embodiment 101: The LNP is a. mRNA nucleic acid with an N / P ratio of 4 to 7, b. The ionizable cationic lipid according to any one of embodiments 1 to 9 or 73 to 88, in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition, c. Cholesterol in a total amount of 25 to 40 mol% of the total lipid content of the LNP composition, d. (L-Serine)PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition, e. DSPC phospholipid in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition, and f. PEG-DMG in a total amount of 0 to 2.5 mol% of the total lipid content of the LNP composition The use according to any one of embodiments 98 to 100, which is a nucleic acid-lipid nanoparticle vaccine composition comprising the above.

[0508] Embodiment 102: The use according to embodiment 101, wherein the nucleic acid is the mRNA of SEQ ID NO: 2.

[0509] Embodiment 103: The LNP composition comprises the ionizable cationic lipid in a total amount of 46 to 65 mol% of the total lipid content of the LNP composition, and is the composition according to any one of embodiments 17 to 63 or 73 to 89.

[0510] Embodiment 104: The LNP composition comprises PS in a total amount of about 5 mol% of the total lipid in the composition, and is the composition according to any one of embodiments 17 to 63 or 73 to 89 or 103.

[0511] Embodiment 105: The LNP composition comprises the complex lipid in a total amount of about 1.5 mol% of the total lipid content of the LNP composition, and is the composition according to any one of embodiments 17 to 63 or 73 to 89 or 103 to 104.

[0512] Embodiment 106: The complex lipid is PEG-DMG, and the PS lipid is selected from the group consisting of DSPS (L-isomer) and DPPS, A composition according to any one of Embodiments 82 to 87.

[0513] Embodiment 107: A composition according to any one of Embodiments 82 to 87, wherein the ionizable cationic lipid is one or more compounds selected from the group consisting of KC3-OA, KC3-PA, KC3-C17(C8:1), and KC3-C15(C8:1).

[0514] Embodiment 108: A composition according to any one of Embodiments 82 to 87, wherein the ionizable cationic lipid is KC3-PA.

[0515] Embodiment 109: A composition according to any one of Embodiments 82 to 87, wherein the ionizable cationic lipid is KC3-OA.

[0516] Embodiment 110: A composition according to any one of Embodiments 82 to 87, wherein the ionizable cationic lipid is KC3-C17(C8:1).

[0517] Embodiment 111: Use of (L-serine) PS lipid in combination with an ionizable cationic lipid according to any one of Embodiments 73 to 87 in an LNP for targeting the LNP to dendritic cells.

[0518] Embodiment 112: The use according to Embodiment 111, wherein the LNP contains mRNA.

[0519] Embodiment 113: The use according to any one of Embodiments 111 to 112, wherein the LNP further contains cholesterol.

[0520] Embodiment 114: The use according to Embodiment 113, wherein the total amount of (L-serine) PS lipid in the LNP is 2.5 to 10 mol% of the total lipid content of the LNP composition.

[0521] Embodiment 115: The use according to Embodiment 114, wherein the LNP further comprises one or more additional phospholipids including DSPC.

[0522] Embodiment 116: The use according to Embodiment 115, wherein the LNP further comprises a composite lipid.

[0523] Embodiment 117: The LNP a. mRNA nucleic acid with an N / P ratio of 3 to 8, b. KC3-PA or KC3-C17(C8:1) ionizable cationic lipid (ICL) in an amount of 40 to 65 mol% of the total lipid content of the LNP composition, c. Cholesterol in an amount of 25 to 40 mol% of the total lipid content of the LNP composition, d. (L-serine)PS lipid in an amount of 2.5 to 10 mol% of the total lipid content of the LNP composition, e. DSPC phospholipid in an amount of 5 to 25 mol% of the total lipid content of the LNP composition, and f. Composite lipid in an amount of 0 to 2.5 mol% of the total lipid content of the LNP composition The use according to Embodiment 111, comprising

[0524] Embodiment 118: The use according to Embodiment 117, wherein the ICL is KC3-PA.

[0525] Embodiment 119: The use according to Embodiment 117, wherein the ICL is KC3-C17(C8:1).

[0526] Embodiment 120: X is an ammonium cation selected from the group consisting of ammonium (NH4 + ), alkylammonium, dialkylammonium, and trialkylammonium salts, and the composition according to any one of Embodiments 90 to 97.

[0527] Embodiment 121: The composition according to Embodiment 120, wherein X is an ammonium cation selected from the group consisting of ammonium, dimethylamine, diethylamine, triethylamine, trimethylamine, 2-(dimethylamino)ethanol, diethanolamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methyl-glucamine, imidazole, histidine, lysine, arginine, 4-(2-hydroxyethyl)-morpholine, piperazine, 1-(2-hydroxyethyl)-pyrrolidine, triethanolamine, and tromethamine (tris(hydroxymethyl)aminomethane).

[0528] Embodiment 122: The composition according to any one of Embodiments 17 - 28 or 82 - 83, comprising an anionic phospholipid selected from the group consisting of DSPG and DPPG in a total amount of 2.5 - 7.5 mol% of the total lipid content of the LNP composition.

[0529] Embodiment 123: The composition according to Embodiment 122, wherein the composition comprises DSPG anionic phospholipid in a total amount of 2.5 - 7.5 mol% of the total lipid content of the LNP composition.

[0530] Embodiment 124: The composition according to Embodiment 122, wherein the composition comprises DPPG anionic phospholipid in a total amount of 2.5 - 7.5 mol% of the total lipid content of the LNP composition.

[0531] Embodiment 125: The use according to Embodiment 114, wherein the LNP further comprises one or more additional phospholipids including DSPC.

[0532] Embodiment 126: a. Nucleic acid, b. KC3 ionizable cationic lipid in a total amount of 40 - 65 mol% of the total lipid content of the LNP composition, c. Cholesterol in a total amount of 23.5 - 43.5 mol% of the total lipid content of the LNP composition, d. (L-serine)PS lipid in a total amount of 2.5 - 10 mol% of the total lipid content of the LNP composition, e. DSPC or HSPC phospholipids in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition, and f. PEG-containing complex lipids in a total amount of 0.5 mol% to 2.5 mol% of the total lipid content of the LNP composition A nucleic acid-lipid nanoparticle (LNP) composition comprising.

[0533] Embodiment 127: The composition according to embodiment 126, wherein the nucleic acid is mRNA.

[0534] Embodiment 128: The composition according to any one of embodiments 126 to 127, wherein the N / P ratio is 3 to 8.

[0535] Embodiment 129: The composition according to any one of embodiments 126 to 127, wherein the KC3 ionizable cationic lipid is selected from the group consisting of KC3-OA, KC3-PA, KC3-C17(8:1), and KC3-C15(C8:1).

[0536] Embodiment 130: The composition according to embodiment 129, wherein the KC3 ionizable cationic lipid is KC3-OA.

[0537] Embodiment 131: The composition according to embodiment 129, wherein the KC3 ionizable cationic lipid is KC3-PA.

[0538] Embodiment 132: The composition according to embodiment 129, wherein the KC3 ionizable cationic lipid is KC3-C17(C8:1).

[0539] Embodiment 133: The composition according to embodiment 129, wherein the KC3 ionizable cationic lipid is KC3-C15(C8:1).

[0540] Embodiment 134: The composition according to any one of embodiments 126 to 133, wherein the complex lipid comprises PEG-DMG or PEG-DSG.

[0541] Embodiment 135: The composition according to Embodiment 134, comprising a PEG-containing complex lipid in a total amount of 0.5 to 2.0 mol% of the total lipid content of the LNP composition.

[0542] Embodiment 136: The composition according to any one of Embodiments 126 to 135, comprising a KC3 ionizable cationic lipid in a total amount of 48 mol% of the total lipid content of the LNP composition.

[0543] Embodiment 137: The composition according to any one of Embodiments 126 to 136, comprising DSPC and DSPS in a total amount of 10 mol% of the total lipid content of the LNP composition.

[0544] Embodiment 138: The composition according to any one of Embodiments 126 to 137, comprising 5% DSPC or HSPC in a total amount of 5 mol% of the total lipid content of the LNP composition.

[0545] Embodiment 139: The composition according to any one of Embodiments 126 to 137, comprising PEG-DMG in a total amount of 1.5 mol% in total of the total lipid content of the LNP composition.

[0546] Embodiment 140: The composition according to any one of Embodiments 126 to 137, comprising cholesterol in a total amount of 40.5 mol% of the total amount of cholesterol of the total lipid content of the LNP composition.

[0547] Embodiment 141: The composition according to any one of Embodiments 126 to 137, comprising DSPC phospholipid in a total amount of 10 mol% of the total lipid content of the LNP composition.

[0548] Embodiment 142: The composition according to any one of Embodiments 126 to 141, wherein the PEG-containing complex lipid is PEG 2000 -DMG.

[0549] Embodiment 143: The composition according to any one of Embodiments 126 to 139, comprising cholesterol in a total amount of 23.5 mol% of the total lipid content of the LNP composition.

[0550] Embodiment 144: The composition according to any one of Embodiments 126 to 139, which contains cholesterol in an amount of 33.5 mol% of the total lipid content of the LNP composition.

[0551] Embodiment 145: The composition according to any one of Embodiments 126 to 139, which contains cholesterol in an amount of 38.5 mol% of the total lipid content of the LNP composition.

[0552] Embodiment 146: The composition according to any one of Embodiments 126 to 139, which contains cholesterol in an amount of 40.5 mol% of the total lipid content of the LNP composition.

[0553] Embodiment 147: The composition according to any one of Embodiments 126 to 139, which contains cholesterol in an amount of 42.7 mol% of the total lipid content of the LNP composition.

[0554] Embodiment 148: The composition according to any one of Embodiments 126 to 139, which contains cholesterol in an amount of 43.5 mol% of the total lipid content of the LNP composition.

[0555] Embodiment 149: The composition according to any one of Embodiments 126 to 139, which contains cholesterol in an amount of 33.5 to 43.5 mol% of the total lipid content of the LNP composition.

[0556] Embodiment 150: The composition according to any one of Embodiments 126 to 149, which contains KC3 ionizable cationic lipid in an amount of 45 to 55 mol% of the total lipid content of the LNP composition.

[0557] Embodiment 151: a. mRNA nucleic acid, b. A KC3 ionizable cationic lipid selected from the group consisting of KC3-OA, KC3-PA, KC3-C17(8:1), and KC3-C15(C8:1) in an amount of 45 to 55 mol% of the total lipid content of the LNP composition, c. Cholesterol in an amount of 33.5 to 43.5 mol% of the total lipid content of the LNP composition, d. (L-serine)DPPS lipid in an amount of 5 mol% of the total lipid content of the LNP composition, e. DSPC or HSPC phospholipid in an amount of 5 mol% of the total lipid content of the LNP composition, and f. PEG-DMG complex lipid in an amount of 1.5 mol% of the total lipid content of the LNP composition A nucleic acid-lipid nanoparticle (LNP) composition comprising.

[0558] Embodiment 152: A lipid nanoparticle (LNP) composition comprising a KC3 ionizable cationic lipid, (L-serine)PS lipid, cholesterol, one or more phospholipids including at least one anionic phospholipid, and a complex lipid, wherein the LNP is obtained by a process comprising the step of dissolving a sodium or ammonium salt of an anionic phospholipid.

[0559] Embodiment 153: The composition according to Embodiment 152, wherein the anionic phospholipid is a salt according to any one of claims 90-97.

[0560] Embodiment 154: The composition according to any one of Embodiments 152-153, comprising a nucleic acid.

[0561] Embodiment 155: The composition according to Embodiment 154, wherein the nucleic acid is mRNA.

[0562] Embodiment 156: The composition according to Embodiment 155, which is a vaccine.

[0563] Embodiment 157: The total amount of phospholipids in the composition is 5-25 mol% of the total lipid content of the LNP composition, the total amount of phosphatidylserine (PS) is 2.5-10 mol% of the total lipid content of the LNP composition, and the total amount of complex lipid in the composition is 0.5-2.5 mol% of the total lipid content of the LNP composition. The composition according to any one of Embodiments 152-156.

[0564] Embodiment 158: 48 mol% KC3 ionizable cationic lipid, 40.5 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, where each mol% refers to the mol% of the total lipid content of the LNP composition, the composition according to any one of Embodiments 152 to 156.

[0565] Embodiment 159: 48 mol% KC3 ionizable cationic lipid, 38.5 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, where each mol% refers to the mol% of the total lipid content of the LNP composition, the composition according to any one of Embodiments 152 to 156.

[0566] Embodiment 160: 46 - 54 mol% KC3 ionizable cationic lipid, and 5 mol% (L-serine) DPPS lipid, where each mol% refers to the mol% of the total lipid content of the LNP composition, the composition according to any one of Embodiments 152 to 156.

[0567] Embodiment 161: 45 mol% of KC3 ionizable cationic lipid, 42.7 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, where each mol% refers to the mol% of the total lipid content of the LNP composition, the composition according to any one of Embodiments 152 to 156.

[0568] Embodiment 162: 50 mol% of KC3 ionizable cationic lipid, 38.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, and including a total phospholipid concentration of 10 mol%, Each mol% refers to the mol% of the total lipid content of the LNP composition. The composition according to any one of Embodiments 152 to 156.

[0569] Embodiment 163: 48 mol% of KC3 ionizable cationic lipid, 40.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, and including a total phospholipid concentration of 10 mol%, Each mol% refers to the mol% of the total lipid content of the LNP composition. The composition according to any one of Embodiments 152 to 156.

[0570] Embodiment 164: 48 mol% of KC3 ionizable cationic lipid, 40.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, 5 mol% DSPC or DPPC, and including a total phospholipid concentration of 10 mol%, Each mol% refers to the mol% of the total lipid content of the LNP composition. The composition according to any one of Embodiments 152 to 156.

[0571] Embodiment 165: 46.5 mol% of KC3 ionizable cationic lipid, 42 mol% cholesterol, including 5 mol% (L-serine) DPPS lipid, Each mol% refers to the mol% of the total lipid content of the LNP composition. The composition according to any one of Embodiments 152 to 156.

[0572] Embodiment 166: The composition according to any one of Embodiments 158 to 165, further comprising 5 mol% in total of DSPC or HSPC of the total lipid content of the LNP composition.

[0573] Embodiment 167: The composition according to any one of Embodiments 158 to 166, further comprising 1.5 mol% of PEG-DMG based on the total lipid content of the LNP composition.

[0574] Embodiment 168: The composition according to any one of Embodiments 158 to 163, comprising 10 mol% of DSPC / DPPC phospholipid based on the total lipid content of the LNP composition.

[0575] Embodiment 169: Phosphatidylserine salts selected from the group consisting of sodium DSPS, sodium DPPS, ammonium DSPS and ammonium DPPS.

[0576] Embodiment 170: Use of DSPS-Na salt or DPPS-NH4 in the preparation of LNPs comprising (L-serine)PS lipid, sterol, complex lipid, phospholipid for targeting dendritic cells. + salt.

[0577] Embodiment 171: A solution comprising ethanol and DSPS or DPPS, obtained by a process comprising the step of dissolving a phosphatidylserine salt in ethanol, wherein the phosphatidylserine salt is selected from the group consisting of sodium DSPS, sodium DPPS, ammonium DSPS and ammonium DPPS.

Examples

[0578] The present disclosure has been described in connection with specific embodiments and has set forth numerous details for purposes of illustration. However, it will be apparent to those skilled in the art that the present disclosure includes additional embodiments and that some of the details described herein may be significantly varied without departing from the present disclosure. The present disclosure includes such additional embodiments, modifications and equivalents. In particular, the present disclosure includes any combination of various exemplary components and features, terms, or elements.

[0579] Unless otherwise specified, the isomeric form of the phosphatidylserine lipid used in the examples is phosphatidyl-L-serine.

[0580] Specific examples are provided below to illustrate various embodiments of the embodiments disclosed herein. Those skilled in the art will appreciate that the various embodiments disclosed herein are not limited to these specific exemplary examples.

[0581] [Example 1A] Synthesis of Ionizable Lipids Scheme 1 Synthesis of Acid Intermediates for AKG-UO-1 to AKG-UO-3. See FIGS. 47A - 47B.

[0582] [Chem.]

[0583] As shown in Error! Reference source not found, the acid intermediates (6Z,12Z)-6,12-octadecadienoic acid and (6Z,12Z)-6,12-hexadecadienoic acid were prepared by general synthesis, which included i) the first Wittig reaction of triphenylphosphonium ylide prepared from 5-bromopentanol and the corresponding aldehyde, ii) conversion of the terminal alcohol to bromide by mesylation and substitution, iii) repetition of the sequence of ylide synthesis and Wittig reaction, and finally iv) periodate oxidation of the terminal alcohol. The obtained acid intermediates were utilized in the synthesis of AKG-UO-1 to AKG-UO-4 below. Scheme 2 Synthesis of Acid Intermediate for AKG-UO-5

[0584] [Chem.] The acid intermediate (9Z,15Z)-9,15-octadecadienoic acid used in the synthesis of AKG-UO-5 was prepared by the general synthesis shown in Scheme 2, which included i) alkylation of silyl-protected 10-hydroxy-1-decyne using (5Z)-1-bromo-5-octene, ii) catalytic hydrogenation of the alkyne to a cis-alkene, iii) removal of the silyl protection on the alcohol, and finally iv) oxidation of the terminal alcohol to the desired acid.

[0585] Synthesis of Acid Intermediates for Schemes 3 AKG-BDG-01 and AKG-BDG-02

Chem.

[0586] The synthesis of two disulfate intermediates used in the synthesis of AKG-BDG-1 and AKG-BDG-2 is shown in Scheme 4. The general synthesis of the acid intermediate for AKG-BDG-1 involves: i) synthesis of 4-mercaptobutyric acid from 4-bromobutyric acid, ii) preparation of 4-(2-pyridyldisulfanyl)butanoic acid by reaction of 4-mercaptobutyric acid with DPS, iii) catalytic hydrogenation of 3-decyne-1-ol to cis-alkene, iv) tosylation of the primary alcohol, v) preparation of the terminal thiol by substitution of the tosyl group using thiourea, and finally vi) preparation of the disulfide containing the acid intermediate by coupling of the terminal thiol using 4-(2-pyridyldisulfanyl)butanoic acid prepared in step ii above. The second acid intermediate used in the synthesis of AKG-BDG-2 was obtained following a similar synthetic sequence starting from 3-dodecyne-1-ol.

[0587] Scheme 4 Synthesis of AKG-UO-1, AKG-UO-4, AKG-UO-5, AKG-BDG-1 and AKG-BDG-2

Chem.

[0588] Scheme 5 Synthesis of AKG-UO-2

Chem.

[0589] Scheme 6 Synthesis of AKG-UO-3

Chem.

[0590] [Example 1B] Synthesis of Ionizable Lipids - Refer to Figure 48 1. 2-((S)-2,2-Bis((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethane-1-amine (AKG-KC2-01, O-12095) 2. 3-((S)-2,2-Bis((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-01, O-12096) 3. 2-((S)-2,2-Bis((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethane-1-amine (AKG-KC2-OA, O-11880) 4. 2-((S)-2,2-Bis((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethane-1-amine (AKG-KC2-PA, O-11879) 5. 3-((S)-2,2-Bis((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-OA, O-11957) 6. 3-((S)-2,2-Bis((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-PA, O-12418) 7. 3-((S)-2,2-di((Z)-heptadec-8-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine (AKG-KC3-C17(C8:1)) 8. (S)-3-(2,2-diheptadecyl-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine (AKG-KC3-C17)

[0591] Synthesis of 2-((S)-2,2-di((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine (AKG-KC2-01, O-12095) 3-((S)-2,2-di((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine (AKG-KC3-01, O-12096) See Figure 48. Experimental procedure

[0592] Synthesis of (6Z,12Z)-1-bromooctadeca-6,12-diene, 2

Chemical formula

[0593] Synthesis of (6Z,12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-ol, 3

Chemical Structure

[0594] Synthesis of (6Z,12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-one, 4

Chemical Structure

[0595] Synthesis of 2-((S)-2,2-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol, 7

Chemical Structure

[0596] Synthesis of 3-((S)-2,2-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)propan-1-ol, 8

Chemical Structure

[0597] Synthesis of 2-((S)-2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine (AKG-KC2-01, O-12095)

Chem.

[0598] Synthesis of 3-((S)-2,2-di((6Z,12Z)-octadeca-6,12-dien-4-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine, AKG-KC3-01, O-12096

Chemical formula

[0599] Synthesis of (Z)-1-bromooctadec-9-ene 3 [Chemical formula] The procedure was as described above. (Z)-1-Bromooctadec-9-ene as a transparent oil (6.4 g, 19.33 mmol). 1 H NMR (300 MHz, CDCl3): 5.36 - 5.32 (m, 2H), 3.41 (t, J = 7.5 Hz, 2H), 2.01 - 1.99 (m, 4H), 1.87 - 1.82 (m, 2H), 1.44 - 1.26 (m, 22H), 0.87 (t, J = 6.6 Hz, 3H).

[0600] (Z)-16-Bromohexadec-7-ene 4 [Chemical formula] 1 H NMR (300 MHz, CDCl3): 5.36 - 5.32 (m, 2H), 3.42 (t, J = 7.5 Hz, 2H), 2.01 - 1.99 (m, 4H), 1.87 - 1.82 (m, 2H), 1.44 - 1.26 (m, 18H), 0.89 (t, J = 6.6 Hz, 3H).

[0601] Synthesis of (9Z,28Z)-heptatriaconta-9,28-dien-19-ol 5 [Chemical formula] The procedure was as described above. (9Z,28Z)-Heptatriaconta-9,28-dien-19-ol as a solid (1.2 g, 2.25 mmol, 47%). 1 H NMR (300 MHz, CDCl3): 5.36 - 5.29 (m, 4H), 3.57 (bs, 1H), 2.01 - 1.97 (m, 8H), 1.42 - 1.26 (m, 53H), 0.89 (t, J = 6.6 Hz, 6H).

[0602] (7Z,26Z)-Tritriaconta-7,26-dien-17-ol 6

Chem.

[0603]

Chem.

[0604] (7Z,26Z)-Tritriaconta-7,26-dien-17-one 8

Chem.

[0605] Synthesis of 2 - ((S)-2,2 - bis((Z)-octadec - 9 - en - 1 - yl)-1,3 - dioxolan - 4 - yl)ethan - 1 - ol 9 [Chemical formula] The procedure was as described above. 2 - ((S)-2,2 - bis((Z)-octadec - 9 - en - 1 - yl)-1,3 - dioxolan - 4 - yl)ethan - 1 - ol as a transparent oil (0.39 g, 0.63 mmol, 74%). 1 1H NMR (300 MHz, CDCl3): δ 5.36 - 5.28 (m, 4H), 4.22 - 4.10 (m, 1H), 4.08 - 4.05 (m, 1H), 3.82 - 3.79 (m, 2H), 3.48 (t, J = 6.8 Hz, 1H), 2.24 - 2.21 (m, 1H), 2.01 - 1.99 (m, 8H), 1.81 - 1.80 (m, 2H), 1.59 - 1.54 (m, 6H), 1.34 - 1.26 (m, 45H), 0.87 (t, J = 6.3 Hz, 6H).

[0606] Synthesis of 2 - ((S)-2,2 - bis((Z)-hexadec - 9 - en - 1 - yl)-1,3 - dioxolan - 4 - yl)ethan - 1 - ol, 10 [Chemical formula] The procedure was as described above. 2 - ((S)-2,2 - bis((Z)-hexadec - 9 - en - 1 - yl)-1,3 - dioxolan - 4 - yl)ethan - 1 - ol as a transparent oil (1.02 g, 1.65 mmol, 51%). 11H NMR (300 MHz, CDCl3): 5.36 - 5.29 (m, 4H), 4.23 - 4.10 (m, 1H), 4.07 - 4.05 (m, 1H), 3.82 - 3.79 (m, 2H), 3.48 (t, J = 6.6 Hz, 1H), 2.24 - 2.12 (m, 1H), 2.01 - 1.97 (m, 8H), 1.84 - 1.78 (m, 2H), 1.57 - 1.55 (m, 8H), 1.34 - 1.29 (m, 35H), 0.87 (t, J = 6.3 Hz, 6H).

[0607] Synthesis of 3 - ((S)-2,2 - bis((Z)-octadec - 9 - en - 1 - yl)-1,3 - dioxolan - 4 - yl)propan - 1 - ol, 11

Chemical Structure

[0608] Synthesis of 3 - ((S)-2,2 - bis((Z)-hexadec - 9 - en - 1 - yl)-1,3 - dioxolan - 4 - yl)propan - 1 - ol

Chemical Structure

[0609] Synthesis of 2-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine (AKG-KC2-OA, O-11880)

Chemical Structure

[0610] Synthesis of 2-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine (AKG-KC2-PA, O-11879)

Chem.

[0611] Synthesis of 3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine (AKG-KC3-OA, O-11957)

Chem.

[0612] Synthesis of 3 - ((S)-2,2 - bis((Z)-hexadec - 9 - en - 1 - yl)-1,3 - dioxolan - 4 - yl)-N,N - dimethylpropan - 1 - amine (AKG - KC3 - PA, O - 12418) [Chemical Structure Diagram] The procedure has been described above. 3 - ((S)-2,2 - bis((Z)-hexadec - 9 - en - 1 - yl)-1,3 - dioxolan - 4 - yl)-N,N - dimethylpropan - 1 - amine (AKG - KC3 - PA, O - 12418) as a transparent oil (300 mg, 0.49 mmol, 32%). 1 1H NMR (300 MHz, CDCl3): δ 5.39 - 5.28 (m, 4H), 4.06 - 4.01 (m, 2H), 3.47 - 3.42 (m, 1H), 2.43 - 2.41 (m, 2H), 2.31 (s, 6H), 2.01 - 1.97 (m, 8H), 1.70 - 1.52 (m, 6H), 1.27 - 1.18 (m, 42H), 0.87 (t, J = 6.6 Hz, 6H). C 44 H 85 MS (APCI) of NO2: 604.6 3-((S)-2,2-di((Z)-heptadeca-8-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine, synthesis of AKG-KC3-C17(C8:1) (S)-3-(2,2-diheptadecyl-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine, synthesis of AKG-KC3-C17 See Figure 50. Experimental procedure

[0613] (9Z,26Z)-pentatriaconta-9,26-dien-18-one, synthesis of 2 [Chemical formula] To a stirred solution of oleoyl chloride (10 g, 33.3 mmol) in toluene (50 mL) was added triethylamine (5.8 mL, 33.3 mmol) at 0 °C. A heavy precipitate formed and the mixture was stirred at room temperature for 8 h. The mixture was quenched with 2% sulfuric acid solution and then extracted with ethyl acetate. The organic matter was washed with brine and then dehydrated with magnesium sulfate and filtered. The filtrate was concentrated under vacuum to give a crude oil. The obtained oil was diluted with ethanol (20 mL) and [2N NaOH] (30 mL) was added. The mixture was heated at 100 °C for 12 h and then cooled. The mixture was diluted with 2N HCl solution until pH 4 was obtained. The mixture was extracted with ethyl acetate. The combined organic matter was washed with brine and then dehydrated with magnesium sulfate and filtered. The filtrate was concentrated under vacuum to give a crude oil. The oil was purified on silica using 10 - 20% ethyl acetate in n-hexane as the eluent to give (9Z,26Z)-pentatriaconta-9,26-dien-18-one, 2 (3.8 g, 44%) as a yellow oil. 1 H NMR (300 MHz, CDCl3): δ ppm 5.35 - 5.31 (m, 4H), 2.39 - 2.34 (m, 4H), 2.0 - 1.85 (m, 8H), 1.57 - 1.52 (m, 4H), 1.27 - 1.25 (m, 40H), 0.88 (t, J = 6.6 Hz, 3H).

[0614] Synthesis of 3-((S)-2,2-di((Z)-heptadec-8-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol, 3

Chem.

[0615] Synthesis of (S)-3-(2,2-diheptadecyl-1,3-dioxolan-4-yl)propan-1-ol, 4

Chem.

[0616] 3 - ((S)-2,2 - bis((Z)-heptadec - 8 - en - 1 - yl)-1,3 - dioxolan - 4 - yl)-N,N - dimethylpropan - 1 - amine, synthesis of AKG - KC3 - C17(C8:1)(O - 12620) [Chemical Structure Diagram] The procedure was described above in the synthesis of AKG - KC2 - 01. 3 - ((S)-2,2 - bis((Z)-heptadec - 8 - en - 1 - yl)-1,3 - dioxolan - 4 - yl)-N,N - dimethylpropan - 1 - amine (AKG - KC3 - C17(C8:1), O - 12620) (290 mg, 0.46 mmol, 40%), clear oily substance. C 42 H 81 MS (APCI of NO2 + ): 632.6 1 1H NMR (300 MHz, CDCl3): δ ppm 5.38 - 5.28 (m, 4H), 4.09 - 3.99 (m, 2H), 3.48 - 3.41 (m, 1H), 2.77 - 2.71 (m, 1H), 2.55 (s, 6H), 2.01 - 1.95 (m, 8H), 1.88 - 1.78 (m, 2H), 1.62 - 1.50 (m, 6H), 1.27 - 1.24 (m, 44H), 0.88 (t, J = 6.8 Hz, 6H).

[0617] (S)-3-(2,2 - diheptadecyl - 1,3 - dioxolan - 4 - yl)-N,N - dimethylpropan - 1 - amine, synthesis of AKG - KC3 - C17(O - 12637) [Chemistry] The procedure was described above in the synthesis of AKG-KC2-01. (S)-3-(2,2-Diheptadecyl-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine (AKG-KC3-C17, O-12637) as a solid (275 mg, 0.43 mmol, 22%). C 42 H 85 MS of NO2 (APCI + ): 636.6 1 1H NMR (300 MHz, CDCl3): δ ppm 4.06 - 4.00 (m, 2H), 3.47 - 3.43 (m, 1H), 2.29 - 2.25 (m, 2H), 2.21 (s, 6H), 1.60 - 1.48 (m, 8H), 1.29 - 1.24 (m, 60H), 0.87 (t, J = 6.6 Hz, 6H).

[0618] [Example 1C] Synthesis of Dilauroyl-(S)-glycerol-mPEG2000 (PEG(2000)-DL) Experimental Procedure

[0619] Synthesis of mPEG2000 Tosylate 2 [Chemistry] To a solution of poly(ethylene glycol) methyl ether 1 (7 g, 3.5 mmol) in dichloromethane (30 mL) at 0 °C, p-toluenesulfonyl chloride (0.9 g, 7 mmol) and triethylamine (1.8 mL, 10.5 mmol) were added. The resulting mixture was stirred at room temperature for 12 hours and then quenched with water. The mixture was extracted with dichloromethane, the organic matter was washed with brine, then dehydrated over magnesium sulfate and filtered. The filtrate was concentrated under vacuum to give a crude oil. The oil was purified on silica using 10% methanol in dichloromethane as the eluent to give mPEG2000 tosylate (5.2 g, 70%) as a white solid. 1H NMR (300 MHz, CDCl3): δ ppm 7.79 - 7.76 (d, 2H), 7.34 - 7.25 (d, 2H), 5.28 (s, 6H), 4.15 - 4.12 (, 2H), 3.87 - 3.81 (m, 1H), 3.71 - 3.43 (m, 188H), 3.47 - 3.36 (m, 5H).

[0620] (S)-(+)-1,2-Isopropylidene glycerol mPEG2000 Synthesis of 4 [Chemical Structure] Sodium hydride (133 mg, 3.33 mmol) was added to a solution of (S)-(+)-1,2-isopropylidene glycerol (440 mg, 3.33 mmol) in tetrahydrofuran (20 mL) at 0 °C. After stirring for 30 minutes, poly(ethylene glycol) methyl tosylate (5.2 g, 2.41 mmol) was added. The mixture was heated at 70 °C for 18 hours and then cooled. The reaction was quenched with water and extracted with dichloromethane. The organic matter was washed with water, then dehydrated with magnesium sulfate and then filtered. The filtrate was concentrated under vacuum to obtain a crude oil. The oil was purified on silica using 1% methanol in dichloromethane as the eluent to obtain (S)-(+)-1,2-isopropylidene glycerol mPEG2000 (3.3 g, 56%) as a clear oil. 1H NMR (300 MHz, CDCl3): δ ppm 4.32 (dd, J = 11.8, 6.3 Hz, 1H), 3.99 (dd, J = 8.2, 6.3 Hz, 1H), 3.85 - 3.74 (m, 1H), 3.68 - 3.51 (m, 190H), 3.39 - 3.36 (m, 5H), 1.35 (s, 3H), 1.28 (s, 3H).

[0621] (R)-3-(mPEG2000)-propane-1,2-diol Synthesis of 5 [Chemical Structure] (S)-(+)-1,2-Isopropylidene glycerol mPEG2000 (3.3 g, 1.56 mmol) and a mixture of 15 mL of [1N HCl] in THF (10 mL) were stirred at room temperature for 1 hour. After 1 hour, the mixture was concentrated under vacuum to obtain (R)-3-(mPEG2000)-propane-1,2-diol (3.4 g, quantitative) as a white solid. It was carried out without further purification. 1H NMR (300 MHz, CDCl3): δ ppm 4.1 - 4.12 (m, 1H), 3.68 - 3.51 (m, 180H), 3.39 - 3.36 (m, 4H).

[0622] Synthesis of dilauroyl-(S)-glycerol-mPEG2000 [Chemical formula] To a solution of (R)-3-(mPEG2000)-propane-1,2-diol (1.56 mmol) in dichloromethane (10 mL) was added lauroyl chloride (0.75 g, 3.43 mmol), N,N-diisopropylethylamine (1.2 mL, 6.8 mmol), and 4-dimethylaminopyridine (0.42 g, 3.43 mmol) at 0 °C. The resulting mixture was stirred at room temperature for 12 hours and then quenched with water. The mixture was extracted with dichloromethane, the organic matter was washed with brine, then dehydrated with magnesium sulfate and filtered. The filtrate was concentrated under vacuum to obtain a crude oil. The oil was purified on silica using 5 - 100% diethyl ether in hexane as the eluent to obtain dilauroyl-(S)-glycerol-mPEG2000 (0.66 g, 18%) as a white solid. MS (MALDI): CHCA matrix: 2431.57 1H NMR (300 MHz, CDCl3): δ ppm 5.23 - 5.18 (m, 1H), 4.32 (dd, J = 11.8, 3.6 Hz, 1H), 4.13 (dd, J = 11.8, 6.3 Hz, 1H), 3.85 - 3.74 (m, 1H), 3.68 - 3.51 (m, 190H), 3.39 - 3.36 (m, 4H), 2.32 - 2.25 (m, 4H), 1.66 - 1.51 (m, 4H), 1.47 - 1.46 (m, 32H), 0.88 - 0.85 (m, 6H).

[0623] [Example 1D] Synthesis of mPEG2000 - DLPE

[0624] [Chemical formula] 4 - Nitrophenyl - 2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131 - tetracontaoctatriaconta - hectane - 133 - yl) carbonate, 2 To a solution of poly(ethylene glycol) methyl ether (4 g, 2 mmol) in dichloromethane (20 mL) at 0 °C was added 4 - nitrophenyl chloroformate (603 mg, 3 mmol) and pyridine (0.5 mL, 6 mmol). The resulting mixture was stirred at room temperature for 3 hours and then quenched with water. The mixture was extracted with dichloromethane, the organic matter was washed with brine, then dehydrated with magnesium sulfate and filtered. The filtrate was concentrated under vacuum to obtain a crude oil. The oil was purified on silica using 5 - 10% methanol in dichloromethane as the eluent to obtain it. 4-Nitrophenyl (2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131tetratetracontaxaoctatriaconta-133-yl) carbonate as a white solid (3.4 g, 82%). 1H NMR (300 MHz, CDCl3): δ ppm 8.28 - 8.25 (d, 2H), 7.39 - 7.38 (m, 2H), 4.43 - 4.41 (m, 1H), 4.00 - 3.90 (m, 2H), 4.00 - 3.80 (m, 5H), 3.68 - 3.36 (m, 188H), 2.03 (s, 3H). (2R)-3-((Hydroxy((135-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131,134-pentatetratetracontaxaocta-136-azaoctatriacontane-138-yl)oxy)phosphoryl)oxy)propane-1,2-diyl didodecanoate, mPEG2000-DLPE 4-Nitrophenyl (2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131-tetracontaoctaxatriacontahectan-133-yl) carbonate 2 (3.4 g, 1.65 mmol), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (1 g, 1.72 mmol), and triethylamine (0.32 mL, 2.28 mmol) in dichloromethane (30 mL) were stirred at room temperature for 12 hours. After 12 hours, the mixture was quenched with water and extracted with dichloromethane. The combined organics were washed with brine, then dried over magnesium sulfate and filtered. The filtrate was concentrated under vacuum to give a crude oil. The oil was purified on silica using 1 - 5% methanol in dichloromethane as the eluent to give (2R)-3-((hydroxy((135-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131,134-pentatetracontaoctaoxa-136-azaoctatriacontahectan-138-yl)oxy)phosphoryl)oxy)propane-1,2-diyl didodecanoate mPEG2000-DLPE (2 g, 46%) as a semi-solid. 1H NMR (300 MHz, CDCl3): δ ppm 5.17 - 5.15 (m, 1H), 4.35 - 4.31 (m, 1H), 4.16 - 4.10 (m, 3H), 4.00 - 3.80 (m, 5H), 3.65 - 3.37 (m, 188H), 3.34 (s, 3H), 3.05 - 3.01 (m, 1H), 2.26 - 2.23 (m, 6H), 1.54 - 1.52 (m, 4H), 1.32 - 1.17 (m, 33H), 0.86 - 0.82 (t, J = 1.3 Hz, 6H).

[0625] [Example 1E] Synthesis of Distearoylphosphatidyl-D-serine (O-12153) - Refer to Figure 51 Benzyl ((benzyloxy)carbonyl)-D-serinate, 2 To a solution of Z-(D)-serine-OH (2.4 g, 10 mmol) in DMF (10 mL), benzyl bromide (1.2 mL, 10 mmol) and cesium carbonate (3.2 g, 10 mmol) were added. The resulting mixture was stirred at room temperature for 12 hours and then quenched with water. The mixture was extracted with dichloromethane, the organic matter was washed with brine, then dehydrated over magnesium sulfate and filtered. The filtrate was concentrated under vacuum to give a crude oil. The oil was purified on silica using 20 - 40% ethyl acetate in hexane as the eluent to give benzyl ((benzyloxy)carbonyl)-D-serinate (2.5 g, 76%) as a white solid. Benzyl O-((benzyloxy)(diisopropylamino)phosphanyl)-N-((benzyloxy)carbonyl)-D-serinate, 4 A mixture of 1-(benzyloxy)-N,N,N’,N’-tetraisopropylphosphanediamine, 3 (2.8 g, 8.4 mmol), ((benzyloxy)carbonyl)-D-serinate 4 (2.5 g, 7.6 mmol) and tetrazole (16.8 mL, 8.4 mmol) in THF (20 mL) was stirred at room temperature for 12 hours. After 12 hours, the reaction was concentrated under vacuum and the resulting oil was purified on silica pretreated with a 0.1% Et3N / n-hexane solution using 10% ethyl acetate in n-hexane as the eluent to give benzyl O-((benzyloxy)(diisopropylamino)phosphanyl)-N-((benzyloxy)carbonyl)-D-serinate 4 (2.0 g, 47%) as a clear oil. MS (APCI+): 567.2 (M+1); 1H NMR (300 MHz, CDCl3): δ ppm 7.33 - 7.30 (m, 15H), 5.81 - 5.61 (dd, J = 11.8, 3.6 Hz, 1H), 5.12 - 5.09 (m, 3H), 4.61 - 4.58 (m, 2H), 4.13 - 4.11 (m, 2H), 3.58 - 3.55 (m, 2H), 1.16 - 1.12 (m, 12H). (2R)-3-(((Benzyloxy)((R)-3-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropoxy)phosphoryl)oxy)propane-1,2-diyl distearate, 6 A mixture of 1,2-distearoyl-sn-glycerol, 5 (1.0 g, 1.6 mmol), benzyl O-((benzyloxy)(diisopropylamino)phosphanyl)-N-((benzyloxy)carbonyl)-D-serinate 4 (0.94 g, 1.6 mmol), and tetrazole (4.3 mL, 1.9 mmol) in THF (25 mL) was stirred at room temperature for 6 h. After 6 h, a solution of tert-butyl hydroperoxide [70% aqueous solution] in THF (3 mL) was added. The resulting mixture was stirred at room temperature for 1 h and then quenched with saturated sodium thiosulfate. The mixture was extracted with ethyl acetate, the organic matter was washed with brine, then dehydrated over magnesium sulfate and filtered. The filtrate was concentrated under vacuum to give a crude oil. The oil was purified on silica using 10 - 20% ethyl acetate in hexane as the eluent to give (2R)-3-(((benzyloxy)((R)-3-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropoxy)phosphoryl)oxy)propane-1,2-diyl distearate 6 (1.4 g, 80%) as a white solid. 1H NMR (300 MHz, CDCl3): δ ppm 7.33 - 7.25 (m, 15H), 5.91 - 5.79 (dd, J = 11.8, 3.6 Hz, 1H), 5.21 - 5.11 (m, 6H), 5.06 - 4.95 (m, 3H), 4.60 - 4.57 (m, 1H), 4.48 - 4.43 (m, 2H), 4.29 - 4.20 (m, 2H), 4.05 - 4.01 (m, 3H), 2.26 - 2.04 (m, 4H), 1.58 - 1.48 (m, 8H), 1.29 - 1.26 (m, 45H), 0.89 - 0.85 (t, J = 1.3 Hz, 6H). O-(((R)-2,3-bis(stearoyloxy)propoxy)(hydroxy)phosphoryl)-D-serine bis(triethylamine) salt A solution of (2R)-3-(((benzyloxy)((R)-3-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropoxy)phosphoryl)oxy)propane-1,2-diyl distearate 6 (800 mg, 0.72 mmol) in a mixture of methanol, acetic acid, and THF (10:2:4, v:v:v, 10 mL) was hydrogenated at 1 atm and room temperature over 10% palladium on carbon (40 mg). After 3 hours, the mixture was degassed, flushed with nitrogen, and filtered through celite. Triethylamine (0.5 mL) was added to the filtrate, which was then concentrated in vacuo to give an oil. The oil was purified on a C18 column using methanol (0.5% triethylamine) as the eluent to give O-(((R)-2,3-bis(stearoyloxy)propoxy)(hydroxy)phosphoryl)-D-serine bis(triethylamine) salt (360 mg, 50%) as a white solid. MS (APCI+): 567.2 (M+1); 1H NMR (300 MHz, CDCl3): δ ppm 5.30 - 5.20 (m, 1H), 4.38 - 4.17 (m, 4H), 3.94 - 3.84 (m, 4H), 3.12 - 3.07 (m, 4H), 2.32 - 2.02 (m, 14H), 1.58 - 1.57 (m, 4H), 1.24 - 0.89 (m, 66H), 0.87 - 0.85 (m, (dd, J = 0.6 Hz, 6H).

[0626] [Example 2] In vitro analysis of cytotoxicity in human hepatocytes or cancer cells The LNPs can be tested in vitro with a series of 10 dilutions to determine the IC50 within human hepatocyte / liver (HepG2; ATCC#HB8065) cells. Since these formulations generally appear to be non-toxic, a positive control of Lipofectamine™ 3000 (ThermoFisher#L3000015) complexed mRNA (2 μL of reagent / 1 μg of mRNA) is included in all tests. The mRNA used is CleanCap FLuc, EGFP, or MCherry reporter gene mRNA (5 moU; Trilink#L - 7202, #L - 7201, or #L - 7203). Data are recorded from the calculation of the total cell survival curves and the actual IC50 values for each compound.

[0627] Grow adherent cells to approximately 80% confluency. Trypsinize the cells by adding 0.25% trypsin - EDTA (Gibco#25200 - 072), subsequently allowing the cells to settle, and adding 5 mL of growth medium (MEM medium; Corning#10010CM) to disperse the cells. Determine the cell density using a hemocytometer. Adjust the cells to an appropriate concentration by adding growth medium (MEM medium containing 10% FBS; Corning#35015CV). Then add 200 μL of cells (5,000 cells / well) to a 96 - well clear flat - bottom plate (Costar#9804) and incubate in the plate at 37 °C in a humidified incubator with 5% CO2 for 24 hours.

[0628] A serial dilution of the LNP formulation is prepared using the growth medium as a solvent. These compounds are produced as a sterile aqueous solution with a concentration of 1 mg / mL mRNA. To prepare the dilutions, each LNP stock was warmed to room temperature. These were further diluted 4-fold in the growth medium to the highest mRNA concentration tested of 250 μg / mL.

[0629] The old medium is aspirated and removed and replaced with 200 μL of LNP-containing medium, and the LNP is serially diluted 1:3 from an initial concentration of 250 μg / mL of each LNP and added to the wells. The plates are incubated for 72 hours at 37 °C with 5% CO2 in a humidified incubator. At the end of the LNP incubation period, the medium in each well is replaced with 100 μL of 1X PrestoBlue Cell Viability reagent (ThermoFisher catalog #A13261). The plates are incubated for 30 minutes to 2 hours at 37 °C with 5% CO2 in a humidified incubator. Read at 30, 60, and 120 minutes. Fluorescence with excitation at 560 nm and emission at 590 nm is read using a SpectraMax M5 plate reader (Molecular Devices). Background is corrected by subtracting the RFU of a control containing only culture medium (background control wells) from the readings of all samples. The percentage of cytotoxicity is calculated using the following formula: % Cytotoxicity = [(RFU 培地 - RFU 処置 ) / RFU 培地 × 100% The IC50 was determined using GraphPad Prism using the following formula: Y = 100 / (1 + 10^((LogIC50 - X) * HillSlope)))

[0630] The cytotoxicity of Lipofectamine™ 3000 (ThermoFisher #L3000015) complexed with mRNA (2 μL of reagent / 1 μg of mRNA) positive control can be 5 to 100 times more toxic than the compounds disclosed herein in some embodiments. This indicates that the disclosed compounds are less toxic than commercially available transfection reagents in the analysis of in vitro hepatocyte cytotoxicity. In some embodiments, the compounds described herein form LNPs with lower toxicity than commercially available transfection reagents in vivo.

[0631] [Example 3] Determination of the pKa of Ionizable Lipids The pKa of ionizable cationic lipids can be calculated in several ways. This is sometimes difficult for lipids because the membrane structure and adjacent lipids in the membrane can affect the dissociation characteristics of the amino group, which can give inaccurate values. In this case, an in situ measurement where the apparent pKa of the ionizable lipid is measured while the lipid is in the intended environment as part of the LNP is ideal (Jayaraman 2012, Sabins 2018).

[0632] For each LNP formulation, the pKa value of the amino lipid is determined by measuring the fluorescence of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) during titration from pH 3 to 12. TNS is an anionic molecule that does not fluoresce in solution but fluoresces upon interaction with the positive lipid membrane, and this property has conventionally been used to examine the surface charge of the membrane. A master buffer stock (10 mM sodium phosphate, 10 mM sodium borate, 10 mM sodium citrate, 150 mM sodium chloride) is prepared for use in preparing buffer solutions of various pH values for determining the apparent pKa. Using 1 M sodium hydroxide and 1 M hydrochloric acid, approximately 20 specific buffer solutions of various pH values from about 3 to 12 are prepared from the master buffer stock. 300 mM sodium 6-(p-toluidino)-2-naphthalenesulfonate (TNS reagent) solubilized in dimethyl sulfoxide (DMSO) is used as a stock. LNPs are prepared and purified to obtain a buffer solution of the desired pH with a final mRNA concentration of 0.04 mg / mL. Using a 96-well plate, pre-inserted with the desired buffer, mRNA-containing LNPs are added such that the final concentration of mRNA becomes 0.7 μg / mL. TNS is added to each well such that the DMSO concentration becomes 1% (v / v). After mixing, the fluorescence of TNS in each well is measured (Ex / Em = 331 nm / 445 nm), and sigmoidal best fit analysis is applied to the fluorescence data. The pKa is determined as the pH that gives a half-maximal fluorescence intensity. The apparent pKa measured for Compounds 1 to 36 is in the range of pH 6.0 to 7.0.

[0633] [Example 4] Measurement of Cellular Uptake of LNP Measurement of LNP cellular uptake is achieved by fluorescence imaging and / or fluorescence quantification. Many suitable fluorescent tracers are available, such as 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), 3,3'-dilinoleyloxacarbocyanine perchlorate (DiO), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD), and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) (Thermo). These lipids exhibit some fluorescence in water but show high fluorescence when incorporated into lipid membranes such as those present in LNP. It is important that the selected lipids are photostable and have a high extinction coefficient.

[0634] LNPs containing these types of lipids can be visualized under a fluorescence microscope. In one method, the LNP lipid formulation contains a fluorescent lipid tracer such as 1,1'-dioctadecyl-3,3,3',3'-tetramethyindodicarbocyanine-5,5'-disulfonic acid (DiI5-DS) at 0.1 - 0.5 mol% of the total lipid. The cells of interest are grown in a suitable cell culture dish such as a 24-well plate (Corning). The cells are seeded the day before the uptake study at 50% confluence and grown overnight under appropriate conditions, e.g., at 37 °C, 5% CO2, and 90 - 100% humidity. The LNP is added to the cell culture medium at 0.1 - 100 μg / mL mRNA, allowed to interact with the cells for a certain period of time (4 - 24 hours), then the cells are washed three times with the medium to remove uninternalized LNP and observed. The cells are observed using a microscope equipped with fluorescence detection capabilities. Untreated cells are used as a background control to determine the relative extent of LNP cellular uptake from the fluorescence intensity signal obtained from the cells. Alternatively, the cells can be pelleted, solubilized using a detergent such as Triton-X100, and the fluorescence can be quantified by a spectrofluorometer or the fluorescent lipid tracer can be quantified by HPLC to achieve a quantitative measurement of the fluorescent cell lipids.

[0635] In a similar manner, quantification of fluorescently labeled mRNA is achieved. For example, both dye-labeled enhanced green fluorescent protein (EGFP) and firefly luciferase (FLuc) mRNA are transcribed with cyanine 5-UTP:5-methoxy-UTP in a ratio of 1:3 and are currently available from Trilink Biotechnologies. Cyanine 5 has an excitation maximum at 650 nm and an emission maximum at 670 nm. The mRNA obtained by substitution at this ratio can be easily visualized and can still be translated in cell culture. By capturing the fluorescently labeled mRNA, intracellular transport of mRNA by the above method can be visualized.

[0636] Intracellular LNP uptake can be achieved by endogenous methods such as ApoE mediation or exogenous methods such as active targeting. LNP systems containing ionizable cationic lipids adsorb apolipoprotein E (ApoE) in the blood (Cullis et al 2017) and are then actively taken up into hepatocytes by a number of receptors containing ApoE-binding ligands (Williams et al. 2010), and it has been found that a "natural" targeting process is utilized. By using non-overlapping fluorophores, it is possible to independently track the intracellular distribution and organelle accumulation kinetics of mRNA and LNP.

[0637] mRNA cell expression levels can be quantified by using reporter systems such as EGFP, FLuc or mCherry available from Trilink Biotechnologies. In one embodiment, EGFP mRNA is encapsulated in LNP and added to the target cells at 0.1 - 100 μg / mL mRNA. After 4 - 24 hours, the medium may be replaced to wash away the non-internalized LNP of the cells. At 24 hours, the GFP signal is quantified by fluorescence microscopy or flow cytometry. In this way, it is possible to distinguish a panel of LNP formulations based on the reporter protein expression level.

[0638] [Example 5] Transfection Selection Index Calculate the transfection selection index (TSI) to determine the relative transfection efficiency in mammalian cells (compared to relative toxicity in the same cells). The selection index was calculated using the following formula: TSI = EF 哺乳類 / IC 50、哺乳類

[0639] where EF 哺乳類 is the transfection efficiency expressed as protein (ng) per million cells, and IC 50、哺乳類 is related to the cell viability of the same formulation converted to the half-maximal inhibitory concentration.

[0640] The LNPs using the compounds (1 - 36) described herein have a TSI 50% higher than the LNPs made using the same LNPs except that the control molecule DLin - MC3 - DMA was used as the ICL.

[0641] [Example 6] Analysis of lipid peroxidation reaction The degree of oxidation can be determined using a forced degradation assay in which LNP samples are treated with 3% H2O2 at 25 °C and lipid oxidation products are sampled on days 0, 1, 3, and 5 (Blessy et al. (2014) Journal of Pharmaceutical Analysis 4, 159 - 165). The oxidation reaction can be deactivated by adding 0.1 M butylated hydroxytoluene (BHT) in ethanol and stored frozen at -80 °C until measurement. Lipid oxidation products can be measured using a 2 - thiobarbituric acid (TBA) reactive assay (Gutteridge (1982) FEBS Letters 150, 454 - 458) to detect malondialdehyde (MDA), the final product of the lipid peroxidation reaction, or it may be by detection using an HPLC assay with evaporative light scattering detection (ELSD) or charged aerosol detection (CAD). The impurity structures of lipid oxidation and isomerization can be assigned based on known prior literature and are predicted to be mixtures of isomers.

[0642] Generally, in the art, lipids having multiple unsaturations in the acyl chain are known to be sensitive to oxidation (see Reis and Spickett (2012) Biochim Biophys Acta 1818, 2374-2387).

[0643] Compounds 1-36 described herein are presumed to be less vulnerable to oxidative damage or degradation when compared to control LNPs containing DLin-KC2-DMA lipid or when compared to control LNPs containing DLin-MC3-DMA. In some embodiments, the compounds provided herein have more than 30%, more than 50%, more than 75%, more than 90%, and more than 95% less oxidative by-products when compared to control LNPs.

[0644] [Example 7] Preparation of Ligand-Targeted LNPs Antibody ligands in the form of antibody Fab’ fragments or single-chain Fv fragments that result in specific uptake of LNPs into target cells such as immune cells are prepared by any method known in the art (e.g., Drummond et al., U.S. Patent Application No. 20180271998, Zhou et al., U.S. Patent No. 10,406,225, Marks et al., U.S. Patent No. 8,974,792, which are incorporated herein by reference). To effect conjugation of the ligand to the LNP, the ligand is constructed with a C-terminal sequence having a cysteine residue (such as CAA or GGSGGC) and is subsequently conjugated to the maleimide-terminated PEG-DSPE anchor as shown in Figure 2. The ligand is expressed in bacteria or eukaryotic cells and isolated from cell pellets or growth media using standard methods such as protein affinity chromatography or metal chelation chromatography. To activate the thiol group of the terminal cysteine residue, the ligand is incubated for 1 hour in the presence of 15 mM cysteine in 10 mM citrate buffer (pH 6.0 - 6.2) containing 140 mM NaCl and purified by gel chromatography using 10 mM citrate buffer (pH 6.0 - 6.2) containing 140 mM NaCl as the eluent on a Sephadex G-25 or similar column. The protein concentration in the purified cysteine-activated ligand solution is determined using UV spectrophotometry at 280 nm. An aqueous solution of the maleimide-terminated PEG-DSPE derivative (mal-PEG(2000)-DSPE, catalog number 880126, Avanti Polar Lipids, Alabama, USA, or Sunbright® DSPE-020MA, NOF Corporation, Japan) is mixed with the antibody ligand at a protein / lipid molar ratio of 4:1 at 1 - 10 mg / mL in the buffer named above.When it is desirable that the distance between the LNP surface and the ligand moiety be long, a Mal-PEG-lipid having a PEG spacer with a molecular weight of 3,400 (Sunbright® DSPE-034MA) or 5,000 (Sunbright® DSPE-050MA) available from NOF Corporation can be used. The solution is incubated at ambient temperature for 2 hours, adju...

Claims

1. Nucleic acid lipid nanoparticle (LNP) composition containing the following: a. Nucleic acid; b. Ionized cationic lipids in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition; c. Sterols in a total amount of 25 to 45 mol% of the total lipid content of the LNP composition; and d. One or more phospholipids comprising 5 to 25 mol% of the total lipid content of the LNP composition, and containing 2.5 to 10 mol% of phosphatidylglycerol (PG) in total; and e. Furthermore, a total amount of composite lipids of 0.5 to 2.5 mol% of the total lipid content of the LNP composition.

2. The composition according to claim 1, wherein the composition comprises an anionic phospholipid selected from the group consisting of DSPG and DPPG.

3. The composition according to claim 1, wherein the ionized cationic lipid is a KC3 ionized cationic lipid.

4. The composition according to claim 3, wherein the ionized cationic lipid has a monounsaturated alkyl chain.

5. Lipid nanoparticles (LNPs) containing PG for use in a therapeutic method, wherein the therapeutic method comprises delivering nucleic acids to human dendritic cells, and comprising the step of bringing the lipid nanoparticles (LNPs) into contact with the human dendritic cells.

6. LNP for use in the therapeutic method according to claim 5, wherein the PG is present in a concentration of 1 mol% to 10 mol% of the total lipid content of the LNP.

7. An LNP for use in the therapeutic method according to claim 5, wherein the acyl chain of the phosphatidylglycerol is completely saturated, and the PG is optionally selected from the group consisting of dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), or distearoyl phosphatidylglycerol (DSPG).

8. An LNP for use in the therapeutic method according to any one of claims 5 to 7, wherein the LNP is defined in any one of claims 1 to 4.

9. A nucleic acid lipid nanoparticle (LNP) composition for use in a therapeutic method for human subjects, wherein the method comprises targeting the LNP to human dendritic cells, and the nucleic acid LNP composition comprises the following: a. Nucleic acid; b. Ionized cationic lipids in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition, with an N / P ratio of 3 to 8 relative to the nucleic acid; c. Sterols in a total amount of 25 to 45 mol% of the total lipid content of the LNP composition; d. One or more phospholipids comprising 5 to 25 mol% of the total lipid content of the LNP composition, and containing 1.0 to 10 mol% of phosphatidylglycerol (PG) in total; and e. A total amount of composite lipids of 0.5 to 2.5 mol% of the total lipid content of the LNP composition.

10. Nucleic acid lipid nanoparticle (LNP) composition containing the following: a. Nucleic acid; b. Ionized cationic lipids in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition, with an N / P ratio of 3 to 8 relative to the nucleic acid; c. Sterols in a total amount of 25 to 45 mol% of the total lipid content of the LNP composition; d. One or more phospholipids in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition; and e. A composite lipid in a total amount of 0.5 to 2.5 mol% of the total lipid content of the LNP composition, comprising a PEG-lipid having a poly(ethylene glycol) chain terminated at the binding portion and two hydrocarbon chains terminated at the same binding portion, wherein the hydrocarbon chains are saturated C12 chains independently selected from an n-dodecyl (lauryl) group and an n-dodecanoyl (lauroyl) group.

11. The composition according to claim 10, wherein the poly(ethylene glycol) chain is methoxy-poly(ethylene glycol) having an average molecular weight of 2000.

12. The composition according to claim 10, wherein the PEG-lipid is mPEG-1,2-dilauroylglycerol (PEG-DLG), mPEG-1,2-dilaurylglycerol (PEG-DLG), PEG-1,2-dilaurylglycerol, PEG-DLPE, PEG-oxycarbonyl-N,N-didodecylamide, or mPEG-N,N-didodecylacetamide.

13. The composition according to any one of claims 10 to 12, wherein the one or more phospholipids comprises a total amount of phosphatidylserine (PS) lipids of 2.5 to 10 mol% of the total lipid content of the LNP composition, and the LNP composition comprises a total amount of phosphatidylserine (PS) lipids of 2.5 to 10 mol% of the total lipid content of the LNP composition.

14. The LNP composition is a human vaccine composition, and: The one or more phospholipids include phosphatidylglycerol (PG) (optionally DSPG or DPPG) in a total amount of 1.0 to 10 mol% of the total lipid content of the LNP composition; and The composite lipid is selected from PEG-DLG and PEG-DLPE. The nucleic acid lipid nanoparticle (LNP) composition according to claim 10.

15. In the preparation of liposome nanoparticle (LNP) compositions, use of ammonia salts of an anionic phospholipid composition having the following chemical structure (V-A-1): 【Chemistry 1】 In the ceremony, X ＋ is an ammonium cation; and a is 14, 15, or 16.

16. The use according to claim 15, wherein the ammonia salt of the anionic phospholipid composition of formula (V-A-1) is selected from the group consisting of ammonium salts of distearoylphosphatidyl-L-serine (DSPS L-isomer) and ammonium salts of DPPS (L-isomer).

17. A method for preparing a nucleic acid delivery composition containing a lipid, wherein the lipid contains phosphatidylserine and optionally contains DPPS, the method comprising the step of dissolving phosphatidylserine in ethanol, and the phosphatidylserine is in the form of an ammonium salt of the phosphatidylserine.

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