Lipid nanoparticles for delivery of nucleic acids and methods of use thereof

Abstract

The present disclosure provides compositions of improved ionizable lipid nanoparticles for delivery of therapeutic nucleic acids to cells. Anionic phospholipids, including phosphatidylserine and phosphatidylglycerol, are included in the lipid nanoparticles to improve transfection efficiency in human dendritic cells. Further incorporation of monounsaturated alkyl chain analogs in dimethylaminopropyl-dioxolane or heterocyclic ketal ionizable lipids in formulations, as compared to other ionizable lipids in the same family, shows high levels of transfection in human dendritic cells, and shows good stability against oxidative damage. Finally, the use of ammonium salts of phosphatidylserine allows efficient production of PS-targeted LNPs.

Description

[0001] Related applications

[0002] This patent application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 345,823, filed on May 25, 2022, U.S. Provisional Patent Application No. 63 / 346,197, filed on May 26, 2022, and U.S. Utility Patent Application No. 18 / 324,097, filed on May 25, 2023, the entire contents of each of which are hereby incorporated by reference in their entirety.

[0003] References to sequence listings

[0004] This specification includes a Sequence Listing filed herewith, comprising a file entitled 191016-010503.xml created on May 24, 2023, having the following size: 17,585 bytes, the contents of which are incorporated herein by reference. Technical Field

[0005] The present disclosure relates to cationic ionizable lipids and lipid nanoparticles (LNPs). In some embodiments, LNPs comprising one or more cationic ionizable lipids can be used for the delivery of nucleic acid compounds, for dendritic cell targeting, or for methods of using these LNP compositions as vaccines. In some embodiments, LNPs can comprise bioreducible ionizable cationic lipids or unconjugated polyolefin ionizable cationic lipids. Background Art

[0006] 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 contain ionizable cationic lipids (ICLs). However, some ICL compounds known in the art are undesirably sensitive to oxidation during storage. Therefore, there is a need for improved ICL compounds that have improved stability to oxidative degradation during storage while also providing desired transfection activity or efficacy in cells when incorporated into LNPs together with therapeutic agents, such as nucleic acids.

[0007] LNP compositions, including stable nucleic acid lipid particles (SNALP) compositions, can be used to deliver nucleic acid therapeutics for a variety of infectious diseases. Infectious diseases such as tuberculosis, HIV / AIDS, malaria and COVID-19 present a major challenge to human health. For example, mycobacteria is a genus of bacteria that causes tuberculosis (TB). According to the World Health Organization, TB is one of the top 10 causes of death and the main cause of death due to a single infectious agent worldwide. Despite current best efforts, there are still major challenges in developing effective vaccines for preventing many infectious diseases. New efforts to identify single antigenic peptides or combinations of antigenic peptides help to improve the efficiency of vaccines. Nevertheless, there are still important opportunities in transforming adjuvants to help effectively deliver and present these antigen sequences to professional antigen-presenting cells such as dendritic cells. The mRNA encoding antigenic peptides or antigenic proteins combined with ionizable cationic lipid nanoparticles represents a particularly promising strategy in vaccine development. There is a need for safe and effective therapeutic agents comprising LNP pharmaceutical compositions, including vaccine compositions, for the delivery of mRNA for the treatment and prevention of a variety of diseases. Summary of the Invention

[0008] Some aspects of the present disclosure relate to lipid nanoparticle (LNP) vaccine 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, wherein the ionizable lipid has the following chemical structure:

[0009]

[0010] Where R1 is a C containing one or two olefins 15 -C 19 alkyl, and wherein the ionizable cationic lipid is present in the LNP vaccine composition in a total amount of 40 to 65 mol% of the total lipid content in the LNP composition, and the ionizable cationic lipid is optionally selected from the following group: 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); (c) sterols, whose total amount is 25 to 45 mol% of the total lipid content in the LNP composition; (d) one or more phospholipids, whose total phospholipid amount is 5 to 25 mol% of the total lipid content in the LNP composition; and (e) conjugated lipids, whose total amount is 0.5 to 2.5 mol% of the total lipid content in the LNP composition.

[0011] In some embodiments, the one or more phospholipids comprise phosphatidylserine (PS) lipids in a total amount of 2.5 to 10 mol % of the total lipid content in the LNP composition. In some embodiments, the PS-lipid is DSPS or DPPS.

[0012] In some embodiments, the second phospholipid is selected from the group consisting of: DSPC, HSPC, DPPC, and sphingomyelin.

[0013] In some embodiments, the ionizable cationic lipid comprises a monounsaturated alkyl chain or a diunsaturated alkyl chain wherein the olefins are separated by at least two methylene groups.

[0014] 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).

[0015] In some embodiments, the composition has an N / P ratio of 5 to 6 relative to nucleic acid.

[0016] 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-methyl pseudouridine.

[0017] Other aspects of the present disclosure relate to lipid nanoparticle (LNP) compositions comprising: (a) a nucleic acid; and (b) an ionizable cationic lipid having an N / P ratio of 3 to 8 relative to the nucleic acid, wherein the ionizable lipid has the following chemical structure:

[0018]

[0019] Where R1 is a C containing one or two olefins 15 -C 19alkyl, and wherein the ionizable cationic lipid is present in the LNP composition in a total amount of 40 to 65 mol% of the total lipid content in the LNP composition, and the ionizable cationic lipid is optionally selected from the group consisting of Compound 3 (Table 1A), Compound 8 (Table 1A), DLin-KC3-DMA, KC3-01, KC33--OA, KC3-PA, KC3-C17 (8:1), and KC3-C15 (C8:1); (c) sterols in a total amount of 25 to 45 mol% of the total lipid content in the LNP composition; (d) one or more phospholipids in a total amount of phospholipids of 5 to 25 mol% of the total lipid content in the LNP composition, and comprising dipalmitoylphosphatidylserine (DPPS) lipids in a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition; and (e) conjugated lipids in a total amount of 0.5 to 2.5 mol% of the total lipid content in the LNP composition.

[0020] In some embodiments, the second phospholipid is selected from the group consisting of: DSPC, HSPC, and sphingomyelin.

[0021] In some embodiments, the N / P ratio of the ionizable cationic lipid relative to the nucleic acid is 5 to 6.

[0022] In some embodiments, the ionizable cationic lipid comprises two monounsaturated alkyl chains or two diunsaturated alkyl chains, and wherein the olefins are separated by at least two methylene groups.

[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).

[0024] 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).

[0025] In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA is chemically modified with N-methylpseudouridine.

[0026] Some 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 relative to the nucleic acid of 3 to 8, in a total amount of 40 to 65 mol% of the total lipid content in the LNP composition; (c) a sterol, in a total amount of 25 to 45 mol% of the total lipid content in the LNP composition; (d) one or more phospholipids, in a total amount of phospholipids of 5 to 25 mol% of the total lipid content in the LNP composition; and (e) a conjugated lipid, in a total amount of 0.5 to 2.5 mol% of the total lipid content in the LNP composition, the conjugated lipid comprising a PEG-lipid having a poly(ethylene glycol) chain terminally attached to a linker moiety and two hydrocarbon chains terminally attached to the same linker moiety, wherein the hydrocarbon chains are saturated C 2-hydroxy-1,1-diols independently selected from n-dodecyl (lauryl) and n-dodecanoyl (lauroyl). 12 chain.

[0027] In some embodiments, the linker moiety is glyceryl, N-oxycarbonylglycerophosphoethanolamidocarbonyl, oxycarbonylamide, or oxyacetamide. In some embodiments, the poly(ethylene glycol) chain is methoxy-poly(ethylene glycol) having an average molecular weight of 2000. In some embodiments, 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-didodecanamide, or mPEG-N,N-didodecanacetamide.

[0028] In some embodiments, the LNPs have a z-average particle size of 60 to 150 nm and are freeze-thaw stable.

[0029] In some embodiments, the one or more phospholipids comprise phosphatidylserine (PS) lipids in a total amount of 2.5 to 10 mol % of the total lipid content in the LNP composition, and wherein the LNP composition comprises phosphatidylserine (PS) lipids in a total amount of 2.5 to 10 mol % of the total lipid content in the LNP composition.

[0030] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) human vaccine 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 in the LNP composition; (c) a sterol, in a total amount of 25 to 45 mol% of the total lipid content in the LNP composition; (d) one or more phospholipids, in a total amount of phospholipids of 5 to 25 mol% of the total lipid content in the LNP composition, and comprising phosphatidylglycerol (PG) in a total amount of 1.0 to 10 mol% of the total lipid content in the LNP composition; and (e) a conjugated lipid, in a total amount of 0.5 to 2.5 mol% of the total lipid content in the LNP composition.

[0031] 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).

[0032] In some embodiments, the sterol is cholesterol; and the phosphatidylglycerol (PG) is an anionic phospholipid selected from distearoylphosphatidylglycerol (DSPG) and dipalmitoylphosphatidylglycerol (DPPG).

[0033] In some embodiments, the nucleic acid is mRNA and the ionizable cationic lipid is present at an N / P ratio of 4 to 7 relative to the nucleic acid.

[0034] In some embodiments, the conjugated lipid is selected from the group consisting of PEG-DMG, PEG-DLG, and PEG-DLPE.

[0035] In some embodiments, the one or more phospholipids comprise a phospholipid selected from distearoylphosphatidylcholine (DSPC) and hydrogenated soy phosphatidylcholine (HSPC).

[0036] Some aspects of the present disclosure relate to methods of preparing a nucleic acid delivery composition comprising a lipid, wherein the lipid comprises phosphatidylserine, the method comprising the step of dissolving the phosphatidylserine in ethanol, wherein the phosphatidylserine is in the form of an ammonium salt of the phosphatidylserine.

[0037] In some embodiments, phosphatidylserine is DPPS. In some embodiments, phosphatidylserine is dissolved in ethanol to a concentration exceeding 0.2 mM. In some embodiments, ammonium salt is a salt comprising ammonium selected from: ammonium, alkylammonium, dialkylammonium, trialkylammonium and tetraalkylammonium. In some embodiments, the ammonium form is selected from: ammonia, dimethylamine, diethylamine, triethylamine, trimethylamine, 2-(dimethylamino) ethanol, diethanolamine, 2-(diethylamino) ethanol, ethanolamine, ethylenediamine, N-methyl-glucosamine, imidazole, histidine, lysine, arginine, 4-(2-hydroxyethyl)-morpholine, piperazine, 1-(2-hydroxyethyl)-pyrrolidine, triethanolamine or tromethamine (tris(hydroxymethyl)aminomethane).

[0038] Some aspects of the present disclosure relate to methods for delivering a nucleic acid into a cell, comprising the steps of contacting a lipid nanoparticle (LNP) with the cell, wherein the cell is a human dendritic cell, wherein the LNP composition is obtained by the methods of some aspects of the present disclosure.

[0039] In some embodiments, the LNP comprises an ionizable cationic lipid of the disclosure.

[0040] In some embodiments, the cell is in a human or animal subject.In some embodiments, the LNP is administered intramuscularly, subcutaneously, intradermally, or topically to the subject.

[0041] Some aspects of the present disclosure relate to LNP compositions comprising: (a) a nucleic acid, wherein the nucleic acid is mRNA; (b) a cholesterol sterol in a total amount of 25 to 40 mol % of the total lipid content in the LNP composition; (c) 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 in the LNP composition; (d) one or more phospholipids in a total amount of phospholipids of 5 to 25 mol % of the total lipid content in the LNP composition, the one or more phospholipids being selected from (i) an ammonium salt of dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS) lipid in a total amount of 2.5 to 10 mol % of the total lipid content in the LNP composition; and (ii) a distearoylphosphatidylcholine (DSPC) phospholipid in a total amount of 5 to 25 mol % of the total lipid content in the LNP composition; and (e) a PEG-containing conjugated lipid in a total amount of 0.5 to 2.5 mol % of the total lipid content in the LNP composition.

[0042] Some aspects of the present disclosure relate to methods for administering a nucleic acid to a subject in need thereof, comprising administering to a human subject a nucleic acid lipid nanoparticle (LNP) composition comprising: (a) a nucleic acid; (b) an ionizable cationic lipid having an N / P ratio relative to the nucleic acid of 3 to 8, in a total amount of 40 to 65 mol% of the total lipid content in the LNP composition; (c) a sterol, in a total amount of 25 to 45 mol% of the total lipid content in the LNP composition; (d) one or more phospholipids, in a total amount of phospholipids of 5 to 25 mol% of the total lipid content in the LNP composition, and the one or more phospholipids comprise phosphatidylglycerol (PG) in a total amount of 1.0 to 10 mol% of the total lipid content in the LNP composition; and (e) a conjugated lipid, in a total amount of 0.5 to 2.5 mol% of the total lipid content in the LNP composition.

[0043] Some 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, the total amount of which is 40 to 65 mol% of the total lipid content in the LNP composition; (c) a sterol, the total amount of which is 25 to 45 mol% of the total lipid content in the LNP composition; (d) one or more phospholipids, the total amount of which is 5 to 25 mol% of the total lipid content in the LNP composition, and including a phosphatidylserine (PS) lipid in an amount of 2.5 to 10 mol% of the total lipid content in the LNP composition; and (e) a conjugated lipid, the total amount of which is 0.5 to 2.5 mol% of the total lipid content in 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 conjugated lipid is a PEG-containing conjugated lipid.

[0044] In some embodiments, the one or more phospholipids comprise at least two phospholipids with mismatched acyl chain lengths.

[0045] In some embodiments, the phosphatidylserine (PS) lipid is dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS).

[0046] In some embodiments, the one or more phospholipids comprise a phospholipid selected from 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 conjugated lipid is PEG (2000)-dimyristoylglycerol (PEG-DMG).

[0047] Some aspects of the present disclosure relate to LNP compositions wherein the ionizable lipid has the following chemical structure:

[0048]

[0049] Where R1 is

[0050]

[0051] 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;

[0052] R2 and R3 are each independently (C1-C4)alkyl optionally substituted with hydroxy; and

[0053] n is an integer equal to 2, 3 or 4.

[0054] In some embodiments, R2 and R3 are each methyl; and n is 3 or 4.

[0055] 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).

[0056] Some aspects of the present disclosure relate to ionizable cationic lipids selected from KC3-PA, KC3-C17(8:1), and KC3-C15(C8:1).

[0057] Some 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 in the LNP composition; (c) a sterol, in a total amount of 25 to 45 mol % of the total lipid content in the LNP composition; (d) one or more phospholipids, in a total amount of phospholipids of 5 to 25 mol % of the total lipid content in the LNP composition, and including a phosphatidylserine (PS) lipid in a total amount of 2.5 to 10 mol % of the total lipid content in the LNP composition; and (d) a conjugated lipid, in a total amount of 0.5 to 2.5 mol % of the total lipid content in the LNP composition.

[0058] In some embodiments, sterol is cholesterol. In some embodiments, one or more phospholipids include phospholipids with mispairing acyl chain length. In some embodiments, PS lipid is dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS). In some embodiments, one or more phospholipids include phospholipids selected from distearoylphosphatidylcholine (DSPC) and hydrogenated soybean phosphatidylcholine (HSPC). In some embodiments, one or more phospholipids include distearoylphosphatidylcholine (DSPC) and phosphatidylserine (PS). In some embodiments, ionizable cationic lipid is 3-((S)-2,2-bis((Z)-octadec-9-ene-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylpropane-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-dimethylpropane-1-amine (KC3-OA). In some embodiments, the one or more phospholipids are composed of distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS), and the phosphatidylserine (PS) is (L-serine) DPPS.

[0059] In some embodiments, the conjugated lipid is a PEG-containing conjugated lipid, and wherein the PEG-containing conjugated lipid is selected from: PEG(2000)-dimyristoylglycerol (PEG-DMG), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-DLPE) and PEG(2000)-dilauroylglycerol (PEG-DLG).

[0060] 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 conjugated lipid comprises polyethylene glycol (PEG). In some embodiments, the conjugated lipid is PEG (2000) - dimyristoylglycerol (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-dimethylpropane-1-amine (KC3-OA). In some embodiments, the one or more phospholipids consist of distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS).

[0061] In some embodiments, dipalmitoylphosphatidyl-L-serine ((L-serine)DPPS) is the ammonium salt of (L-serine)DPPS.

[0062] In some embodiments, (a) the nucleic acid is mRNA; (b) the sterol is a cholesterol sterol and its total amount is 25 to 45 mol% of the total lipid content in the LNP composition; (c) the ionizable cationic lipid has an N / P ratio relative to the nucleic acid of 3 to 8 and its total amount is 40 to 65 mol% of the total lipid content in the LNP composition; (d) the total amount of phospholipids of the one or more phospholipids is 5 to 25 mol% of the total lipid content in the LNP composition, and the one or more phospholipids consists of the following: (i) dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS) lipid, its total amount is 2.5 to 10 mol% of the total lipid content in the LNP composition; and (ii) distearoylphosphatidylcholine (DSPC) phospholipid, its total amount is 5 to 25 mol% of the total lipid content in the LNP composition; (e) the conjugated lipid is a PEG-containing conjugated lipid, its total amount is 0.5 to 2.5 mol% of the total lipid content in the LNP composition.

[0063] In some embodiments, the PEG-containing conjugated lipid is selected from: PEG(2000)-dimyristoylglycerol (PEG-DMG), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-DLPE) and PEG(2000)-dilauroylglycerol (PEG-DLG).

[0064] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) human vaccine 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, the total amount of which is 40 to 65 mol% of the total lipid content in the LNP composition; (c) a sterol, the total amount of which is 25 to 45 mol% of the total lipid content in the LNP composition; (d) one or more phospholipids, the total amount of which is 5 to 25 mol% of the total lipid content in the LNP composition, and the total amount of which is 1.0 to 10 mol% of the total lipid content in the LNP composition. mo 1% phosphatidylglycerol (PG); and (e) conjugated lipids, the total amount of which is 0.5 to 2.5 mol% of the total lipid content in the LNP composition.

[0065] In some embodiments, the sterol is cholesterol; and the phosphatidylglycerol (PG) is an anionic phospholipid selected from distearoylphosphatidylglycerol (DSPG) and dipalmitoylphosphatidylglycerol (DPPG).

[0066] In some embodiments, the nucleic acid is mRNA and the ionizable cationic lipid is present at an N / P ratio of 4 to 7 relative to the nucleic acid.

[0067] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) compositions comprising an ionizable cationic lipid in a total amount of 40 to 65 mol % of the total lipid content in 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:

[0068]

[0069]

[0070] Some 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 relative to the nucleic acid of 3 to 8, in a total amount of 40 to 65 mol% of the total lipid content in the LNP composition; (c) a sterol, in a total amount of 25 to 45 mol% of the total lipid content in the LNP composition; (d) one or more phospholipids, in a total amount of phospholipids of 5 to 25 mol% of the total lipid content in the LNP composition; and (d) a conjugated lipid, in a total amount of 0.5 to 2.5 mol% of the total lipid content in the LNP composition, wherein the conjugated lipid is a PEG-lipid having a poly(ethylene glycol) chain terminally attached to a linker moiety and two hydrocarbon chains terminally attached to the same linker moiety, wherein the two hydrocarbon chains are saturated C 2-hydroxy-1,1-diols independently selected from n-dodecyl (lauryl) and n-dodecanoyl (lauroyl). 12 chain.

[0071] In some embodiments, the linker moiety is glyceryl, N-oxycarbonylglycerophosphoethanolamidocarbonyl, oxycarbonylamide, or oxyacetamide. In some embodiments, the poly(ethylene glycol) chain is methoxy-poly(ethylene glycol) having an average molecular weight of 2000. In some embodiments, 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-didodecanamide, or mPEG-N,N-didodecanacetamide. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The mechanism of oxidative degradation of linoleic acid lipid esters containing conjugated polyunsaturates that are particularly sensitive to oxidation is delineated.

[0073] Figure 2Shown is the reaction of the reduced c-terminal cysteine ​​of a Fab' antibody fragment with maleimide-terminated poly(ethylene glycol) 2000-derivatized distearoylphosphatidylethanolamine. R1 and R2 are stearic acid. The final antibody-lipopolymer conjugate is an intermediate that is subsequently inserted into the outer lipid layer of lipid nanoparticles for their active targeting.

[0074] Figure 3A uses DLin-KC2-DMA as the mCherry mRNA LNP formulated with ionizable cationic lipids, and the effect of 0 to 2.5 mol% DSPS inclusion on the transfection efficiency of dendritic cells (MutuDC1940). ICL is maintained at 50 mol%, cholesterol is maintained at 38.5 mol%, PEG-DMG is maintained at 1.5 mol%, and DSPS content is varied. DSPS is included by reducing the DSPC content to the same mol% as the added DSPS. Cells were incubated for 24 hours with each preparation at a concentration of 1 μg mRNA / mL. The UT sample corresponds to cells to which LNP was not added. Lipofect refers to samples treated with Lipofectamine.

[0075] FIG3B uses DLin-KC2-DMA as the mCherry mRNA LNP prepared with ionizable cationic lipids, and the effect of 0 to 7.5 mol% DSPS inclusion on the transfection efficiency of dendritic cells (MutuDC1940). ICL is maintained at 50 mol%, cholesterol is maintained at 38.5 mol%, PEG-DMG is maintained at 1.5 mol%, and DSPS content is varied. DSPS is included by reducing DSPC content to the same mol% as the added DSPS. Cells are incubated for 24 hours with each preparation at a concentration of 1 μg mRNA / mL. UT samples correspond to cells to which LNPs are not added. Lipofectamine transfection refers to samples treated with lipofectamine.

[0076] Fig. 3 C uses DLin-KC2-DMA as the mCherry mRNA LNP that can be prepared with ionizable cationic lipid, and the influence of DSPS inclusion of 0 to 7.5mol% on the transfection efficiency of dendritic cells (MutuDC1940).ICL is maintained at 50mol%, cholesterol is maintained at 38.5mol%, PEG-DMG is maintained at 1.5mol%, and DSPS content changes.DSPS is included by reducing DSPC content to the same mol% as added DSPS.Cells are incubated for 24 hours with each preparation that concentration is 0.3μg mRNA / mL.UT sample corresponds to cells without LNP addition.

[0077] Fig. 3 D uses DLin-KC2-DMA as the mCherry mRNA LNP prepared with ionizable cationic lipids, and the influence of DSPS inclusions of 0 to 7.5mol% on the transfection efficiency of dendritic cells (MutuDC1940). ICL is maintained at 50mol%, cholesterol is maintained at 38.5mol%, PEG-DMG is maintained at 1.5mol%, and DSPS content changes. DSPS is included by reducing DSPC content to the same mol% as added DSPS. Cells are incubated for 24 hours with each preparation having a concentration of 0.1 μg mRNA / mL. UT samples correspond to cells without LNP addition.

[0078] Figure 4 shows the transfection of murine dendritic cells (MutuDC1940) with LNPs containing various ICLs (KC2, KC2-OA, KC3-OA, and SM-102) and 5 mol% DSPS, compared with LNPs containing Glu-DSPE or Suc-DSPE instead of DSPS. UT samples correspond to cells to which no LNPs were added.

[0079] Figure 5. DSPS or DPPS enhances transfection of mCherry LNPs with ICLs containing KC2, KC2-01, KC2-PA, KC3-01, and KC3-OA. UT samples correspond to cells to which no LNPs were added.

[0080] FIG6A compares the transfection of murine dendritic cells using various chemical forms of phosphatidylserine and LNPs containing AKG-UO-1. UT samples correspond to cells to which no LNPs were added. Lipo refers to Lipofectamine MessengerMax (ThermoFisher) used according to the manufacturer's instructions at the same dose level as the LNPs.

[0081] FIG6B compares DSPS with other negatively charged phospholipids in the context of transfection of murine dendritic cells using LNPs containing AKG-UO1. UT samples correspond to cells to which no LNPs were added. Lipo refers to Lipofectamine MessengerMax (ThermoFisher) used according to the manufacturer's instructions at the same dose level as the LNPs.

[0082] Figure 7 Effect of DSPS concentration in LNPs containing AUG-UO-1 on transfected dendritic cells. UT samples correspond to cells to which no LNPs were added.

[0083] Figure 8 Effect of PEG-DMG concentration in LNPs containing AUG-UO-1 on transfected dendritic cells with and without 5 mol% DSPS. The Y axis shows the % PEG used in the composition, followed by the concentration of mRNA added to the cells (0.11, 0.33, or 1 μg / mL). The UT sample corresponds to cells to which no LNPs were added.

[0084] FIG9A Oxidative degradation of lipid suspensions of ICLs with a single methylene group between two alkenes (KC2, KC3, and O-11769) and ICLs with four methylene groups between two alkenes (KC2-01, KC3-01, and UO-1).

[0085] FIG9B shows oxidative degradation of liposomes containing O-11769, an ICL with a single methylene group between two olefins, and liposomes containing UO-1, an ICL with four methylene groups between two olefins.

[0086] Figure 10A shows the effect of N / P on mCherry expression in murine dendritic cells in response to 1 μg / ml of LNPs containing KC2-01. The UT sample corresponds to cells to which no LNPs were added.

[0087] Figure 10B shows the effect of N / P on mCherry expression in mouse dendritic cells in response to 0.33 μg / ml of LNPs containing KC2-01. The UT sample corresponds to cells to which no LNPs were added.

[0088] Figure 11 Transfection efficiency of LNPs with and without DSPS (7.5 mol%) and containing different ionizable cationic lipids. The UT sample corresponds to cells without LNP addition.

[0089] Figure 12 Transfection efficiency of LNP formulations containing various concentrations of DOPS (0, 10 and 25 mol%, as % of total lipids) and mCherry mRNA in murine dendritic cells.

[0090] Figure 13A The mRNA sequence of VRN-029, which is the sequence producing the SARS-COV2 spike protein.

[0091] FIG13B Effect of PEG-DMG (C14) concentration (mol%) on LNP vaccine immunogenicity. Total anti-spike antibody titers and CD4 responses from mice immunized with mRNA-LNPs using 7.5% DSPS and ionizable lipid UO1 with increasing mol% of PEG-DMG. The middle graph shows the endpoint antibody titers at day 34. The right graph shows the corresponding CD4 T cell responses.

[0092] FIG13C Effect of PEG-DPPE (C16) concentration (mol%) on LNP vaccine immunogenicity. Total anti-Spike antibody titers from mice immunized with mRNA-LNPs using 7.5% DSPS and the ionizable lipid UO1 with increasing mol% of PEG-DPPE. The middle graph shows the endpoint antibody titers at day 34. The mol% of PEG-DPPE inversely affects antibody levels. The right graph shows the corresponding CD4 T cell responses.

[0093] Figure 13D Total anti-spike antibody titers and CD4 responses from mice immunized with mRNA-LNPs containing 7.5% DSPS and the ionizable lipid KC2OA and 1.5 mol% PEG-DMG (14C) or PEG-DSG (18C). The left graph shows the endpoint antibody titers at day 34. The right graph shows the corresponding CD4 T cell responses.

[0094] Figure 13E Total anti-spike antibody titers and CD4 responses from mice immunized with mRNA-LNPs containing 7.5% DSPS and ionizable lipid UO1 and 1.5 mol% PEG-DMG (14C) or PEG-DSG (18C). The left graph shows the endpoint antibody titers at day 34. The right graph shows the corresponding CD4 T cell responses.

[0095] Figure 13F Effect of phosphatidylserine incorporation on mRNA-LNP immunogenicity. Total anti-Spike antibody titers (A) and Spike-specific CD4 T cell responses from mice immunized with mRNA-LNPs using various ionizable lipids and PEG-lipids plus / minus 7.5 mol% DSPS. Antibody data were log-transformed and analyzed using a two-way ANOVA with Sidak's multiple comparison test. CD4 T cell data were analyzed using a REML mixed-effects model with Sidak's multiple comparison test.

[0096] Figure 13G Effect of phosphatidylserine lipid tail (DPPS vs DSPS) composition on B cell responses (Panel A) and T cell responses (Panel B) of mRNA-LNP primes. Antibody data were log-transformed and subsequently analyzed. Data were analyzed using one-way ANOVA and Tukey's multiple comparison test.

[0097] FIG. 14A compares mCherry expression by KC2-01 LNPs, 7.5 mol% DSPS (D isomer), and DSPS (L isomer) at 1 μg / mL mRNA for 24 hours.

[0098] FIG. 14B compares mCherry expression by KC2-01 LNPs, 7.5 mol% DSPS (D isomer), and DSPS (L isomer) at 0.33 μg / mL mRNA for 24 hours.

[0099] FIG15 compares mCherry expression in KC2 LNPs containing 5 and 7.5 mol% DSPS (L-isomer) with LNPs prepared with SM-102 or ALC-0315 at 1 μg / mL mRNA for 24 hours. The Y axis is mean fluorescence intensity (MFI). UT samples correspond to cells without LNP addition.

[0100] Figure 16 compares mCherry expression in UO1, UO6, and UO7 alone or with the addition of 7.5 mol% D-isomer of DSPS at 1 μg / mL mRNA for 24 hours. UT samples correspond to cells without LNP addition.

[0101] FIG17 compares mCherry expression at 1 μg / mL mRNA for 24 hours for UO1, SM102, ALC-0315 alone or with the addition of DSPS. Lipo refers to Lipofectamine MessengerMax (ThermoFisher) used according to the manufacturer's instructions at the same dose level as LNP. UT samples correspond to cells without LNP addition.

[0102] Figure 18 Oxidative degradation of liposomes containing KC3 (DLin-KC3-DMA), a polyunsaturated ICL with a single methylene group between two olefins, and liposomes containing ICLs with monounsaturated alkyl chains (KC3-OA, KC3-PA, or KC3-C17 (C8:1)) and fully saturated ICL (KC3-C17). The effect of hydrogen peroxide on the stability of single ionizable cationic lipids as measured by CAD-HPLC.

[0103] Figure 19 compares mCherry expression in human dendritic cells at 0.1 and 1 μg / mL mRNA for 24 hours for UO1, UO1A, and KC3-OALNP preparations alone or with the addition of DSPS. Untreated DC samples correspond to human dendritic cells without LNP addition.

[0104] FIG20 shows the effect of LNPs containing polyunsaturated KC3, monounsaturated KC3-OA, KC3-PA or KC3C17 (C8:1) and fully saturated KC3C17 in the presence or absence of DPPS (NH4 + Comparison of mCherry expression in the presence of 0.3 or 1 μg / mL mRNA for 24 hours in the presence of saline. The UT sample corresponds to cells without LNP addition.

[0105] FIG21 shows the effect of LNPs containing polyunsaturated KC2 or KC3 with a single methylene group between two olefins, polyunsaturated KC3-01 with four methylene groups between two olefins, monounsaturated KC3-OA, KC3-PA or KC3C17 (C8:1) and ALC-0315 in human dendritic cells (all LNPs except ALC-0315 had DPPS (NH4 + Comparison of mCherry expression in the presence of 0.1 or 1 μg / mL mRNA for 24 hours. Untreated samples correspond to cells without LNP addition.

[0106] Figure 22 Comparison of mCherry expression in human dendritic cells at 0.1 and 1 μg / mL mRNA for 24 hours with LNP formulations containing 5 mol% DSPS and 46 to 54 mol% KC3-OA compared to ALC-0315 and SM-102 LNP controls. Untreated DC samples correspond to human dendritic cells without LNP addition.

[0107] FIG23 compares mCherry expression in human dendritic cells at 0.1 μg / mL mRNA for 24 hours using LNP formulations with 0 or 5 mol% DSPS and 50 mol% KC2-O1 at N / P ratios of 4 to 7. These are also compared to LNPs containing KC3-OA and 5 mol% DSPS at an N / P ratio of 5. Untreated DC samples correspond to human dendritic cells to which no LNPs were added.

[0108] FIG24A compares LNP formulations containing polyunsaturated KC3 with monounsaturated KC3-OA and KC3-PA in terms of vaccine immunogenicity. Total anti-spike antibody titers from mice immunized with mRNA-LNPs containing KC3, KC3-OA, or KC3-PA targeted with 5 mol% DSPS or DPPS. For KC3-OA and KC3-PALNP, each formulation was also evaluated with either C16 DPPC or C18 DSPC neutral phosphatidylcholine components. All LNPs contained 1.5 mol% PEG-DMG. The figure shows the endpoint antibody titers at day 21 following the initial prime injection of 1 μg mRNA / mouse.

[0109] FIG24B compares LNP formulations containing polyunsaturated KC3 with monounsaturated KC3-OA and KC3-PA in terms of vaccine immunogenicity. Total anti-spike antibody titers from mice immunized with mRNA-LNPs containing LNPs targeting KC3, KC3-OA, or KC3-PA with 5 mol% DSPS or DPPS. For KC3-OA and KC3-PALNP, each formulation was also evaluated with either C16 DPPC or C18 DSPC neutral phosphatidylcholine components. All LNPs contained 1.5 mol% PEG-DMG. The figure shows the endpoint antibody titers at day 34 after a primary immunization of 1 μg mRNA / mouse followed by a boost at day 21.

[0110] FIG25A compares mCherry expression in human dendritic cells at 1 μg / mL of mRNA for 24 hours using LNP formulations containing 5 mol% DSPS and 43 to 48 mol% KC3-OA. KC3-OA LNPs prepared with 45 mol% KC3-OA and 5 mol% DSPS of total lipids were also compared at N / P ratios of 5, 5.5, 6.0, and 6.5. Finally, LNPs containing 45 mol% KC3-OA at N / P ratios of 5 and 6 were evaluated using 1 or 3 mol% PEG-SA instead of 1.5 mol% PEG-DMG. Untreated DC samples correspond to human dendritic cells to which no LNPs were added.

[0111] FIG25B compares mCherry expression in human dendritic cells (DCs) at 0.1 μg / mL of mRNA for 24 hours using LNP formulations containing 5 mol% DSPS and 43 to 48 mol% KC3-OA. KC3-OA LNPs prepared with 45 mol% KC3-OA and 5 mol% DSPS of total lipids were also compared at N / P ratios of 5, 5.5, 6.0, and 6.5. Finally, LNPs containing 45 mol% KC3-OA at N / P ratios of 5 and 6 were evaluated using 1 or 3 mol% PEG-SA instead of 1.5 mol% PEG-DMG. Untreated DC samples correspond to human DCs without LNP addition.

[0112] Figure 26 A is hatched after 24 hours under 1 μ g / mL mRNA, and the mCherry expression of the LNP preparation with 7.5mol% multiple anionic phospholipids comprising 50mol%UO-1 in human dendritic cells is compared.All LNPs comprise 2.5mol%DSPC, 50mol%UO-1 and 1.5mol%PEG-DMG.Anionic phospholipids include phosphatidylserine, DOPS, DSPS, DPPS and DMPS, distearoylphosphatidylglycerol (DSPG) and N-glutaryl-distearoylphosphatidylethanolamine (Glu-DSPE) and N-succinyl-distearoylphosphatidylethanolamine (Suc-DSPE), and it is included with 7.5mol%, except that DSPG is compared with 5 and 7.5mol%.

[0113] Figure 26 B is hatched after 24 hours under 0.1 μ g / mL mRNA, and the mCherry expression of the LNP preparation with 7.5mol% multiple anionic phospholipids comprising 50mol%UO-1 is compared in human dendritic cells.All LNPs comprise 2.5mol%DSPC, 50mol%UO-1 and 1.5mol%PEG-DMG.Anionic phospholipids include phosphatidylserine, DOPS, DSPS, DPPS and DMPS, distearoylphosphatidylglycerol (DSPG) and N-glutaryl-distearoylphosphatidylethanolamine (Glu-DSPE) and N-succinyl-distearoylphosphatidylethanolamine (Suc-DSPE), and it is included with 7.5mol%, except that DSPG is compared with 5 and 7.5mol%.

[0114] FIG27A compares mCherry expression in human dendritic cells from LNP formulations containing UO-1 or KC3-01 with 0 to 10 mol% DSPG after 24 hours of incubation at 1 μg / mL mRNA. Also included are ALC-0315 and SM-102 LNP controls at 1 μg / mL mRNA, and untreated DC samples correspond to human dendritic cells to which no LNPs were added.

[0115] FIG27B compares mCherry expression in human dendritic cells of LNP formulations containing UO-1 or KC3-01 with 0 to 10 mol% DSPG after 24 hours of incubation at 0.1 μg / mL mRNA. ALC-0315 and SM-102 LNP controls at 0.1 μg / mL mRNA are also included, and untreated DC samples correspond to human dendritic cells to which no LNPs were added.

[0116] Figure 28 Comparison of mCherry expression in murine dendritic cells after 24 hours of incubation with 1 μg / mL mRNA for LNPs containing UO1, UO6, or UO7 and 7.5 mol% DSPS. The UT sample corresponds to cells without LNP addition.

[0117] FIG29 compares LNP formulations containing dilinoleyl KC2, monounsaturated KC3-OA, and four methylene-intercalated polyunsaturated ICLs (KC3-01, AKG-UO1, and AKG-UO9) for vaccine immunogenicity. LNPs containing ALC-0315 were included as a control. All LNPs contained 1.5 mol% PEG-DMG. Total anti-Spike antibody titers from mice immunized with mRNA-LNPs were determined on day 21 following an initial priming injection of 1 μg mRNA / mouse on day 1.

[0118] Figure 30 A is a comparison of mCherry expression of LNP formulations with 5mol% various anionic phospholipids comprising 48mol% KC3-OA in human dendritic cells after incubation for 24 hours at 1 μg / mL mRNA. All LNPs contain 2.5mol% DSPC, 50mol% UO-1, and 1.5mol% PEG-DMG. Anionic phospholipids include phosphatidylglycerol, DOPG, DSPG, DPPG, DMPG, and DSPS. In some LNPs, DSPG and DSPS are combined alone, or combined together with DSPC. In this study, two donors were used to produce human dendritic cells, and the untreated DC sample corresponded to human dendritic cells to which LNPs were not added.

[0119] Figure 30B is a comparison of mCherry expression of LNP formulations with 5mol% various anionic phospholipids comprising 48mol% KC3-OA in human dendritic cells after incubation for 24 hours at 0.1 μg / mL mRNA. All LNPs contain 2.5mol% DSPC, 50mol% UO-1, and 1.5mol% PEG-DMG. Anionic phospholipids include phosphatidylglycerol, DOPG, DSPG, DPPG, DMPG, and DSPS. In some LNPs, DSPG and DSPS are combined alone, or combined together with DSPC. In this study, two donors were used to produce human dendritic cells, and the untreated DC sample corresponded to human dendritic cells to which LNPs were not added.

[0120] FIG31 shows the effect of KC3-OA LNPs and 5 mol% DSPS (Na) on the expression of KC3-OA LNPs in mouse dendritic cells after 24 hours of incubation with 1 μg / mL mRNA. + salt) or 5 mol% DPPS (NH4 +Figure 3. Comparison of mCherry expression in LNPs treated with LNPs containing 1 μg / mL of saline. ALC-0315 and SM-102 LNP controls at 1 μg / mL of mRNA were also included. The UT sample corresponds to cells without LNP addition.

[0121] Figure 32 . Plot of LNP uptake over time in MutDC1940 cells incubated with various LNP formulations of mCherry mRNA (0.2 μg / mL) labeled with the fluorescent lipid marker DiIC18(5)-DS (DiI5-DS) (Example 47). At the indicated times, LNP uptake was quantified by flow cytometry of the marker (RL-1 fluorescence channel) and plotted as median cell fluorescence intensity. Data are the mean of triplicate runs. Error bars are standard deviations. Formulations: AKG+PS, KC3OA+PS; AKG-PS, KC3OA-PS; SM102; ALC-0315. Untreated refers to cells not treated with LNPs.

[0122] Figure 33 . Plot of LNP mRNA expression over time in MutuDC1940 cells incubated with various LNP formulations of mCherry mRNA (0.2 μg / mL) labeled with the fluorescent lipid marker DiIC18(5)-DS (DiI5-DS) (Example 47). At the indicated times, mCherry protein expression was quantified by flow cytometry with protein fluorescence (YL-2 fluorescence channel) and plotted as median cell fluorescence intensity. Data are the mean of triplicate runs. Error bars are standard deviations. Formulations: AKG+PS, KC3OA+PS; AKG-PS, KC3OA-PS; SM102; ALC-0315. Untreated refers to cells not treated with LNPs.

[0123] Figure 34 .The effect of pre-treatment of cells with "blocking liposomes" at 10×LNP lipid concentration on the uptake of DiI5-DS labeled LNPs by MutuDC1940 cells (Example 48). Liposomes were added 15 minutes before LNPs. After 1 hour of LNP exposure, the uptake of LNPs was measured by flow cytometry (RL1-A channel) using DiI5-DS fluorescence and plotted as median cell fluorescence intensity. The data are the average of four runs. The error bars are standard deviations. The LNP type is shown at the horizontal axis. The legend indicates the amount and nature of the PS component in the "blocking liposomes". "%" indicates the mol% of PS relative to the total lipids in the blocking liposome preparation.

[0124] Figure 35Effect of PS targeting agents on mCherry expression in MutuDC1940 cells after 3 hours of incubation with mCherry mRNA-LNP constructs (0.3 μg / mL mRNA) prepared with various ICLs (Example 49). Expression was quantified by flow cytometry using mCherry protein fluorescence (YL2-A fluorescence channel) and plotted as median cell fluorescence intensity. Data are the average of four replicate runs. Error bars are standard deviations. ICLs: UO1, KC2, KC30A. Targeted PSs: DPPS, DSPS, D-DSPS, no targeting (NT). UT refers to cells not treated with LNPs.

[0125] Figure 36 Effect of PS targeting agents on mCherry expression in MutuDC1940 cells after 24 hours of incubation with mCherry mRNA-LNP constructs (0.3 μg / mL mRNA) prepared with various ICLs (Example 49). Expression was quantified by flow cytometry using mCherry protein fluorescence (YL2-A fluorescence channel) and plotted as median cell fluorescence intensity. Data are the average of four replicate runs. Error bars are standard deviations. ICLs: UO1, KC2, KC30A. Targeted PSs: DPPS, DSPS, D-DSPS, no targeting (NT). UT refers to cells not treated with LNPs.

[0126] Figure 37 Effect of PS targeting agents on LNP uptake in MutuDC1940 cells after 3 hours of incubation with mCherry mRNA-LNP constructs (0.3 μg / mL mRNA) prepared with various ICLs (Example 49). Expression was quantified by flow cytometry using DiI5-DS (DiI) lipid-labeled fluorescence (RL1A fluorescence channel) and plotted as median cell fluorescence intensity. Data are the mean of quadruplicate runs. Error bars are standard deviations. ICLs: UO1, KC2, KC30A. Targeted PSs: DPPS, DSPS, D-DSPS, untargeted (NT). UT refers to cells not treated with LNPs.

[0127] Figure 38Effect of PS targeting agents on LNP uptake in MutuDC1940 cells after 24 hours of incubation with mCherry mRNA-LNP constructs (0.3 μg / mL mRNA) prepared with various ICLs (Example 49). Expression was quantified by flow cytometry using DiI5-DS (DiI) lipid-labeled fluorescence (RL1A fluorescence channel) and plotted as median cell fluorescence intensity. Data are the mean of four replicate runs. Error bars are standard deviations. ICLs: UO1, KC2, KC30A. Targeted PSs: DPPS, DSPS, D-DSPS, untargeted (NT). UT refers to cells not treated with LNPs.

[0128] Figure 39 . Expression of mCherry mRNA delivered to MutuDC1940 cells by LNP formulation of mCherry mRNA (0.3 μg / mL) formulated in KC3OA / DPPS LNPs, prepared with PEG-DMG and various amounts of PEG-DLG (Example 50). 24 hours after LNP transfection at 1.0 and 0.1 μg / mL mRNA, expression was quantified by flow cytometry of mCherry fluorescence. PBMCs were taken from two donors, and each donor was run in triplicate. The bars are the averages of the combined donor replicates. The LNP composition is shown at the X-axis. "%" refers to the mol% of the PEG-lipid component relative to the total LNP lipid.

[0129] Figure 40. Anti-SARS-COV-2 spike protein antibody titers in serum of Balb / C mice immunized with VRN029 spike mRNA formulated in KC3-PA / DSPC / DPPS-NH4 / Chol / PEG-lipid LNPs containing PRG-DMG or PEG-DLG (N=5) (Example 51). Serum was collected 2 weeks after the booster dose of 1 μg mRNA / mouse (day 35 after the initial dose). Titers were determined using 4× background as the endpoint of the ELISA test. The bars represent the geometric mean titer in each group, the error intervals are the standard deviations of the log-transformed data, and the markers show the individual animal titers. The numbers at the PEG-lipid designations are the mol% of PEG-lipid relative to the total lipids in the LNP. The dotted line shows the upper limit of detection in the titer assay.

[0130] Figure 41A. Anti-SARS-COV-2 spike protein antibody titers in serum of Balb / C mice immunized with VRN029 spike mRNA formulated in KC3-OA / DSPC / DPPS-NH4 / Chol / PEG-lipid LNPs containing PEG-DMG or PEG-DLG (N=5) (Example 52). Serum was collected 3 weeks after the initial mRNA-LNP dose of 1 μg mRNA / mouse. Titers were determined using 4× background as the endpoint of the ELISA test. The bars represent the geometric mean titer in each group, the error intervals are the standard deviations of the log-transformed data, and the markers show the individual animal titers. The differences between the groups were statistically significant. The dotted line shows the lower limit of detection in the titer assay.

[0131] Figure 41B. Anti-SARS-COV-2 spike protein antibody titers in serum of Balb / C mice immunized with VRN029 spike mRNA formulated in KC3-OA / DSPC / DPPS-NH4 / Chol / PEG-lipid LNPs containing various amounts of DPPS and PEG-DMG or PEG-DLG (N=5) (Example 52). Serum was collected 2 weeks after a boost mRNA-LNP dose of 1 μg mRNA / mouse was administered 3 weeks after the initial immunization. Titers were determined using 4× background as the endpoint of the ELISA test. The bars represent the geometric mean titer in each group, the error intervals are the standard deviations of the log-transformed data, and the markers show the individual animal titers. The dotted line shows the lower limit of detection in the titer assay. "%" represents the mol% of the lipid component relative to the total LNP lipid.

[0132] Figure 42 . Expression of mCherry mRNA delivered to human PBMC-derived dendritic cells by KC3OA / DSPC / DPPS-NH4 / Chol / PEG-lipid LNPs prepared with various amounts of PEG-DMG or PEG-DLPE (Example 53). 24 hours after LNP transfection at 0.3 μg / mL mRNA, expression was quantified by flow cytometry of mCherry fluorescence. The bars show the average median cell fluorescence intensity of the replicates. The error intervals are standard deviations. The mol% of PEG-lipid refers to the mol% relative to the total LNP lipid.

[0133] Figure 43A. Anti-SARS-COV-2 spike protein antibody titers in sera of Balb / C mice immunized with VRN029 spike mRNA formulated in KC3-OA / DSPC / DPPS-NH4 / Chol / PEG-DMG LNPs containing various amounts of DPPS (N=5) (Example 54). Sera were collected on day 20 after a priming mRNA dose of 0.3 μg mRNA / mouse. Titers were determined using 4× background as the endpoint of the ELISA test. Bars represent the geometric mean titer in each group, error intervals are the standard deviation of the log-transformed data, and labels show individual animal titers. Asterisks indicate statistical significance of the difference between groups at p=0.05. The group with a p-value of 0.059 is also shown. The dotted line shows the lower limit of detection in the titer assay. The numbers at the LNP lipid composition markers are the mol% of each component relative to the total LNP lipid.

[0134] Figure 43B Anti-SARS-COV-2 spike protein antibody titers in serum of Balb / C mice immunized with VRN029 spike mRNA formulated in KC3-OA / DSPC / DPPS-NH4 / Chol / PEG-lipid LNPs, and in serum of Balb / C mice immunized with VRN029 spike mRNA in a non-targeted LNP formulation based on ALC-0315 containing various amounts of PEG-DMG or PEG-DLG (N=5) (Example 54). Sera were collected 2 weeks after a boost injection 3 weeks after a priming mRNA-LNP dose of 0.3 μg mRNA / mouse. Titers were determined using 4× background as the endpoint of the ELISA assay. Bars represent the geometric mean titer in each group, error bars are the standard deviation of the log-transformed data, and markers indicate individual animal titers. The dotted line shows the lower limit of detection in the titer assay. The numbers at the LNP lipid composition markers are the mol% of each component relative to the total LNP lipids.

[0135] Figure 43C. Anti-SARS-COV-2 spike protein antibody titers in sera of Balb / C mice immunized with VRN029 spike mRNA formulated in KC3-OA / / PS / Chol / PEG-lipid LNPs containing 5 mol% of DSPS-Na or DPPS-NH4 as the PS component relative to the total LNP lipids and 1.5 mol% of PEG-DMG or PEG-DLG as the PEG-lipid component relative to the total LNP lipids (N=5) (Example 54). Sera were collected 2 weeks after the boost injection 3 weeks after the priming mRNA-LNP dose of 0.3 μg mRNA / mouse (35 days after the priming). Titers were determined using 4× background as the endpoint of the ELISA test. Bars represent the geometric mean titer in each group, error intervals are the standard deviation of the log-transformed data, and markers show individual animal titers. The dotted line shows the lower limit of detection in the titer assay. The numbers at the LNP lipid composition markers are the mol% of PEG-lipid relative to the total LNP lipid.

[0136] Figure 44 . Anti-SARS-COV-2 spike protein antibody titers in serum of Balb / C mice immunized with various SARS-COV-2 spike mRNAs (VRN029, VRN118, VRN119) formulated in KC3-OA / DSPC / DPPS-NH4 / Chol / PEG-DMG LNPs or ALC-0315LNPs (N=5) (Example 55). Sera were collected on day 20 after the initial mRNA-LNP dose of 0.3 μg mRNA / mouse. Titers were determined using 4× background as the endpoint of the ELISA test. The bars represent the geometric mean titer in each group, the error interval is the standard deviation of the log-transformed data, and the markers show the individual animal titers. The dotted line shows the lower limit of detection in the titer assay.

[0137] Figure 45A. Anti-SARS-COV-2 spike protein antibody titers in sera of Balb / C mice immunized with VRN029 spike mRNA formulated in ALC-0315-based LNPs, SM-102-based LNPs, or KC3-OA / DSPC / DPPS-NH4 / Chol / PEG-DMG LNPs (N=5) (Example 56). Sera were collected 2 weeks after a boost injection 3 weeks after a priming mRNA-LNP dose of 0.1, 0.3, or 1.0 μg mRNA / mouse (day 35 after priming). Titers were determined using 4× background as the endpoint of the ELISA test. Bars represent the geometric mean titer in each group, error intervals are the standard deviation of the log-transformed data, and markers show individual animal titers. The dotted line shows the lower limit of detection in the titer assay. The numbers at the LNP lipid composition markers are the mol% of PEG-lipid relative to the total LNP lipid.

[0138] Figure 45B . Anti-SARS-COV-2 spike protein antibody titers in sera of Balb / C mice immunized with VRN029 spike mRNA formulated in KC3-OA / DSPC / DPPS-NH4 / Chol / PEG-DMG LNPs with different amounts of PEG-DMG and administered at different doses (N=5) (Example 56). Sera were collected 2 weeks after a boost injection 3 weeks after the initial mRNA-LNP dose of 0.1, 0.3 or 1.0 μg mRNA / mouse (35 days after the initial immunization). Titers were determined using 4× background as the endpoint of the ELISA test. The bars represent the geometric mean titer in each group, the error intervals are the standard deviations of the log-transformed data, and the markers show the individual animal titers. The dotted line shows the lower limit of detection in the titer assay. The numbers at the LNP lipid composition markers are the mol% of the corresponding lipid component relative to the total LNP lipid.

[0139] Figure 46AAnti-SARS-COV-2 spike protein antibody titers in serum of Balb / C mice immunized with VRN029 spike mRNA formulated in KC3-OA / DSPC / DPPS-NH4 / Chol / PEG-DMG (targeted KC3-OA) LNPs, KC3-OA / DSPC / Chol / PEG-DMG (non-targeted, KC3-OA) LNPs, or ALC-0315 LNPs (Example 57). Sera were collected on day 20 after the initial mRNA-LNP dose of 0.1 μg mRNA / mouse. Titers were determined using 4× background as the endpoint of the ELISA test. The bars represent the geometric mean titer in each group, the error intervals are the standard deviations of the log-transformed data, and the markers show individual animal titers. The dotted line shows the lower limit of detection in the titer assay.

[0140] Figure 46B Anti-SARS-COV-2 spike protein antibody titers in serum of Balb / C mice immunized with VRN029 spike mRNA formulated in KC3-OA / DSPC / DPPS-NH4 / Chol / PEG-DMG (targeted KC3-OA) LNPs, KC3-OA / DSPC / Chol / PEG-DMG (non-targeted, KC3-OA) LNPs, or ALC-0315 LNPs (Example 57). Sera were collected 2 weeks after a boost injection 3 weeks after the initial mRNA-LNP dose of 0.1 μg mRNA / mouse (day 35 after the initial immunization). Titers were determined using 4× background as the endpoint of the ELISA test. The bars represent the geometric mean titer in each group, the error interval is the standard deviation of the log-transformed data, and the markers show the individual animal titers. The dotted line shows the lower limit of detection in the titer assay.

[0141] Figure 47A and Figure 47B .Synthesis scheme of the UO-1 series of ionizable cationic lipids.

[0142] Figure 48 Synthetic schemes of KC2-01 and KC3-01 ionizable cationic lipids.

[0143] Figure 49 .Synthetic scheme of monounsaturated KC2-0A, KC2-PA and KC3-OA ionizable cationic lipids.

[0144] Figure 50 .Synthesis schemes of monounsaturated KC3-C17(C8:1) and fully unsaturated KC3-C17 ionizable cationic lipids.

[0145] Figure 51 Synthesis scheme of distearoylphosphatidyl-D-serine. DETAILED DESCRIPTION

[0146] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the compositions and methods of the disclosure.

[0147] Liposomal nanoparticle (LNP) compositions may comprise ionizable lipids, sterols, and one or more phospholipids. In some embodiments, the LNP compositions further comprise nucleic acids, such as mRNA, for use in pharmaceutical compositions, such as vaccines. In some embodiments, the LNP compositions optionally further comprise conjugated lipids.

[0148] Lipid nanoparticle (LNP) compositions comprising mRNA include stable nucleic acid lipid particles (SNALP) used as carriers for systemic delivery of mRNA or other nucleic acid therapeutics. The SNALP compositions comprise cationic lipids such as MC3 or KC2, which comprise protonatable tertiary amine head groups connected to a pair of straight 18 carbon aliphatic chains, wherein the straight 18 carbon aliphatic chains comprise a pair of carbon-carbon double bonds (such as linoleic acid) separated by a single methylene group. However, although the structure of these hydrocarbon chains comprising a pair of double bonds separated by a single methylene group each gives the biological properties expected for the SNALP compositions, this chemical substructure has also caused such undesirable problems: the sensitivity of the compound to oxidative degradation is improved. For example, Figure 1 The oxidative degradation mechanism of lipid esters of linoleic acid containing conjugated polyunsaturates that are particularly susceptible to oxidation is delineated.What is needed are new cationic lipids suitable for use in SNALP compositions but with enhanced resistance to oxidative degradation.

[0149] Disclosed herein are compounds, compositions and methods relevant 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 bioreducible ionizable lipids. These lipids are cationic (i.e., positively charged) at acidic pH, such as those encountered intracellularly after endocytosis or phagocytosis by the cell. The same lipids and compositions comprising them have a charge close to neutral when present at pH 7.4. These lipids may also have a single olefin group present in their alkyl or acyl groups.

[0150] Some aspects of the present disclosure relate to methods for synthesizing new ionizable lipids.

[0151] Other aspects relate to compositions comprising lipid nanoparticles comprising ionizable cationic lipids, wherein the lipid nanoparticles comprise nucleic acids. In some embodiments, the nucleic acids are encapsulated in the lipid nanoparticles.

[0152] Some 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 improve transfection efficiency in dendritic cells. Compared to other ionizable lipids in the same family, the further incorporation of geminal disubstituted ionizable lipids with a monounsaturated alkyl chain (monoolefin) at the 2nd position of 1,3-dioxolane or ketal in the LNP formulation showed high levels of transfection in human dendritic cells and showed good stability to oxidative damage.

[0153] definition

[0154] For convenience, certain terms used in the specification, examples and appended claims are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which this disclosure belongs.

[0155] As used herein, the following terms and phrases are intended to have the following meanings:

[0156] As used herein, nouns without quantifiers are used to refer to one or more than one (ie, at least one) grammatical object. For example, "element" means one element or more than one element.

[0157] As used herein, the terms "comprising" or "including" are used to refer to compositions, methods, and their corresponding components as present in a given embodiment, but are also open ended to include unspecified elements.

[0158] As used herein, the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the embodiment of the disclosure.

[0159] The term "consisting of refers to the compositions, methods, and corresponding components thereof as described herein, excluding any elements not recited in the description of the embodiment.

[0160] The term "comprising" as used in this specification includes "consisting of" and "consisting essentially of.

[0161] If "as mentioned above" or "mentioned above" or "above" is mentioned in this specification, it refers to any of the present disclosures on any of the preceding pages in this specification.

[0162] If in this specification reference is made to "as described herein", "described herein", "provided herein" or "as described in this text" or "illustrated herein", this refers to any of the present disclosure on any of the preceding or following pages of this specification.

[0163] As used herein, the term "about" means acceptable variations within 20%, within 10%, and within 5% of the stated value. In certain embodiments, "about" can mean variations of + / - 1%, 2%, 3%, 4%, 5%, 10%, or 20%.

[0164] As used herein, the term "effective amount" with respect to a compound or composition means an amount of the active compound (also referred to herein as an active agent or drug) sufficient to produce a bactericidal or bacteriostatic effect. In some embodiments, the effective amount is a "therapeutically effective amount," which means an amount of the active compound sufficient to alleviate the symptoms of the bacterial infection being treated.

[0165] As used herein, the term "subject" (or alternatively, "patient") refers to an animal, preferably a mammal, most preferably a human, receiving prophylactic or therapeutic treatment.

[0166] The term "administer" as used herein or its variations include all means by which a compound or pharmaceutical composition is introduced into an object in need thereof, including but not limited to oral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, inhalation, buccal, eye, sublingual, vaginal, rectal, etc. The administration of a compound or composition is suitably parenteral. For example, a compound or composition may preferably be administered intravenously, but may also be administered intraperitoneally or by inhalation, similar to the liposomal amikacin (amikacin) currently used clinically for Mycobacterium avium (mycobacterium avium) treatment. (See Shirley et al., Amikacin Liposome Inhalation Suspension: A Review in Mycobacterium avium Complex Lung Disease. Drugs. 2019 Apr; 79 (5): 555-562).

[0167] As used herein, the terms "treat" and "treatment" refer to therapeutic or prophylactic measures, such as those described herein.

[0168] The term "pharmaceutically acceptable salt" refers to the relatively non-toxic, inorganic or organic acid addition salts of compounds of the present disclosure, which salts possess the desired pharmacological activity.

[0169] The term "alkyl" means a saturated carbon chain having from one to twenty carbon atoms, which may be linear or branched, or a combination thereof, unless otherwise specified. Some examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl and tert-butyl, pentyl, hexyl, heptyl, octyl, and the like. Unless otherwise specified in the specification, an alkyl group is optionally substituted.

[0170] The term "phosphatidylserine" and any of its acyl chain components refers to the L-isomer of serine in the head group, unless otherwise indicated in a specific instance.

[0171] The term "lipid conjugate" refers to a lipid that is conjugated to suppress the aggregation of lipid particles. Such lipid conjugates include, but are not limited to: polysarcosine (see, for example, WO2021191265A1, which is incorporated herein by reference in its entirety for all purposes); polyamide oligomers (such as ATTA-lipid conjugates); PEG-lipid conjugates, such as PEG coupled to a dialkoxypropyl group, PEG coupled to diacylglycerol, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, PEG conjugated to ceramide (see, for example, U.S. Patent No. 5,885,613, the disclosure of which is incorporated herein by reference in its entirety for all purposes); cationic PEG lipids; and mixtures thereof. PEG can be directly conjugated to lipid, or it can be connected to lipid by a linker moiety. Any linker moiety suitable for coupling PEG to lipid can be used, including, for example, non-ester-containing linker moieties and ester-containing linker moieties. In some preferred embodiments, non-ester-containing linker moieties are used.

[0172] In the Examples, the abbreviations for the ionizable cationic lipids may be taken from the abbreviations used in the Tables, for example, AKG-UO-1 or AKG-KC2-01 may be referred to as UO1 or KC2-01.

[0173] The abbreviation UT used in various studies refers to untreated samples.

[0174] The term "lipid nanoparticle" or "LNP" refers to particles with a diameter of about 5 to 500 nm. In some embodiments, the lipid nanoparticle contains one or more active agents. In some embodiments, the lipid nanoparticle contains nucleic acids. In some embodiments, the nucleic acids are condensed inside the nanoparticle, which has a cationic lipid, polymer or multivalent small molecule and an outer lipid coating that interacts with the biological environment. Due to the repulsive forces between the phosphate groups, nucleic acids are naturally rigid polymers and are preferably in an elongated configuration. In cells, in order to cope with volume constraints, DNA can accumulate itself with the help of ions and other molecules under appropriate solution conditions. Generally, DNA condensation is defined as the collapse of extended DNA chains into compact, ordered particles containing only one or several molecules. By binding to phosphate groups, cationic lipids can condense DNA by neutralizing the phosphate charge and allowing close packing.

[0175] In some embodiments, the active agent is encapsulated in LNP. In some embodiments, the active agent can be an anionic compound, such as, but not limited to: DNA; RNA; natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA and small interfering RNA); nucleoprotein; peptide; nucleic acid; ribozyme; DNA-containing nucleoprotein, such as complete or partially deproteinized virus particles (virions); oligomeric and polyanionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins). In some embodiments, the active agent can be mixed with excipients.

[0176] In LNP vaccine products, the active agent is typically contained within the LNP. In some embodiments, the active agent comprises a nucleic acid. Typically, the water-soluble nucleic acid is condensed with a cationic lipid or polycationic polymer within the particle interior, and the particle surface is enriched with a neutral lipid or PEG-lipid derivative. Additional ionizable cationic lipids may also be located on the surface and respond to acidification in the environment by becoming positively charged, promoting endosomal escape.

[0177] About LNP, ionizable lipids can have different properties or functions. Due to the pKa of amino groups, lipid molecules can become positively charged under acidic conditions. Under these conditions, lipid molecules can be electrostatically bound to the phosphate groups of nucleic acids, which allows the formation of LNP and the entrapment of nucleic acids. In some embodiments, the pKa can be low enough so that the surface charge of LNP in biological fluids such as blood at physiological pH values ​​is essentially neutral. High LNP surface charge is associated with toxicity, rapid clearance from circulation by fixed and free macrophages, hemolytic toxicity (including immune activation) (Filion et alBiochim Biophys Acta.1997Oct 23; 1329 (2): 345-56).

[0178] In some embodiments, the pKa can be high enough so that the ionizable cationic lipid can adopt a positively charged form at acidic endosomal pH. In this way, the cationic lipid can be combined with an endogenous endosomal anionic lipid to promote membrane lysis non-bilayer structure such as hexagonal HII phase (hexagonal HII phase), thereby resulting in more effective intracellular delivery. In some embodiments, the pKa ranges from 6.2 to 6.5. For example, the pKa can be about 6.2, about 6.3, about 6.4, about 6.5. The unsaturated tail also contributes to the ability of the lipid to adopt a non-bilayer structure. (Jayaraman et al., Angew Chem Int Ed Engl. 2012 Aug 20; 51 (34): 8529-33).

[0179] Release of nucleic acids from LNP formulations, as well as characteristics such as liposomal clearance and circulation half-life, can be improved 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 lipids included as part of the formulation.

[0180] The term "bioreducible" refers to a compound that undergoes accelerated degradation due to the cleavage of disulfide bonds in a reducing environment. Unlike other nucleic acid therapeutics such as siRNA, the success of mRNA-based treatment depends on the availability of a safe and effective delivery vehicle for encapsulating mRNA. mRNA is fragile and requires a protective coating for it to maintain activity until it reaches its target site. LNPs containing mRNA are promising vaccine options for Covid-19 immunity (Jackson et al., Preliminary Report. N Engl J Med. 2020 Nov 12; 383 (20): 1920-1931). The efficiency and tolerance of LNPs are attributed to amino lipids and are different from many biomaterial applications that can have the required service life of several weeks or months. Functional LNP-mediated mRNA delivery occurs within a few hours, avoiding the need for persistent lipids. In fact, this will be particularly important in applications requiring long-term administration. It has been shown that LNPs enter cells by endocytosis and accumulate in the endolysosomal compartment. Ionizable cationic lipids (ICLs) are able to effectively deliver mRNA to the cytosol after endocytosis, while being susceptible to enzymatic hydrolysis by lipase in late endosomes / lysosomes or the effects of hydrolysis triggered by the reducing environment of the lysosomes and allowing complete biodegradation. The extracellular space is a relatively oxidizing environment, while the intracellular space is a reducing environment, which allows disulfide-linked molecules to remain intact in the extracellular space, but quickly reduce once internalized (Huang et al., Mol Ther. 2005 Mar; 11 (3): 409-17, 2005). Some embodiments provide bioreducible disulfide-linked ICL molecules (see compounds 29 to 36, Table 2), which are stable in LNP formulations and stable in circulation, but undergo cleavage in the reducing environment of the lysosomes. Such compounds and compositions may promote rapid biodestruction of lipids and prevent the potentially toxic accumulation of ICL lipids (as observed in rats treated with DLin-MC3-DMA (Sabins et al., Mol Ther. 2018 Jun 6; 26(6): 1509-1519).

[0181] As used herein, the terms "encapsulation" and "encapsulation" refer to the incorporation of mRNA, DNA, siRNA or other nucleic acid agents into or association with lipid nanoparticles. As used herein, the term "encapsulation" refers to complete encapsulation or partial encapsulation. siRNA may be able to selectively knock down or downregulate the expression of a target gene. For example, siRNA may be selected to silence a gene associated with a specific disease, disorder or condition after administering a nanoparticle composition comprising the siRNA to a subject in need thereof. The siRNA may comprise a sequence complementary to the mRNA sequence encoding the target gene or protein.

[0182] The term "mol%" with respect to cholesterol refers to the molar amount of cholesterol expressed as a percentage relative to the sum of the molar amounts of cholesterol and non-PEGylated phospholipids. For example, "55 mol% cholesterol" in a liposome comprising cholesterol and HSPC refers to a composition of 55 mol parts cholesterol / 45 mol parts HSPC.

[0183] The term "mol%" with respect to PEG-lipid refers to the ratio of the molar amounts of PEG-lipid to non-PEGylated phospholipid expressed as a percentage. For example, "5 mol.% PEG-DSPE" in an LNP comprising HSPC and PEG-DSPE means a composition having 5 mol. parts PEG-DSPE per 100 mol. parts HSPC.

[0184] In some embodiments, "mol %" with respect to cholesterol refers to the molar amount of cholesterol expressed as a percentage relative to the sum of the molar amounts of total lipids. For example, "40.5 mol % cholesterol" in an LNP composition comprising cholesterol, ICL, DSPC, PS, cholesterol, and PEG-DMG refers to a composition of 40.5 mol parts cholesterol per 59.5 mol parts of the combined components of ICL, DSPC, PS, and PEG-DMG.

[0185] In some embodiments, "mol %" with respect to conjugated lipids refers to the ratio of the molar amounts of the conjugated lipids expressed as a percentage. For example, "1.5 mol % PEG-DMG" in an LNP comprising cholesterol, ICL, DSPC, PS, cholesterol, and PEG-DMG refers to a composition of 1.5 mol parts PEG-DMG per 98.5 mol parts of the combined ICL, DSPC, PS, and cholesterol components.

[0186] In some embodiments, "mol %" with respect to PS lipids refers to the ratio of the molar amounts of PS lipids expressed as percentages. For example, "5 mol % DPPS" in an LNP comprising cholesterol, ICL, DSPC, DPPS, cholesterol, and PEG-DMG refers to a composition of 5 mol parts PEG-DMG per 95 mol parts of the combined ICL, DSPC, PEG-DMG, and cholesterol components.

[0187] In some embodiments, "mol %" with respect to ICL refers to the ratio of the molar amounts of ICL expressed in percentage points. For example, "48 mol % PEG-DMG" in an LNP comprising cholesterol, KC3-OA (e.g., an example of ICL), DSPC, PS, cholesterol, and PEG-DMG refers to a composition of 48 mol parts ICL per 52 mol parts of the combined PEG-DMG, DSPC, PS, and cholesterol components.

[0188] As used herein, the term "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that has been approved by the United States Food and Drug Administration for use in humans or domestic animals.

[0189] Various aspects and embodiments are described in further detail in the following subsections.

[0190] Ionizable cationic lipids

[0191] Provided herein are compounds that can be used to prepare lipid nanoparticle (LNP) compositions.

[0192] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I):

[0193]

[0194] Where R1 is

[0195]

[0196] 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;

[0197] R2 and R3 are each independently (C1-C4)alkyl optionally substituted with hydroxy; and

[0198] n is an integer equal to 2, 3 or 4.

[0199] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having a chemical structure of formula (I), wherein 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 .

[0200] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having a 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 a 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 a 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 a chemical structure of formula (I), wherein a is 0 and b is 3.

[0201] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having a 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 a 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 a 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 a chemical structure of formula (I), wherein a is 1 and b is 3.

[0202] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having a chemical structure of formula (I), wherein R 10 and R 12 In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having a chemical structure of formula (I), wherein R 10 and R 12 Each is a (C1-C4) alkyl group optionally substituted with a hydroxyl group. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having a chemical structure of formula (I), wherein R 10 and R12 In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having a chemical structure of formula (I), wherein R 10 and R 12 In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having a chemical structure of formula (I), wherein R 10 and R 12 In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having a chemical structure of formula (I), wherein R 10 and R 12 In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having a chemical structure of formula (I), wherein R 10 and R 12 Each is independently selected from methyl, ethyl, -(CH2)(CH2)OH and -(CH2)2(CH2)OH.

[0203] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having a 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 a 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 a 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 a chemical structure of formula (I), wherein a is 0 or 1; b is 1, 2, 3 or 4, with the proviso 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.

[0204] In some embodiments, the ionizable cationic lipid comprises the chemical structure of Formula (II):

[0205]

[0206] or a pharmaceutically acceptable salt thereof, wherein:

[0207] Y is

[0208] n is an integer 2, 3, or 4;

[0209] R 22 is a single olefin with a total length of C 15 -C 18 hydrocarbon chains, and

[0210] R 10 and R 12 Each is independently a (C1-C4)alkyl group optionally substituted with hydroxy.

[0211] In some aspects, R in formula (II) 22 is a polyolefin chain of formula A.

[0212] In some aspects, 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 aspects, in formula (II), R 10 and R 12 are each independently methyl. In some aspects, in formula (II) R 10 and R 12 In some aspects, in formula (II), R 10 and R 12 In some aspects, in formula (II), at least one of R 10 is a methyl group, and R 12 is selected from methyl, ethyl, -(CH2)(CH2)OH and -(CH2)2(CH2)OH. In some aspects, in formula (II), R 10 is a methyl group, and R 12 In some aspects, in the compound comprising the chemical structure of formula (II), R 10 is a methyl group, and R 12 is selected from -(CH2)(CH2)OH and -(CH2)2(CH2)OH. In some aspects, in formula (II), R 10 and R 12 are independently selected from methyl or ethyl optionally substituted with one or more hydroxyl groups. In some aspects, in formula (II), R 10 and R 12 One or both of are -(CH2)(CH2)OH or -(CH2)2(CH2)OH. In some aspects, in formula (II), R 10 is a methyl group, and R12 is a methyl or ethyl group substituted with a hydroxyl group. In some aspects, R in formula (II) 10 One or both of them are methyl, and in formula (II) R 12 is -(CH2)(CH2)OH. In some aspects, R in formula (II) 10 One or both of them are methyl, and in formula (II) R 12 It is -(CH2)2(CH2)OH.

[0213] In some embodiments, the compound has the structure of the compound listed in the following table. Table 1A and Table 1B show some examples of cationic lipids. Table 2 shows some examples of bioreducible cationic lipids.

[0214] Table 1A. Exemplary cationic lipids

[0215]

[0216] Table 1A. Exemplary Cationic Lipids (Continued)

[0217]

[0218] Table 1A. Exemplary Cationic Lipids (Continued)

[0219]

[0220] Table 1A. Exemplary Cationic Lipids (Continued)

[0221]

[0222] Table 1B Additional Exemplary Cationic Lipids

[0223]

[0224] Table 2. Exemplary bioreducible cationic lipids

[0225]

[0226] Table 2. Exemplary bioreducible cationic lipids (continued)

[0227]

[0228] In some embodiments, the ionizable lipids encapsulate nucleic acids. In some embodiments, the ionizable lipids encapsulate nucleic acids in LNP formulations. In some embodiments, the nucleic acids are siRNA molecules. In some embodiments, the nucleic acids are mRNA molecules. In some embodiments, the nucleic acids are DNA molecules.

[0229] In some embodiments, compositions are provided that further comprise a ligand, such as an antibody conjugate, that targets the lipid nanoparticles to dendritic cells in a highly specific manner against cell surface receptors. In some embodiments, the compositions further comprise a targeting ligand, wherein the targeting ligand is directed toward the exterior of the nanoparticle. In some embodiments, the targeting ligand is an antibody.

[0230] In some embodiments, the lipid nanoparticles are in an aqueous medium.

[0231] In some embodiments, nucleic acids are encapsulated in lipid nanoparticles with compounds disclosed herein, including compounds of Formula I, II, III, IV-B, VA-1, or combinations thereof, wherein the nucleic acids are RNA or DNA. In some embodiments, nucleic acids are encapsulated in lipid nanoparticles with compounds disclosed herein, including compounds disclosed herein, or combinations thereof, wherein the nucleic acids are RNA or DNA. In some embodiments, the nucleic acids are mRNA. In some embodiments, the nucleic acids are siRNA. In some embodiments, the nucleic acids are DNA.

[0232] In some embodiments, the lipid nanoparticle comprises a membrane containing phosphatidylcholine and a sterol. In some embodiments, the sterol is cholesterol. In some embodiments, the lipid nanoparticle comprises a membrane containing phosphatidylcholine and an ionizable cationic lipid (ICL). In some embodiments, the ICL has the structure of Formula I, II, III, IV-B, VA-1, and cholesterol, wherein the membrane separates the interior of the lipid nanoparticle from the aqueous medium. In some embodiments, the ICL has the structure 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 ionizable cationic lipid to cholesterol is about 65:35 to 40:60. In some embodiments, the molar ratio of ICL to cholesterol is about 60:40 to about 45:55.

[0233] In some embodiments, the molar ratio of phosphatidylcholine to cholesterol is from about 1:5 to about 1:2.

[0234] In some embodiments, the membrane further comprises a polymer-conjugated lipid.

[0235] In some embodiments, the lipid nanoparticle comprises ICL, DSPC, cholesterol, and polymer-conjugated lipid in a molar ratio of about 49.5:10.3:39.6:2.5.

[0236] In some embodiments, the polymer-conjugated lipid is PEG (2000)-dimyristoylglycerol (PEG-DMG) or PEG (molecular weight 2,000)-dimyristoylphosphatidylethanolamine (PEG-DMPE).

[0237] In some embodiments, the percentage of oxidative degradation products of ionizable lipids is less than 50% of the percentage of oxidative degradation products of a control formulation of DLin-KC2-DMA or DLin-MC3-DMA.

[0238] In some embodiments, the composition is a liquid pharmaceutical formulation for parenteral administration.

[0239] In some embodiments, the composition is a liquid pharmaceutical formulation for subcutaneous, intramuscular, or intradermal administration.

[0240] In some embodiments, the composition is in the form of a lyophilized powder, which is subsequently reconstituted with an aqueous medium prior to administration.

[0241] Other aspects of the present disclosure relate to methods for preventing bacterial or viral infection, comprising administering to a subject in need thereof an effective amount of a composition provided herein to elicit an immune response. Some embodiments provide methods for vaccinating a subject in need thereof, comprising administering a composition comprising a nucleic acid encoding an antigenic protein.

[0242] In some embodiments, the composition is administered subcutaneously, intramuscularly, or intradermally.

[0243] In some embodiments, the bacterial infection is a Mycobacterium tuberculosis infection. In some embodiments, the bacterial infection is in the form of nontuberculous mycobacteria.

[0244] In some embodiments, the viral infection is a coronavirus. In some embodiments, the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2.

[0245] In some embodiments, the viral infection is HIV / AIDS.

[0246] In some embodiments, the lipid nanoparticles are administered parenterally.

[0247] In some embodiments, the lipid nanoparticle composition is administered as part of a single injection.

[0248] The present disclosure is characterized in that lipid nanoparticles, it comprises: nucleic acid such as DNA, mRNA, siRNA, antisense oligonucleotides, CRISPR components such as guide RNA (gRNA or sgRNA) and CRISPR associated nuclease (Cas protein), and lipid. Some exemplary lipids include ionizable cationic lipids (ICL), phospholipids, sterol lipids, alkylene glycol lipids (e.g., polyethylene glycol lipids), sphingolipids, glycerolipids, glycerophospholipids, pregnenolone (prenol) lipids, glycolipids, fatty acids and polyketides. In some embodiments, LNP comprises a single type of lipid. In some embodiments, LNP comprises multiple (e.g., two or more) lipids. LNP can include one or more of ionizable cationic lipids, phospholipids, sterols or alkylene glycol lipids (e.g., polyethylene glycol lipids).

[0249] In one embodiment, LNP comprises ionizable cationic lipid. "Ionizable cationic lipid" as used herein, "ionizable lipid" and "ICL" are used interchangeably. ICL is a lipid comprising an ionizable portion capable of carrying an electric charge (e.g., a positive charge, such as a cationic lipid) under specific conditions (e.g., within a specific pH range, e.g., under physiological conditions). The ionizable portion may comprise an amine, and preferably a substituted amine. The ionizable lipid may be a cationic lipid or an anionic lipid. In addition to the ionizable portion, the ionizable lipid may comprise an alkyl or alkenyl group, such as an alkyl or alkenyl group having a length greater than 6 carbon atoms (e.g., a length greater than about 8 carbons, 10 carbons, 12 carbons, 14 carbons, 16 carbons, 18 carbons, 20 carbons or more). Additional ionizable lipids that may be included in the LNPs 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. Patent Nos. 8,710,200 and 8,754,062, each of which is incorporated herein by reference in its entirety.

[0250] In some embodiments, the LNP further comprises an ionizable lipid having the structure of Formula (IV-A), or a pharmaceutically acceptable salt thereof,

[0251]

[0252]

[0253] where R 10 and R 12 are each independently (C1-C4)alkyl optionally substituted with hydroxy; v is 0 or 1; q1 is 1 or 2; Y is R 22 yes a is 1, 2, 3, 4, or 5; and c is 4, 5, 6, 7, or 8.

[0254] In some embodiments, for compounds of formula (III), v is equal to 0. In some embodiments, for compounds of formula (III), v is equal to 1. In some embodiments, for compounds of formula (III), v is equal to 1 and q1 is equal to 1. In some embodiments, for compounds of formula (III), v is equal to 1 and q1 is equal to 2.

[0255] In some embodiments, for compounds of formula (III), R 22 Where the sum of a and c is 6, 7, 8 or 9. In some embodiments, for the compound of formula (III), R 22 In some embodiments, for the compound of formula (III), the sum of a and c is 6. 22 In some embodiments, for the compound of formula (III), the sum of a and c is 7. 22 The sum of a and c is 9.

[0256] In some embodiments, for compounds of formula (III), v is equal to 0, and R 22 Where the sum of a and c is 6, 7, 8 or 9. In some embodiments, for the compound of formula (IV-B), v is equal to 0, and R 22 Where the sum of a and c is 6. In some embodiments, for the compound of formula (III), v is equal to 0, and R 22 Where the sum of a and c is 7. In some embodiments, for the compound of formula (III), v is equal to 0, and R 22 The sum of a and c is 9.

[0257] In some embodiments, for compounds of formula (III), R 10 and R 12 In some embodiments, for compounds of formula (III), R 10 and R 12 Each is a methyl group, and R 22 Where the sum of a and c is 6, 7, 8 or 9. In some embodiments, for the compound of formula (III), R 10 and R 12 Each is methyl, v is 0, and R 22 The sum of a and c is 6, 7, 8 or 9.

[0258] In some embodiments, for compounds of formula (IV-B), v is equal to 0, and R 22 yes In some embodiments, for compounds of formula (III), v is equal to 0, and R 22 yes and the sum of a and c is 7 or 9. In some embodiments, for the compound of formula (III), v is equal to 0, and R 22 yes and a is 4 and c is 5. In some embodiments, for compounds of formula (III), v is equal to 0, and R 22 yes and a is 1 and c is 8. In some embodiments, for compounds of formula (III), v is equal to 0, and R 22 yes And a is 2 and c is 5.

[0259] LNP can comprise the ionizable lipid of about 0.1mol% that concentration is greater than the total lipid content of for example LNP.In one embodiment, LNP comprises the ionizable lipid of about 1mol%, about 2mol%, about 4mol%, about 8mol%, about 20mol%, about 40mol%, about 50mol%, about 60mol%, about 80mol% that concentration is greater than the total lipid content of for example LNP.In one embodiment, LNP comprises the ionizable lipid of about 20mol%, about 40mol% or about 50mol% that concentration is greater than.In one embodiment, LNP comprises the ionizable lipid of about 1mol% to about 95mol% that concentration is the total lipid content of for example LNP.In one embodiment, LNP comprises the ionizable lipid of about 2mol% to about 90mol%, about 4mol% to about 80mol%, about 10mol% to about 70mol%, about 20mol% to about 60mol%, about 40mol% to about 55mol% that concentration is the total lipid content of for example LNP. In one embodiment, the LNP comprises an ionizable lipid at a concentration of about 20 mol% to about 60 mol%. In one embodiment, the LNP comprises an ionizable lipid at a concentration of about 40 mol% to about 55 mol%.

[0260] In one embodiment, LNP comprises phospholipid.Phospholipid is the lipid comprising phosphate group and at least one alkyl, alkenyl or heteroalkyl chain.Phospholipid can be naturally occurring or non-naturally occurring (for example, synthetic phospholipid).Phospholipid can comprise amine, amide, ester, carboxyl, choline, hydroxyl, acetal, ether, carbohydrate, sterol or glycerol.In some embodiments, phospholipid can comprise phosphorylcholine, sphingomyelin or plasmalogen. Some 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), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPC). The phosphocholine phosphate (PSP) is a phosphatidylcholine phosphate (PSP), which contains 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (DAPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (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-phosphocholine (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 incorporated herein by reference in its entirety.

[0261] 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).

[0262] Phosphatidylserine incorporation

[0263] LNPs (e.g., as described herein) may comprise one or more of the following components: (i) an ionizable cationic lipid (ICL) comprising 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 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%), wherein the phospholipid further comprises a C16 or C18 alkyl or alkenyl group; (iii) a cholesterol at a concentration of about 1 mol% to about 95 mol% (or any value therebetween, e.g., about 20 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, and (v) lipids containing polyethylene glycol (PEG) -2000 (e.g., DPG-PEG2000, DPPE-PEG2000, DMPE-PEG2000, DMG-PEG2000) at a concentration of about 0.1 mol% to about 5 mol% (or any value therebetween, e.g., about 1 mol% to about 2.5 mol%). In some embodiments, the LNP comprises two of (i) to (v). In some embodiments, the LNP comprises three of (i) to (v). In some embodiments, the LNP comprises four of (i) to (v). In some embodiments, the LNP comprises each of (i) to (v). In some embodiments, LNP comprises (i) and (ii). In some embodiments, LNP comprises (i) and (iii). In some embodiments, LNP comprises (i) and (v). In some embodiments, LNP comprises (ii) and (iii). In some embodiments, LNP comprises (ii) and (v). In some embodiments, LNP comprises (iii) and (iv). In some embodiments, LNP comprises (iii) and (v). In some embodiments, LNP comprises (i), (ii) and (iii). In some embodiments, LNP comprises (i), (ii) and (v). In some embodiments, LNP comprises (ii), (iii) and (v). In some embodiments, LNP comprises (ii), (iii), (iv) and (v). In one embodiment, LNP consists of four of (i) to (v) or consists essentially of four of (i) to (v). In one embodiment, LNP consists of each of (i) to (v) or consists essentially of each of (i) to (v).In some embodiments, LNP consists of (i) and (ii) or consists essentially of (i) and (ii). In some embodiments, LNP consists of (i) and (iii) or consists essentially of (i) and (iii). In some embodiments, LNP consists of (i) and (v) or consists essentially of (i) and (v). In some embodiments, LNP consists of (ii) and (iii) or consists essentially of (ii) and (iii). In some embodiments, LNP comprises (ii) and (v). In some embodiments, LNP consists of (iii) and (iv) or consists essentially of (iii) and (iv). In some embodiments, LNP consists of (iii) and (v) or consists essentially of (iii) and (v). In some embodiments, LNP consists of (i), (ii) and (iii) or consists essentially of (i), (ii) and (iii). In some embodiments, LNP consists of (i), (ii) and (v) 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).

[0264] LNP can comprise the phospholipid of about 0.1mol% that concentration is greater than the total lipid content of for example LNP.In one embodiment, LNP comprises the phospholipid of about 0.5mol%, about 1mol%, about 1.5mol%, about 2mol%, about 3mol%, about 4mol%, about 5mol%, about 6mol%, about 8mol%, about 10mol%, about 12mol%, about 15mol%, about 20mol%, about 50mol% that concentration is greater than the total lipid content of for example LNP.In one embodiment, LNP comprises the phospholipid of about 1mol%, about 5mol% or about 10mol%.In one embodiment, LNP comprises the phospholipid of about 0.1mol% to about 50mol% that concentration is greater than the total lipid content of for example LNP. In one embodiment, LNP comprises the phospholipid of about 0.5mol% to about 40mol%, about 1mol% to about 30mol%, about 5mol% to about 25mol%, about 10mol% to about 20mol%, about 10mol% to about 15mol% or about 15mol% to about 20mol% of the total lipid content that concentration is such as LNP.In one embodiment, LNP comprises the phospholipid of about 5mol% to about 25mol%.In one embodiment, LNP comprises the phospholipid of about 10mol% to 20mol%.

[0265] In one embodiment, LNP comprises sterol or ionizable sterol molecule.Sterol is the lipid comprising polycyclic structure and optional hydroxyl or ether substituent, and can be naturally occurring or non-naturally occurring (for example, synthetic sterol).Sterol can not comprise double bond, comprises single double bond or multiple double bonds.Sterol can also comprise alkyl, alkenyl, halogen, ester, ketone, hydroxyl, amine, polyether, carbohydrate or ring part.The exemplary list of sterol comprises cholesterol, dehydroergosterol, ergosterol, campesterol, β-sitosterol, stigmasterol, lanosterol, dihydrolanosterol, chain sterol (desmosterol), vegetable sterol (brassicasterol), 7-cholestenol (lathosterol), yeast sterol (zymosterol), 7-dehydrochain sterol (7-dehydrodesmosterol), avenasterol, campesterol, lupeol and cycloartenol. In some embodiments, the sterol comprises cholesterol, dehydroergosterol, ergosterol, campesterol, β-sitosterol, or stigmasterol. Additional sterols that may be included in the LNPs described herein are disclosed in Fahy, E. et al. (J. Lipid. Res. 46: 839-862 (2005).

[0266] Ionizable sterols

[0267] In some embodiments, LNP comprises sterol. In some embodiments, sterol is cholesterol. In some embodiments, sterol is dehydroergosterol. In some embodiments, sterol is ergosterol. In some embodiments, sterol is campesterol. In some embodiments, sterol is β-sitosterol. In some embodiments, sterol is stigmasterol. In some embodiments, sterol is a corticosteroid (e.g., corticosterone, hydrocortisone, cortisone or aldosterone).

[0268] In some embodiments, the ionizable lipid can be a branched chain ionizable lipid selected from ALC-0315 and SM-102:

[0269]

[0270] LNP can comprise the sterol of about 0.1mol% that concentration is greater than the total lipid content of for example LNP.In one embodiment, LNP comprises the sterol of about 0.5mol%, about 1mol%, about 5mol%, about 10mol%, about 15mol%, about 20mol%, about 25mol%, about 35mol%, about 40mol%, about 45mol%, about 50mol%, about 55mol%, about 60mol%, about 65mol% or about 70mol% that concentration is greater than the total lipid content of for example LNP.In one embodiment, LNP comprises the sterol of about 10mol%, about 15mol%, about 20mol% or about 25mol% that concentration is greater than.In one embodiment, LNP comprises the sterol of about 1mol% to about 95mol% that concentration is greater than the total lipid content of for example LNP. In one embodiment, LNP comprises the sterol of about 5mol% to about 90mol%, about 10mol% to about 85mol%, about 20mol% to about 80mol%, about 20mol% to about 60mol%, about 20mol% to about 50mol% or about 20mol% to 40mol% of the total lipid content that concentration is for example LNP.In one embodiment, LNP comprises the sterol that concentration is about 20mol% to about 50mol%.In one embodiment, LNP comprises the sterol that concentration is about 30mol% to about 60mol%.

[0271] In some embodiments, LNP comprises the lipid containing alkylene glycol.The lipid containing alkylene glycol is the lipid comprising at least one alkylene glycol moiety, for example methylene glycol or ethylene glycol moiety.In some embodiments, the lipid containing alkylene glycol comprises polyethylene glycol (PEG).The lipid containing alkylene glycol can be the lipid containing PEG. Polymer-conjugated lipids can 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 portion of the PEG-lipid component may also vary from 500 to 10,000 g / mol, 1,500 to 6000 g / mol, but is preferably about 2,000 MW. Other polymers for conjugation to lipid anchors may include poly (2-methyl-2- Oxazoline) (PMOZ), poly (2-ethyl-2- Examples of the compounds include poly(hydroxyethyl L-asparagine) (PEOZ), poly-N-vinylpyrrolidone (PVP), polyglycerol, poly(hydroxyethyl L-asparagine) (PHEA), and poly(hydroxyethyl L-glutamine) (PHEG).

[0272] The lipid containing PEG can also include amine, amide, ester, carboxyl, phosphate, choline, hydroxyl, acetal, ether, heterocycle or carbohydrate.The lipid containing PEG can include at least one alkyl or alkenyl, such as length is greater than 6 carbon atoms (for example, length is greater than about 8 carbon, 10 carbon, 12 carbon, 14 carbon, 16 carbon, 18 carbon, 20 carbon or more) alkyl or alkenyl, such as except PEG part.In one embodiment, the lipid containing PEG includes containing following PEG part: at least 20 PEG monomers, such as, 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, PEG-lipids include PEG-DMG (e.g., DMG-PEG2k), PEG-c-DMG, PEG-DSG, and PEG-DPG. Additional PEG-lipids that may be included in the LNPs described herein are disclosed in Fahy, E. et al. (J. Lipid. Res. 46: 839-862 (2005), which is incorporated herein by reference in its entirety.

[0273] 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)propoxy]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.

[0274] LNP can comprise the lipid containing alkylene glycol of the approximately 0.1mol% that concentration is greater than the total lipid content of for example LNP.In one embodiment, LNP comprises the lipid containing alkylene glycol of the approximately 0.5mol% that concentration is greater than the total lipid content of for example LNP, about 1mol%, about 1.5mol%, about 2mol%, about 3mol%, about 4mol%, about 5mol%, about 6mol%, about 8mol%, about 10mol%, about 12mol%, about 15mol%, about 20mol%, about 50mol%.In one embodiment, LNP comprises the lipid containing alkylene glycol of the approximately 0.1mol% that concentration is greater than the total lipid content of for example LNP, about 50mol%. In one embodiment, LNP comprises the lipid containing alkylene glycol of about 0.5mol% to about 40mol%, about 1mol% to about 35mol%, about 1.5mol% to about 30mol%, about 2mol% to about 25mol%, about 2.5mol% to about 20%, about 3mol% to about 15mol%, about 3.5mol% to about 10mol% or about 4mol% to 9mol% of the total lipid content that concentration is such as LNP. In one embodiment, LNP comprises the lipid containing alkylene glycol of about 3.5mol% to about 10mol%. In one embodiment, LNP comprises the lipid containing alkylene glycol of about 4mol% to 9mol% of concentration.

[0275] In some embodiments, LNP comprises at least two types of lipids. In one embodiment, LNP comprises two of ionizable lipids, phospholipids, sterols and the lipid containing alkylene glycols. In some embodiments, LNP comprises at least three types of lipids. In one embodiment, LNP comprises three of ionizable lipids, phospholipids, sterols and the lipid containing alkylene glycols. In some embodiments, LNP comprises at least four types of lipids. In one embodiment, LNP comprises each of ionizable lipids, phospholipids, sterols and the lipid containing alkylene glycols.

[0276] LNP (e.g., as described herein) may comprise 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) a phospholipid at a concentration of 0.1 mol% to about 50 mol% (e.g., about 2.5 mol% to about 20 mol%); (iii) a sterol at a concentration of about 1 mol% to about 95 mol% (e.g., about 20 mol% to about 80 mol%); and (iv) a PEG-containing lipid 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 comprises one of (i) to (iv). In some embodiments, the LNP comprises two of (i) to (iv). In some embodiments, the LNP comprises three of (i) to (iv). In some embodiments, the LNP comprises each of (i) to (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).

[0277] LNPs (e.g., as described herein) may comprise one or more of the following components: (i) an ionizable cationic lipid (ICL) 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%); and (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 comprises two of (i) to (iv). In some embodiments, the LNP comprises three of (i) to (iv). In some embodiments, the LNP comprises each of (i) to (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 (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 (i), (ii) and (iv). In some embodiments, the LNP comprises (ii), (iii) and (iv).

[0278] In one embodiment, the LNP comprises an ionizable lipid to phospholipid ratio 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 comprises an ionizable lipid to phospholipid ratio of about 15: 2. In one embodiment, the LNP comprises an ionizable lipid to phospholipid ratio of about 5: 1. In one embodiment, the LNP comprises 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 comprises an ionizable lipid to alkylene-containing lipid ratio 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 comprises 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 comprises 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).

[0279] In some embodiments, LNP (e.g., described herein) comprises two of an ionizable lipid, a phospholipid, a sterol, and a lipid containing an alkylene glycol (e.g., a lipid containing PEG). In another embodiment, LNP (e.g., described herein) comprises three of an ionizable lipid, a phospholipid, a sterol, and a lipid containing an alkylene glycol (e.g., a lipid containing PEG). In some embodiments, LNP (e.g., described herein) comprises each of an ionizable lipid, a phospholipid, a sterol, and a lipid containing an alkylene glycol (e.g., a lipid containing PEG).

[0280] In some embodiments, the diameter of LNP described herein is 5 to 500nm, for example, 10 to 400nm, 20 to 350nm, 25 to 325nm, 30 to 300nm, 50 to 250nm, 60 to 200nm, 75 to 190nm, 80 to 180nm, 100 to 200nm, 200 to 300nm and 150 to 250nm. The diameter of LNP can be determined by any method known in the art, such as dynamic light scattering, transmission electron microscopy (transmission electron microscopy, TEM) or scanning electron microscopy (scanning electron microscopy, SEM). In some embodiments, the diameter of LNP is 50 to 100nm, 70 to 100nm and 80 to 100nm. In one embodiment, the diameter of LNP is about 90nm. In some embodiments, the diameter of LNP described herein is greater than about 30nm. In some embodiments, the diameter of LNP is greater than about 35nm, about 40nm, about 45nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm, about 120nm, about 140nm, about 160nm, about 180nm, about 200nm, about 225nm, about 250nm, about 275nm or about 300nm. In one embodiment, the diameter of LNP is greater than about 70nm. In one embodiment, the diameter of LNP is greater than about 90nm. In one embodiment, the diameter of LNP is greater than about 180nm.

[0281] In some embodiments, the average diameter of multiple LNP described herein is about 40nm to about 180nm. In some embodiments, the average diameter of multiple LNP described herein is about 50nm to about 150nm. In some embodiments, the average diameter of multiple LNP described herein is about 50nm to about 120nm. In some embodiments, the average diameter of multiple LNP described herein is about 60nm to about 120nm. In some embodiments, the average diameter of multiple LNP described herein is about 40nm, about 45nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm, about 120nm, about 140nm, about 160nm, about 180nm.

[0282] In some embodiments, the nanoparticle or nanoparticles described herein have an average neutral to negative surface charge of less than -100 mv, e.g., less than -90 mv, -80 mv, -70 mv, -60 mv, -50 mv, -40 mv, -30 mv, and -20 mv. In some embodiments, the nanoparticle or nanoparticles have a neutral to negative surface charge of between -100 mv and 100 mv, between -75 mv and 0, or between -50 mv and -10 mv.

[0283] 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 nanoparticles or nanoparticles have an average neutral to negative surface charge of less than -100 mv. In some embodiments, the average surface charge of the nanoparticles or nanoparticles at pH 7.4 is between -20 mv and +20, between -10 mv and +10 mv, or between -5 mv and +5 mv. Neutral charged LNPs have improved pharmacokinetic and biological properties compared to cationic LNPs.

[0284] Preparation of lipid nanoparticles (LNP)

[0285] The method for preparing LNP can comprise mixing the first solution with the second solution.Standard liquid mixing technology can be used, such as propeller (propellor) mixing, vortex solution or preferably by microfluid mixing or efficient T mixing to achieve mixing.In some embodiments, the first solution comprises lipid or multiple lipids, and nucleic acid, and wherein all components are dissolved in water / solvent system.Solvent can be any water miscible solvent (for example, ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane or tetrahydrofuran).In some embodiments, the first solution comprises water or pH buffered water of small percentage.The first solution can comprise the water of up to at least 60 volume %, for example, up to at least about 0.05 volume %, 0.1 volume %, 0.5 volume %, 1 volume %, 2 volume %, 3 volume %, 4 volume %, 5 volume %, 10 volume %, 15 volume %, 20 volume %, 25 volume %, 30 volume %, 35 volume %, 40 volume %, 45 volume %, 50 volume %, 55 volume % or 60 volume % water. In one embodiment, the first solution comprises about 0.05% to 60% by volume water, eg, about 0.05% to 50% by volume, about 0.05% to 40% by volume, or about 5% to 20% by volume water.

[0286] In some embodiments, the first solution comprises a single type of lipid, for example, ionizable lipids, phospholipids, sterols or the lipid containing PEG. In some embodiments, the first solution comprises multiple lipids. In some embodiments, multiple lipids comprise ionizable lipids, phospholipids, sterols or the lipid containing PEG. In some embodiments, multiple lipids comprise cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DMG-PEG2k) or α-(3'-{[1,2-bis(myristyloxy)propoxy]carbonylamino}propyl)-ω-methoxy, polyoxyethylene (PEG2000-C-DMG) and ionizable lipids. Multiple lipids can exist in any ratio. In one embodiment, multiple lipids comprise the ionizable lipids or sterols, phospholipids, sterols, the lipid containing PEG or the combination of its specific ratio (for example, ratio described herein) in the above-mentioned lipids.

[0287] In some embodiments, the second solution is water.In some embodiments, the second solution is an aqueous buffer solution of 3 to 6 (for example, pH is about 3, about 4, about 5 or about 6).The second solution can include a load component, such as nucleic acid (such as mRNA).The second solution can include a water-miscible organic solvent of small percentage.The second solution can include at least one water-miscible organic solvent up to at least 60 volume %, for example, up to at least about 0.05 volume %, 0.1 volume %, 0.5 volume %, 1 volume %, 2 volume %, 3 volume %, 4 volume %, 5 volume %, 6 volume %, 7 volume %, 8 volume %, 9 volume %, 10 volume %, 15 volume %, 20 volume %, 25 volume %, 30 volume %, 35 volume %, 40 volume %, 45 volume %, 50 volume %, 55 volume %, 60 volume % or at least one organic solvent (for example, a water-miscible organic solvent) of any volume percentage therebetween. In one embodiment, the second solution comprises an organic solvent of about 0.05% to 60% by volume, for example, an organic solvent (e.g., a water-miscible organic solvent) of about 0.05% to 50% by volume, about 0.05% to 40% by volume, or about 5% to 20% by volume. The aqueous buffer solution can be a citrate buffer solution. In some embodiments, the aqueous buffer solution is a citrate buffer solution having a pH of 4 to 6 (e.g., a pH of about 4, about 5, or about 6). In one embodiment, the aqueous buffer solution is a citrate buffer solution having a pH of about 6.

[0288] In some embodiments, the solution comprising the mixture of the first and second solutions can be diluted, and the mixture comprises an LNP suspension. In some embodiments, the pH of the solution comprising the mixture of the first and second solutions can be adjusted, and the mixture comprises an LNP suspension. The dilution of the LNP suspension or the regulation of the LNP suspension pH can be achieved by adding water, acid, alkali, or aqueous buffer. In some embodiments, the dilution of the LNP suspension or the regulation of the LNP suspension pH are not carried out. In some embodiments, both the dilution of the LNP suspension and the regulation of the LNP suspension pH are carried out.

[0289] In some embodiments, excessive reagents, solvents, and unencapsulated nucleic acids can be removed from the LNP suspension by tangential flow filtration (TFF) (e.g., diafiltration). Organic solvents (e.g., ethanol) and buffer can also be removed from the LNP suspension by TFF. In some embodiments, the LNP suspension is dialyzed rather than TFF. In some embodiments, the LNP suspension is dialyzed rather than TFF. In some embodiments, the LNP suspension is dialyzed and TFF is performed.

[0290] In one aspect, the disclosure features a method that includes treating a sample of LNPs containing nucleic acids 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-octylglucoside, or Zwittergent 3-14) for a period of time suitable to degrade the lipid layer and thereby release the encapsulated and / or entrapped nucleic acids. In one embodiment, the method further includes analyzing the sample for the presence, absence, and / or amount of the released nucleic acid.

[0291] Ligand-containing LNPs

[0292] Some aspects of the present disclosure relate to LNPs comprising a ligand having binding specificity for a cell surface antigen (also referred to herein as a targeting ligand), wherein binding of the ligand to the antigen induces internalization of the ligand. Some embodiments relate to compositions comprising LNPs comprising a ligand as described herein.

[0293] LNP targeting can also be achieved by adding lipids to the formulation. For example, it is known that phosphatidylserine is redistributed to the outer surface of the plasma membrane during apoptosis and is a molecular signal (cue) for phagocyte attraction (Fadok et al. Curr Biol. 2003 Aug 19; 13 (16): R655-7). Phosphatidylserine (PS) and phosphatidylglycerol (PG) are recognized by dendritic cells and can induce the uptake and activation of dendritic cells. LNP targeting can also be achieved by adding certain anionic phospholipids to the formulation (Table 3A). For example, it is known that phosphatidylserine is redistributed to the outer surface of the plasma membrane during apoptosis and is a molecular signal (Fadok et al. Curr Biol. 2003 Aug 19; 13 (16): R655-7) for phagocyte attraction. Phosphatidylserine (PS) and phosphatidylglycerol (PG) are recognized by dendritic cells and can induce their uptake and activation (Caronni et al., Nat Comm. 2021 April 14; 12: 2237-2253; Ischihashi et al., PLOS One 2013). Although anionic phospholipids have been used previously in the context of liposomes, their inclusion in lipid nanoparticles containing condensed nucleic acids was unexpected because the anionic head group can compete with the phosphate backbone of the mRNA for binding sites of the ionizable cationic lipid, can inhibit intracellular escape by altering the surface charge, or can cause LNPs to aggregate during formation or storage.

[0294] Table 3A. Anionic phospholipid targeting moieties

[0295]

[0296] Table 3B. Phosphatidylserine targeting moieties

[0297]

[0298] In some embodiments, the anionic targeting 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 comprises the L-isomer of serine. In another embodiment, the acyl chain of phosphatidylserine is fully saturated, such as in the case of dimyristoylphosphatidyl-L-serine (DMPS), dipalmitoylphosphatidyl-L-serine (DPPS), or distearoylphosphatidyl-L-serine (DSPS). In a preferred embodiment, the PS used is the L-isomer of DPPS or the L-isomer of DSPS. Phosphatidylserine may also comprise an asymmetric acyl chain composition, such as where one acyl chain is stearic acid and the other is palmitic acid.

[0299] In some embodiments, the anionic phospholipids are selected from the group except 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.

[0300] Table 3C. Non-phosphatidylserine anionic phospholipids

[0301]

[0302] In some embodiments, the anionic phospholipid used is phosphatidylglycerol. In another embodiment, the acyl chains of the phosphatidylglycerol are fully saturated, such as in the case of dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), or distearoylphosphatidylglycerol (DSPG). In a preferred embodiment, the PG used is DPPS or DSPS. Phosphatidylglycerol may also contain an asymmetric acyl chain composition, for example, one acyl chain is stearic acid and the other is palmitic acid.

[0303] 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 when being greater than 0.5mg / ml, greater than 1mg / mL, greater than 5mg / mL, greater than 10mg / mL or greater than 20mg / mL. In some embodiments, at a temperature of 22 ° C or lower, as measured in 200 standard ethanol (200proofethanol) by a flask shaking method, the salt form of phosphatidylglycerol or phosphatidylserine is soluble when being at least 0.3mM, at least 0.4mM, at least 0.5mM, at least 0.6mM or at least 0.8mM. 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 salt or a substituted ammonium salt. Substituted ammonium salts can be monoalkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium having alkyl groups, each of which has one to six, one to four, one to three, one, two, or three carbon atoms. One or more alkyl groups can be normal alkyl groups, branched alkyl groups (e.g., such as isopropyl), or form a ring (e.g., such as cyclohexyl). The alkyl group and the nitrogen ammonium atom can form a heterocycle. Substituted ammonium salts can also be formed from alkylenediamines. Tris(hydroxymethyl)aminomethane and triethanolamine can also be used as amine bases to form PS salts. 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, the targeting lipid is an ammonium salt of DPPS.

[0304] Table 3D. Ammonium and sodium salt forms of dipalmitoyl-phosphatidylserine or distearoyl-phosphatidylserine.

[0305]

[0306] To obtain phosphatidylserine in the form of an ammonium salt or a substituted ammonium salt, any method known in the art can be used. In one embodiment, the sodium salt of phosphatidylserine (PS) is dissolved in a single-phase system of chloroform, methanol, and water containing an ammonium or substituted ammonium chloride salt (Bligh-Dyer single phase), and the system is brought to a two-phase state by adding additional water and / or methanol containing ammonium 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 salt (substituted ammonium salt) of PS is obtained by evaporating the chloroform-rich phase. Optionally, the obtained ammonium salt or substituted ammonium salt of PS is vacuum dried or dissolved in cyclohexane and lyophilized. In another embodiment, the sodium or potassium salt of PS is dissolved in a water-immiscible organic solvent, such as chloroform or a chloroform-methanol mixture, and washed with a dilute aqueous solution of an acid (e.g., HCl) to obtain the free acid form of PS, which is then neutralized with a substituted amine or ammonium hydroxide in its free base form. In yet another embodiment, an organic solution of the sodium or potassium salt of PS is treated with a cation exchange resin of ammonium in its substituted ammonium form. In yet another embodiment, PS is prepared as a calcium or magnesium salt and treated with an ammonium or substituted ammonium salt of a chelating agent (e.g., EDTA), or with ammonium phosphate or substituted ammonium phosphate in the presence of an organic solvent, resulting in the replacement of the calcium or magnesium ions with a chelate or a less soluble phosphate, which is separated, for example, by filtration, while the ammonium or substituted ammonium salt of PS remains in the organic (e.g., ethanol) solution.

[0307] In one embodiment, PS or PG are added to the LNP lipid formulations at a concentration of about 0.1mol% to about 20mol%, about 0.1mol% to about 10mol%, about 0.1mol% to about 5mol%, about 0.5mol% to about 20mol%, about 0.5mol% to about 10mol%, about 0.5mol% to about 5mol%, about 1mol% to about 20mol%, about 1mol% to about 10mol% or about 1mol% to about 5mol%. In one embodiment, PS is added to the LNP lipid formulations at a concentration of about 1mol% to about 20mol%, about 2.5mol% to about 10mol%, about 3mol% to about 9mol% or about 4mol% to about 8mol% of the total lipid content of LNP.

[0308] In one embodiment, the PS or PG lipid is included in an LNP composition comprising ionizable cationic lipids 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.

[0309] In another embodiment, the PS lipid is included in an LNP composition comprising an ICL of Formula I, II, III, IV-B, VA-1, a combination thereof, or a pharmaceutical salt 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.

[0310] In some aspects, a method for delivering nucleic acid to a cell is provided, comprising contacting the cell with a composition comprising an LNP containing a ligand (also referred to herein as a targeting ligand) having binding specificity to a cell surface antigen, wherein the internalization of the ligand in combination with the antigen is induced by the binding of the ligand. In some embodiments, the targeting ligand can be, but is not limited to, an internalizing antibody or fragment thereof, a small molecule conjugate, or a glycoconjugate. In some embodiments, when contacted and incubated with a cell expressing at least 100,000 or at least 1,000,000 molecules of antigen under internalization conditions, the binding of the targeting ligand to a specific cell surface antigen is induced by the internalization of the LNP.

[0311] Table 4A. Exemplary dialkyl and branched-chain ionizable cationic lipids

[0312]

[0313] Table 4B. Exemplary dialkyl and branched ionizable cationic lipids (continued)

[0314]

[0315] Composition

[0316] In some embodiments, the lipid nanoparticle composition comprises a lipid and a nucleic acid, the lipid nanoparticle comprising a compound of Formula I, II, III, IV-B, VA-1, a combination thereof, or a pharmaceutically acceptable salt thereof.

[0317] Other aspects of the present disclosure relate to the use of these ionizable lipids or lipid nanoparticle compositions comprising ionizable lipids in vaccines for preventing infectious diseases or cancer. In some embodiments, infectious diseases can be bacterial or viral infections. In some embodiments, compositions described herein can be used to prevent infections related to tuberculosis, HIV / AIDS, malaria or coronavirus-related infections (e.g., COVID-19). In other embodiments, infection is influenza, hepatitis B, hepatitis C, dengue fever, human papillomavirus (human papillomavirus, HPV), norovirus, mumps, measles, meningococcal disease (Meningococcal disease), pneumococcal disease (pneuomococcal disease), polio, rotavirus (rotovirus), respiratory syncytial virus (respiratory syncytial virus, RSV), rubella, herpes zoster virus (shingles / herpes zostervirus), tetanus or whooping cough.

[0318] In some embodiments, the compounds and compositions described herein can promote the effective uptake and transfection of target cells (including tissue macrophages and dendritic cells). The result is the effective delivery of nucleic acids encoding antigens specific to infectious viruses or bacteria, and the subsequent presentation of the antigens to elicit the desired immune response to prevent the corresponding infection. In some embodiments, the nucleic acid can be a synthetic nucleic acid encoding an epitope of a coronavirus such as SARS-CoV, MERS-CoV or SARS-CoV-2 (e.g., a modified codon-optimized mRNA). In some embodiments, the nucleic acid can be a synthetic nucleic acid encoding an S protein (spike protein) of a coronavirus such as SARS-CoV, MERS-CoV or SARS-CoV-2 or a fragment thereof (e.g., a modified codon-optimized mRNA).

[0319] In some embodiments, the vaccine is used to prevent mycobacterial infection. In some embodiments, the vaccine can be used to prevent tuberculosis, nontuberculous mycobacteria (NTM), nontuberculous lung disease, leprosy, mycobacterium avium-intracellulare, mycobacterium kansasii, mycobacterium marinum, mycobacterium ulcerans, mycobacterium chelonae, mycobacterium fortuitum, mycobacterium abscessus, mycobacterium thunbergii, mycobacterium ulcerans, mycobacterium chelonae, mycobacterium fortuitum, mycobacterium thunbergii ... 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 fever, 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), and AIDS. The list includes human metapneumovirus (hMPV), human papillomavirus (HPV), Lassa, Lyme, malaria, Marburg, measles, meningococcal disease, mumps, Nipahvirus, 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.

[0320] In some embodiments, the composition further comprises a pharmaceutical excipient.

[0321] In some embodiments, the lipid nanoparticles are in an aqueous medium.

[0322] In some embodiments, the nucleic acid is encapsulated in the lipid nanoparticle by the ionizable cationic lipid compound provided herein or a combination thereof, wherein the nucleic acid is 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.

[0323] In some embodiments, the lipid nanoparticle comprises a membrane containing phosphatidylcholine and a sterol. In some embodiments, the sterol is cholesterol. In some embodiments, the lipid nanoparticle comprises a membrane containing phosphatidylcholine and an ionizable cationic lipid (ICL). In some embodiments, the ICL has a structure of formula I and cholesterol, wherein the membrane separates the interior of the lipid nanoparticle from the aqueous medium. In some embodiments, the ICL has the structure 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 ionizable cationic lipid to cholesterol is about 65:35 to 40:60. In some embodiments, the molar ratio of ICL to cholesterol is about 60:40 to about 45:55.

[0324] In some embodiments, the molar ratio of phosphatidylcholine to cholesterol is from about 1:5 to about 1:2.

[0325] In some embodiments, the membrane further comprises a polymer-conjugated lipid.

[0326] In some embodiments, the lipid nanoparticle comprises ICL, DSPC, cholesterol, and polymer-conjugated lipid in a molar ratio of about 49.5:10.3:39.6:2.5.

[0327] In some embodiments, the polymer-conjugated lipid is PEG (2000)-dimyristoylglycerol (PEG-DMG) or PEG (molecular weight 2,000)-dimyristoylphosphatidylethanolamine (PEG-DMPE).

[0328] The compositions of the present disclosure can be administered by a variety of routes, for example, by intravenous, parenteral, intraperitoneal or topical routes to achieve systemic delivery. The compositions can be administered intravenously, subcutaneously or intraperitoneally to a subject. In some embodiments, the present disclosure provides methods for delivering nucleic acids in vivo to a subject.

[0329] In some embodiments, the composition is a liquid pharmaceutical formulation for parenteral administration.

[0330] In some embodiments, the composition is a liquid pharmaceutical formulation for subcutaneous, intramuscular, or intradermal administration.

[0331] In some embodiments, the composition is in the form of a lyophilized powder, which is subsequently reconstituted with an aqueous medium prior to administration.

[0332] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I):

[0333]

[0334] Where R1 is

[0335] 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;

[0336] R2 and R3 are each independently (C1-C4)alkyl optionally substituted with hydroxy; and

[0337] n is an integer equal to 2, 3 or 4.

[0338] Wherein a and b in the two R1 hydrocarbon chains are the same or different, or one of the two R1 hydrocarbon chains is a saturated C 12 -C 18 alkyl.

[0339] In some embodiments, ionizable cationic lipid compositions are provided. In some embodiments, lipid nanoparticle (LNP) compositions comprise ionizable cationic lipids having a chemical structure of formula (IA):

[0340]

[0341] Where R1 is

[0342]

[0343] 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;

[0344] R2 and R3 are each independently (C1-C4)alkyl optionally substituted with hydroxy; and

[0345] n is an integer equal to 2, 3 or 4.

[0346] In some embodiments, a and b of the two R1 hydrocarbon chains are the same.

[0347] In some embodiments, a and b of the two R1 hydrocarbon chains are different.

[0348] In some embodiments, one of the two R1 hydrocarbon chains is a saturated C 12 -C 18 alkyl.

[0349] In some embodiments, ionizable cationic lipid compositions are provided. In some embodiments, lipid nanoparticle (LNP) compositions comprise ionizable cationic lipids having a chemical structure of formula (IA):

[0350]

[0351] Where R1 is

[0352]

[0353] 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;

[0354] R2 and R3 are each methyl; and

[0355] n is an integer equal to 3.

[0356] In some embodiments, a and b of the two R1 hydrocarbon chains are the same.

[0357] In some embodiments, a and b of the two R1 hydrocarbon chains are different.

[0358] In some embodiments, one of the two R1 hydrocarbon chains is a saturated C 12 -C 18 alkyl.

[0359] In some embodiments, ionizable cationic lipid compositions are provided. In some embodiments, lipid nanoparticle (LNP) compositions comprise ionizable cationic lipids having a chemical structure of formula (IA):

[0360]

[0361] in:

[0362] R1 is a saturated C15-C18 hydrocarbon chain,

[0363] R2 and R3 are each methyl; and

[0364] n is an integer equal to 3.

[0365] In some embodiments, a and b of the two R1 hydrocarbon chains are the same.

[0366] In some embodiments, a and b of the two R1 hydrocarbon chains are different.

[0367] In some embodiments, one of the two R1 hydrocarbon chains is a saturated C 12 -C 18 alkyl.

[0368] How to use

[0369] Targeting dendritic cells

[0370] Dendritic cells (DCs) are specialized antigen-presenting cells that play a central role in initiating and regulating adaptive immunity. Due to their efficient antigen (Ag) presentation and ability to generate diverse T cell responses, efficient and specific delivery of Ag to DCs is the cornerstone for generating Ag-specific effector and memory cells against tumors or pathogens.

[0371] Dendritic cells can be generated from human blood monocytes by adding granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4 and IFN-γ to differentiate monocyte-derived DCs in vitro. The cells in culture exhibit both dendritic and veiled morphologies, the former being adherent and the latter being suspended. Phenotypically, they are

[0372] CD1a- / dim, CD11a+, CD11b++, CD11c+, CD14dim / -, CD16a- / dim, CD18+, CD32dim / -, CD33+, CD40+, CD45R0+, CD 50+, CD54+, CD64- / dim, CD68+, CD71+, CD80dim, CD86+ / ++, MHCI class++ / , HLA-DR++ / , HLA-DP+, and HLA-DQ (Geiseler et al. Dev Immunol. 1998;6(1-2):25-39).

[0373] As an alternative, a human primary blood dendritic cell line has been developed and is commercially available from Creative Biolabs.

[0374] CD8+ T cells can produce IL2, IFN-γ and TNF, which are known to play a key role during mycobacterium tuberculosis infection. Importantly, CD8+ T cells have cell lysis functions to kill mycobacterium tuberculosis infected cells through granule-mediated functions (through perforin, granzymes and granulysin), or Fas-Fas ligand interactions to induce apoptosis. In humans, CD8+ T cells can produce granulysin, which can directly kill mycobacterium tuberculosis. Therefore, it is expected that the antigen-producing mRNA LNPs delivered to DCs will stimulate CD8+ T cell responses to fight mycobacterium tuberculosis infection.

[0375] CD8+ T cells can recognize mycobacterium tuberculosis (M.tuberculosis) specific antigens (as peptides) presented by classical and non-classical MHC molecules. Classical restricted CD8+ T cells have been identified, and they recognize antigens presented by antigen-presenting cells in the context of classical MHC Ia (HLA-A, HLA-B, HLA-C) molecules. Non-classical restricted CD8+ T cells include those CD8+ T cells that can recognize Mg antigens in the context of HLA-E molecules (non-MHC 1a), glycolipids associated with class 1 CD1 molecules, and MHC I related molecules (MR1), such as mucosal associated invariant T cells (mucosal associated invariant T cell, MAIT). Finally, γδ T cells represent a separate population of CD8 (and CD4) T cells, which have both inherent and adaptive functions in terms of the response to mycobacterium tuberculosis infection. CD8+ T cells have been shown to play a direct role in the response to M. tuberculosis infection, but they also play an important role in coordinating many different functions in the overall host immune response (e.g., interacting to provide optimal CD4 T cell function).

[0376] In one embodiment, LNP can be added to the human dendritic cells of culture with appropriate concentration (for example, 1 to 5 μg / mL mRNA).After a period of time allowing cellular uptake and antigen expression, human T cells (HemaCare) can be added, and at different times, cell culture medium is sampled for the INF-γ by Elisa (R&D Systems, DIF50C). As an alternative, the CD8+ markers or intracellular INFγ of cells can be analyzed by flow cytometry to produce (PE anti-human IFN-γ antibodies, Biolegend).

[0377] In one embodiment, LNPs can be administered to a subject at a dose of 0.01 to 5 mg / kg of mRNA via any of the routes of administration outlined above. According to some embodiments, a portion of the LNPs is taken up by DC cells, while the majority accumulates in the liver and spleen. DC cells can express the antigenic peptide, process it for MHC I presentation, and travel to the lymph nodes for presentation to naive T cells, which induces the education of memory T cells against the antigen.

[0378] In one embodiment, the LNP modified with targeting ligands such as anti-DEC205-PEG-DSPE can be administered to the subject at a dosage of 0.01 to 5 mg / kg of mRNA. According to some embodiments, a higher ratio of LNP can be absorbed by DC cells, which allows improving the generation of antigenic peptides (compared with non-targeted LNP) and the more effective vaccination against pathogens. The other targeting ligands of dendritic cells include but are not limited to CLEC9A, CLEC4A, XCR1, CD141 and HLD-DR. For example, the reactivity of the antigen produced in vivo by assessment CD8+ can be accomplished by measuring INF γ plasma levels by the species-specific IFN-γ Quantikine ELISA test kit from R&D Systems.

[0379] In some embodiments, LNP compositions provide desired pharmacokinetic properties, such as extended plasma half-life and stable mRNA encapsulation. Plasma half-life can be measured as the percentage of the injected dose (injected dose, ID) retained in the blood after 6 or 24 hours after intravenous injection in immunocompetent mice. The encapsulation stability of mRNA after 24 hours in plasma can be determined by the change of mRNA and lipid ratio (mRNA / L ratio) after iv administration in mice. In some embodiments, at 6 hours, the percentage of the encapsulated mRNA retained in the blood is greater than 20% of the injected dose, preferably greater than 30%, and most preferably greater than 40%. The percentage retained in the blood after 24 hours is preferably greater than 10% of the injected dose, and more preferably greater than 20%.

[0380] Disclosed herein are methods for preventing infection with mycobacteria (eg, Mycobacterium tuberculosis) or Gram-positive bacteria (eg, 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, viridans group streptococci, Listeria monocytogenes, monocytogenes), Nocardia, and Corynebacterium.

[0381] The vaccine administered to induce the second immune response may provide an epitope presented by class II MHC that is capable of eliciting a CD4+ helper T cell response against cells expressing the antigen, the epitope presented by MHC being derived from the antigen. Alternatively or in addition, the vaccine administered to induce the second immune response may provide an epitope presented by class I MHC that is capable of eliciting a CD8+ T cell response against cells expressing the antigen, the epitope presented by MHC being derived from the antigen. In addition, the vaccine administered to induce the second immune response may provide one or more new epitopes (including known new epitopes) and one or more epitopes that do not contain 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, the vaccine administered to induce a secondary immune response provides a new epitope that is an epitope presented by MHC class II and / or capable of eliciting a CD4+ helper T cell response against cells expressing the antigen from which the epitope presented by MHC is derived; and provides an epitope that is free of cancer-specific somatic mutations that is an epitope presented by MHC class I and / or capable of eliciting a CD8+ T cell response against cells expressing the antigen from which the epitope presented by MHC is derived. In one embodiment, the epitope does not contain cancer-specific somatic mutations.

[0382] "Cellular immune response," "cellular response," "cellular response to an antigen," or similar terms are intended to include cellular responses directed against cells characterized by presentation of the antigen by class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes, which act as "helper cells" or "killer cells." Helper T cells (also known as CD4+ T cells) play a central role by regulating the immune response, and killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill diseased cells, such as cancer cells, thereby preventing the generation of more diseased cells. In some preferred embodiments, the present disclosure is directed to stimulating anti-M. tuberculosis CTL responses against mycobacteria that express one or more expressed antigens, and preferably such expressed antigens are presented by class I MHC.

[0383] According to the present disclosure, "antigen" encompasses any substance that will trigger an immune response. In particular, "antigen" refers to any substance that specifically reacts with antibodies or T lymphocytes (T cells), preferably peptides or proteins. The term "antigen" as used herein comprises any molecule containing at least one epitope. Preferably, in the context of the present disclosure, an antigen is a molecule that, optionally after processing, induces an immune response that is preferably specific for the antigen (including cells expressing the antigen). According to the present disclosure, any suitable antigen as an immune response candidate can be used, wherein the immune response is preferably a cellular immune response. In the context of the embodiments of the present disclosure, in the context of MHC molecules, the antigen is preferably presented by cells, preferably by antigen presenting cells (including diseased cells), particularly cancer cells, which results in an immune response against the antigen. Antigen is preferably a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens may include tumor antigens.

[0384] As used herein, "antigenic peptide" refers to a portion or fragment of an antigen that is capable of stimulating an immune response, preferably a cellular response, against the antigen or against cells characterized by expressing the antigen and preferably characterized by presenting the antigen (e.g., diseased cells, particularly cancer cells). Preferably, the antigenic peptide is capable of stimulating a cellular response against cells characterized by presenting the antigen with class I MHC, and preferably is capable of stimulating antigen-reactive cytotoxic T lymphocytes (CTLs). Preferably, the antigenic peptide according to the present disclosure is a peptide presented by class I MHC and / or class II MHC, or can be processed to produce a peptide presented by class I MHC and / or class II MHC. Preferably, the antigenic peptide comprises an amino acid sequence that substantially corresponds to the amino acid sequence of the antigenic fragment. Preferably, the antigenic fragment is a peptide presented by class I MHC and / or class II MHC. Preferably, the antigenic peptide according to the present disclosure comprises an amino acid sequence that substantially corresponds to the amino acid sequence of such a fragment, and is processed to produce such a fragment, i.e., a peptide presented by class I MHC and / or class II MHC derived from the antigen. If the peptide is presented directly, i.e. without processing, in particular without cleavage, its length is suitable for binding to MHC molecules, in particular MHC class I molecules, and is preferably 7 to 20 amino acids in length, more preferably 7 to 12 amino acids in length, more preferably 8 to 11 amino acids in length, in particular 9 or 10 amino acids in length.

[0385] The main type of professional antigen presenting cell is the dendritic cell with the widest antigen presentation range, and may be the most important antigen presenting cell, macrophage, B cell and some activated epithelial cells. Dendritic cells (DC) are leukocyte populations that present antigens captured in peripheral tissues to T cells through both class II MHC and class I MHC antigen presentation pathways. Dendritic cells are known to be effective inducers of immune responses, and the activation of these cells is a key step in inducing anti-tumor immunity. Dendritic cells are conveniently classified into "immature" and "mature" cells, which can be used as a simple method to distinguish between two well-characterized phenotypes.

[0386] In some embodiments, the present invention relates to the differentiation of immature dendritic cells.Dendritic cells are characterized by high antigen uptake and processing capacity (this is related to the high expression of Fc γ receptors and mannose receptors) antigen presenting cells. Generally, the mature phenotype is characterized by the lower expression of these markers, but the cell surface molecules responsible for T cell activation are such as class I and class II MHC, adhesion molecules (such as CD54 and CD11) and costimulatory molecules (such as CD40, CD80, CD86 and 4-1BB) high expression. Dendritic cell maturation is considered to be the state of dendritic cell activation, and in this state such antigen presenting dendritic cells cause T cell first immunity, and the presentation of immature dendritic cells causes tolerance.Dendritic cell maturation is mainly caused by the following: there is the biomolecule (bacterial DNA, viral RNA, endotoxin etc.) of the microbial characteristics detected by intrinsic receptors, proinflammatory cytokines (TNF, IL-1, IFN), the connection of CD40 and CD4OL on the dendritic cell surface and the material released from the cell of experience stress cell death. Dendritic cells can be obtained by culturing bone marrow cells in vitro 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 the class II MHC proteins required for interaction with naive T cells; these are only expressed when certain cytokines such as IFNγ stimulate the non-professional antigen-presenting cells. "Antigen-presenting cells" can be loaded with peptides presented by class I MHC by transducing the cells with a nucleic acid encoding the peptide or a polypeptide comprising the peptide to be presented, preferably mRNA (e.g., a nucleic acid encoding an antigen).

[0387] In some embodiments, a pharmaceutical composition comprising a gene delivery vehicle targeting dendritic cells or other antigen presenting cells can be administered to a patient, resulting in transfection in vivo. As used herein, "nucleic acid" is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, most preferably in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present disclosure, nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. According to the present disclosure, nucleic acids can exist as single-stranded or double-stranded, as well as linear or covalently circularly closed molecules. According to the present disclosure, nucleic acids can be isolated. According to the present disclosure, the term "isolated nucleic acid" means that the nucleic acid (i) is amplified in vitro, for example, by polymerase chain reaction (PCR), (ii) is produced by cloning recombination, (iii) is purified, for example, by cutting and separating by gel electrophoresis, or (iv) is synthesized, for example, by chemical synthesis. The nucleic acid can be used to introduce into cells, i.e., transfect cells, particularly in the form of RNA that can be prepared by in vitro transcription from a DNA template. In addition, the RNA can be modified by stabilizing sequences, capping, and polyadenylation before use.

[0388] As used herein, the term "RNA" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the BD-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially or completely purified RNA, substantially pure RNA, synthetic RNA, and recombinantly produced RNA, such as modified RNA that is different from naturally occurring RNA by the addition, deletion, replacement, and / or change of one or more nucleotides. Such changes may include adding a non-nucleotide substance to, for example, the end of the RNA or adding it to the interior, such as at one or more nucleotides of the RNA. The nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0389] The term "RNA" as used herein includes and preferably relates to "mRNA". The term "mRNA" means "messenger-RNA" and refers to a "transcript" that is produced using a DNA template and encodes a peptide or polypeptide. Typically, mRNA comprises 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 produced by in vitro transcription from a DNA template. In the context of RNA used in the present disclosure, the term "modification" includes any modification of non-naturally occurring RNA in the RNA. In one embodiment of the present disclosure, the RNA used in accordance with the present disclosure does not have an uncapped 5'-triphosphate. Removal of such an uncapped 5'-triphosphate can be achieved by treating the RNA with a phosphatase. The RNA according to the present disclosure may have modified ribonucleotides to improve its stability and / or reduce cytotoxicity. For example, in one embodiment, in the RNA used in accordance with the present disclosure, 5-methylcytidine is partially or completely, preferably completely, replaced with cytidine. Alternatively or additionally, in one embodiment, in the RNA used according to the present disclosure, pseudouridine is partially or completely, preferably completely, replaced by uridine.

[0390] In one embodiment, the term "modification" refers to providing an RNA with a 5'-cap or a 5'-cap analog. The term "5-cap" refers to a cap structure present at the 5' end of an mRNA molecule and is typically composed of a guanosine nucleotide linked to the mRNA by an unusual 5' to 5 triphosphate linkage. In one embodiment, the guanosine is methylated at the 7-position. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, preferably a 7-methylguanosine cap (m'G). The term "5'-cap" as used herein includes 5'-cap analogs that are similar to RNA cap structures and are modified to have the ability to stabilize RNA and / or enhance RNA translation (if attached thereto), preferably in vivo and / or in cells.

[0391] According to the present disclosure, the stability and translation efficiency of RNA can be modified as required.For example, by one or more modifications with stabilization and / or improving RNA translation efficiency, RNA can be stabilized and its translation can be improved.For example, such modification is described in PCT / EP2006 / 009448, which is incorporated herein by reference. In order to improve the expression of the RNA used according to the present disclosure, it can be modified in the coding region (i.e., the sequence encoding the expressed peptide or protein), preferably the sequence of the expressed peptide or protein is not changed, so that the GC content is improved to improve mRNA stability and to carry out codon optimization, and therefore the translation in the enhanced cell is enhanced.

[0392] Some aspects of the present disclosure relate to methods of preventing bacterial or viral infection, comprising administering to a subject in need thereof an effective amount of a composition provided herein to elicit an immune response.

[0393] Some aspects of the present disclosure provide methods for vaccinating a subject, comprising administering to the subject a single dose of a composition described herein comprising a nucleic acid (e.g., mRNA) encoding a polypeptide of interest for vaccination. In some embodiments, the nucleic acid is formulated within a cationic lipid nanoparticle. In some embodiments, the lipid nanoparticle composition is administered as a single injection.

[0394] In some embodiments, the bacterial infection is a Mycobacterium tuberculosis infection.

[0395] In some embodiments, the viral infection is a coronavirus. In some embodiments, the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2.

[0396] In some embodiments, the viral infection is HIV / AIDS.

[0397] In some embodiments, lipid nanoparticles are administered parenterally.

[0398] Generally, the drug can be administered to the patient by intradermal injection. However, injection can also be performed intranodally into the lymph nodes (Maloy et al. (2001), Proc Natl Acad Sci USA 98: 3299-3033). The cells produced present the target complex and are recognized by the autologous cytotoxic T lymphocytes that subsequently proliferate.

[0399] In some embodiments, the composition is administered by inhalation.In some embodiments, the composition is formulated as a nasal spray and / or aerosol.

[0400] Actual dosage levels of the active agents in the pharmaceutical compositions disclosed herein may be varied without toxicity to the patient in order to obtain an amount of the active agent that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration.

[0401] As used herein, "parenteral" in the context of administration means modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0402] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral (i.e., through the digestive tract) and topical administration, typically by injection or infusion, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, inhalation, subcapsular, subarachnoid, respiratory mucosal, intraspinal, epidural, and intrasternal injection and infusion. Intravenous injection and infusion are often (but not exclusively) used for liposomal drug administration.

[0403] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response).For example, one or more doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0404] In some embodiments, the dose comprises 0.01 to 5 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 to 5 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 to 3 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 to 3 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 to 1 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 to 1 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 to 0.5 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 to 0.5 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 to 1 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 to 0.1 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 to 0.05 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 to 0.1 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 to 0.05 mg / kg of mRNA.

[0405] The dosage of the compound and / or its pharmaceutically acceptable salt or LNP comprising the compound and / or its pharmaceutically acceptable salt can vary within wide limits and should naturally be adjusted in each particular case to the individual conditions and the pathogen to be controlled.

[0406] In some embodiments, ionizable cationic lipids (ICLs) are provided. Cationic lipids are engineered to have improved stability to oxidative degradation during storage while maintaining high transfection activity or efficacy in cells. Aspects of the present disclosure are based, in part, on the discovery that LNP compositions comprising mRNA and certain ionizable cationic lipids (ICLs) enhance mRNA expression in human dendritic cells.

[0407] In some embodiments, LNP compositions include targeting ligands for cell surface receptors for targeting lipid nanoparticles to dendritic cells in a highly specific manner. In some embodiments, LNP compositions include phosphatidyl-L-serine compounds as targeting ligands, such as dipalmitoylphosphatidyl-L-serine (DPPS) or distearoylphosphatidyl-L-serine (DSPS). In some embodiments, LNP compositions include phosphatidyl-L-serine compounds and anionic phospholipids as targeting ligands. In some embodiments, LNP compositions include compounds containing phosphatidylglycerol as targeting ligands, such as distearoylphosphatidylglycerol (DSPG) or dipalmitoylphosphatidylglycerol (DPPG), for enhancing expression in human dendritic cells. In some embodiments, LNP compositions include phosphatidyl-L-serine compounds as targeting ligands and distearoylphosphatidylcholine (DSPC) as a second phospholipid. In some embodiments, the LNP composition comprises both a phosphatidyl-L-serine compound as a targeting ligand and distearoylphosphatidylcholine (DSPC) as a second phospholipid, but does not comprise dipalmitoylphosphatidylcholine (DPPC).

[0408] Some aspects of the present disclosure are based, in part, on the discovery that the selection of certain cationic ionizable lipids can enhance transfection of human dendritic cells. For example, in LNP compositions, KC3 cationic ionic lipids were more active in transfecting human dendritic cells than KC2 or diacyl ionizable lipids (UO series). Among LNP compositions comprising KC3 ionizable cationic lipids, those containing ionizable cationic lipids with monounsaturated alkyl chains were surprisingly more active and more stable to oxidative degradation than those containing ionizable cationic lipids with dilinoleyl alkyl chains. In addition, reduced transfection activity in human dendritic cells was observed in LNP compositions comprising monounsaturated lipids, for example, for diacyl ionizable lipids (UO series).

[0409] In some embodiments, some salt of phosphatidylserine targeting lipid is provided.For example, in some embodiments, phosphatidylserine targeting lipid can be provided as the ammonium salt of DPPS, and it has improved biophysical properties and higher solubility in the presence of ethanol (preferred solvent for the preparation of LNP). DSPS or the sodium salt of DSPS are insoluble in ethanol, and need to exist methanol and heat both to allow it to form, and the ammonium salt of DSPS is also the same. Other ammonium salts of expected phosphatidylserine will produce identical advantages aspect solubility and biophysical properties.

[0410] In some embodiments, an ionizable cationic lipid composition is provided that can be used to prepare a liposome nanoparticle (LNP) composition. In some embodiments, a liposome composition comprising an ionizable cationic lipid is provided, wherein the ionizable cationic lipid has (a) a linear C 16 or C 18 The hydrocarbon chain pairs, each chain comprising a single unsaturated olefinic double bond in each polyolefin chain, are covalently bound to a head group comprising a dialkylaminoalkyl group. In some embodiments, the head group of the ionizable cationic lipid has a dialkylamino group with a pKa of about 6.3 to 7.5. In some embodiments, the head group of the ionizable cationic lipid comprises a heterocyclic or alkyl moiety covalently bound to the dialkylamino group. In some embodiments, the head group of the ionizable cationic lipid optionally further comprises 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 total length of 15, 16, 17, or 18 carbons.

[0411] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I):

[0412]

[0413] Where R1 is

[0414]

[0415] 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;

[0416] R2 and R3 are each independently (C1-C4)alkyl optionally substituted with hydroxy; and

[0417] n is an integer equal to 2, 3 or 4.

[0418] In some embodiments, ionizable cationic lipid compositions are provided. In some embodiments, lipid nanoparticle (LNP) compositions include ionizable cationic lipids with a 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.

[0419] In some embodiments, ionizable cationic lipid compositions are provided. In some embodiments, lipid nanoparticle (LNP) compositions comprise ionizable cationic lipids having a chemical structure of formula (IA):

[0420]

[0421] Where R1 is

[0422]

[0423] 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;

[0424] R2 and R3 are each methyl; and

[0425] n is an integer equal to 3.

[0426] In some embodiments, the LNP composition comprises an ionizable cationic lipid comprising an identical pair of lipid hydrocarbon tails having a total of 15, 16, 17, or 18 carbons and comprising a single olefin group or a pair of olefin groups. In some embodiments, the LNP composition comprises an ionizable cationic lipid selected from the group consisting of:

[0427]

[0428] 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).

[0429] 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.

[0430] The salt form of the targeting lipid can affect its solubility in the alcohol-containing solvent used in preparing 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; one or more phospholipids comprising a phosphatidylserine (PS) lipid; and optionally further comprises a conjugated 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 DSPC, DPPC and DOPC; and a PS lipid selected from DPPS, DSPS and DOPS; and optionally further comprises a conjugated lipid containing PEG.

[0431] In some embodiments, the ionizable lipid is AKG-UO-1A:

[0432] In some embodiments, the ionizable lipid is AKG-UO-1B:

[0433] In some embodiments, the ionizable lipid is AKG-UO-2

[0434] In some embodiments, the ionizable lipid is AKG-UO-4:

[0435] In some embodiments, the ionizable lipid is AKG-UO-4A:

[0436] In some embodiments, the ionizable lipid is AKG-UO-5:

[0437]

[0438] 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:

[0439]

[0440] In some embodiments, the LNP composition may include anionic phospholipids. In some embodiments, the LNP composition is prepared using a sodium salt or an ammonium salt of anionic phospholipids. In some embodiments, the anionic phospholipid salt is a compound of formula (VA-1) having the following chemical structure:

[0441]

[0442] in:

[0443] X + It is ammonium (NH4 + ) or sodium (Na + ) cations; and

[0444] a is 14, 15 or 16.

[0445] In some embodiments, the anionic phospholipid salt is selected from:

[0446]

[0447] In some embodiments, the anionic phospholipid salt is DSPS (L-isomer) sodium salt. In some embodiments, the anionic phospholipid salt is DSPS (L-isomer) ammonium salt. In some embodiments, the anionic phospholipid salt is DPPS (L-isomer) sodium salt. In some embodiments, the anionic phospholipid salt is DPPS (L-isomer) ammonium salt. In some embodiments, the targeting lipid is a sodium salt or ammonium salt of dipalmitoylphosphatidyl-L-serine (DPPS) or distearoylphosphatidyl-L-serine (DSPS). In some embodiments, the targeting lipid is a sodium salt or ammonium salt of dipalmitoylphosphatidyl-L-serine (DPPS) or distearoylphosphatidyl-L-serine (DSPS).

[0448] In some embodiments, the LNP composition may comprise an anionic phospholipid selected from the group consisting of:

[0449]

[0450] In some embodiments, the salt form of phosphatidylserine is highly soluble in ethanol. In some embodiments, it is soluble when being greater than 0.5mg / ml, greater than 1mg / mL, greater than 5mg / mL, greater than 10mg / mL or greater than 20mg / mL. In some embodiments, salt is an ammonium salt. In some embodiments, salt is ammonium itself, alkylammonium, dialkylammonium or trialkylammonium salt. In some embodiments, amine is selected from ammonia, dimethylamine, diethylamine, triethylamine, trimethylamine, 2-(dimethylamino) ethanol, diethanolamine, 2-(diethylamino) ethanol, ethanolamine, ethylenediamine, N-methyl-glucosamine, imidazoles, histidine, lysine, arginine, 4-(2-hydroxyethyl)-morpholine, piperazine, 1-(2-hydroxyethyl)-pyrrolidine, triethanolamine and tromethamine (tris(hydroxymethyl)aminomethane). In some embodiments, the targeting lipid is the ammonium salt of DPPS.

[0451] The anionic phospholipids separated from phosphatidyl-L-serine are also considered to be the targeting lipids of LNP. These include phosphatidylglycerol (PG), phosphatidic acid (PA), N-glutaryl-phosphatidylethanolamine (N-Glu-PE), N-succinyl-phosphatidylethanolamine (N-Suc-PE) and cardiolipin. In some embodiments, LNP includes the anionic phospholipids separated from phosphatidyl-L-serine, which can be used as the targeting lipids of LNP. In some embodiments, LNP includes an anionic phospholipid selected from: 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.

[0452] In some embodiments, a lipid nanoparticle (LNP) composition comprising an ionizable cationic lipid composition is provided. In some embodiments, a lipid nanoparticle (LNP) composition comprising an ionizable cationic lipid is provided. In some embodiments, the LNP composition comprises an mRNA nucleic acid. In some embodiments, the lipid nanoparticle (LNP) composition further comprises a PS lipid having a total amount of 2.5 to 10 mol% of the total lipid in the LNP composition. In some embodiments, the lipid nanoparticle (LNP) composition further comprises a PS lipid selected from DSPS (L-isomer) and DPPS. In some embodiments, the lipid nanoparticle (LNP) composition comprises a conjugated lipid having a total amount of 0.5 to 2.0 mol% of the total lipid content in the LNP composition. In some embodiments, the lipid nanoparticle (LNP) composition comprises a conjugated lipid having a total amount less than 2 mol% of the total lipid content in the LNP composition, and the conjugated lipid is PEG-DMG.

[0453] In some embodiments, lipid nanoparticle (LNP) compositions comprise nucleic acid; ionizable lipid disclosed herein; sterol; one or more phospholipids comprising phosphatidylserine (PS) lipids; and optionally further comprise conjugated lipids. In some embodiments, lipid nanoparticle (LNP) compositions comprise mRNA nucleic acid; ionizable lipid disclosed herein; cholesterol; one or more phospholipids selected from SM, DSPC, HSPC, DPPC and DOPC; and PS lipids selected from DPPS and DSPS; and optionally further comprise conjugated lipids containing PEG. In some embodiments, the nucleic acid lipid nanoparticle (LNP) composition comprises: a nucleic acid; an ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content in the LNP composition; a sterol in a total amount of 25 to 45 mol% of the total lipid content in the LNP composition; and one or more phospholipids in a total amount of phospholipids of 5 to 25 mol% of the total lipid content in the LNP composition, and comprising phosphatidylserine (PS) in a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition; and optionally further comprises a conjugated lipid in a total amount of 0.5 to 2.5 mol% of the total lipid content in the LNP composition.

[0454] 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).

[0455] Some aspects of the present disclosure relate to lipid nanoparticle (LNP) compositions comprising ionizable lipids having the following chemical structure:

[0456]

[0457] Where R1 is

[0458]

[0459] 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;

[0460] R2 and R3 are each independently (C1-C4)alkyl optionally substituted with hydroxy; and

[0461] n is an integer equal to 2, 3 or 4.

[0462] 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.

[0463] In some embodiments, R1 is

[0464]

[0465] a is 0 or 1, and b is 1 or 3; R2 and R3 are each methyl; and n is 2 or 3. In some embodiments, n is 3.

[0466] In some embodiments, the present invention comprises a nucleic acid; an ionizable lipid as described herein, a sterol; one or more phospholipids comprising a phosphatidylserine (PS) lipid; and optionally a conjugated lipid.

[0467] In some embodiments, the nucleic acid is mRNA.

[0468] In some embodiments, the sterol is cholesterol.

[0469] In some embodiments, the one or more phospholipids consist of: one or more phospholipids selected from SM, DSPC, HSPC, DPPC, and DOPC; and a PS lipid selected from DPPS and DSPS.

[0470] In some embodiments, the one or more phospholipids consist of DSPC; and the one or more PS lipids are selected from (L-serine)DPPS and (L-serine)DSPS.

[0471] In some embodiments, the composition comprises PS lipids in a total amount of 2.5 to 10 mol % of the total lipids in the composition.

[0472] In some embodiments, the conjugated lipid comprises PEG.

[0473] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) compositions comprising: a nucleic acid; an ionizable cationic lipid in a total amount of 40 to 65 mol % of the total lipid content in the LNP composition; a sterol in a total amount of 25 to 45 mol % of the total lipid content in the LNP composition; and one or more phospholipids in a total amount of phospholipids of 5 to 25 mol % of the total lipid content in the LNP composition and including phosphatidylserine (PS) in a total amount of 2.5 to 10 mol % of the total lipid content in the LNP composition; and optionally a conjugated lipid in a total amount of 0.5 to 2.5 mol % of the total lipid content in the LNP composition.

[0474] In some embodiments, the nucleic acid is mRNA.

[0475] In some embodiments, the sterol is cholesterol.

[0476] In some embodiments, the one or more phospholipids consist of DSPC and L-serine PS.

[0477] In some embodiments, the composition comprises PS in a total amount of 2.5 to 7.5 mol% of the total lipids in the composition.

[0478] In some embodiments, the conjugated lipid comprises PEG. In some embodiments, the conjugated lipid is PEG-DMG.

[0479] In some embodiments, the LNP comprises a total amount of conjugated lipids that is 0.5 to 2.0 mol% of the total lipid content in the LNP composition. In some embodiments, the total amount of conjugated lipids is less than 2 mol% of the total lipid content in the LNP composition.

[0480] In some embodiments, the nucleic acid is mRNA, the total amount of ionizable cationic lipids is 45 to 55 mol% of the total lipid content in the LNP composition; the sterol is cholesterol in a total amount of 35 to 45 mol% of the total lipid content in the LNP composition; the total amount of phospholipids is 7 to 15 mol% of the total lipid content in the LNP composition; the one or more phospholipids are composed of DSPC and PS lipids, wherein the PS lipids are one or more lipids selected from DPPS and DSPS in the L-serine configuration; and the total amount of PS lipids is about 5 mol% of the total lipid content in the LNP composition.

[0481] In some embodiments, the composition comprises PS lipids in a total amount selected from the group consisting of 1.25 mol%, 2.5 mol%, 5 mol%, 7.5 mol%, and 10 mol% of the total lipid content in the LNP composition.

[0482] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) compositions comprising: a nucleic acid, wherein the nucleic acid is mRNA; an ionizable cationic lipid, the total amount of the ionizable cationic lipid being 45 to 55 mol % of the total lipid content in the LNP composition; a sterol, wherein the sterol is cholesterol in a total amount being 35 to 45 mol % of the total lipid content in the LNP composition; one or more phospholipids, wherein the one or more phospholipids have a total amount of phospholipids of 10 mol % of the total lipid content in the LNP composition and comprise phosphatidylserine (PS) in a total amount being 3 to 9 mol % of the total lipid content in the LNP composition; and a conjugated lipid, the total amount of the conjugated lipid being 0.5 to 2.0 mol % of the total lipid content in the LNP composition.

[0483] 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).

[0484] In some embodiments, the conjugated lipid is PEG-DMG; and the PS lipid is selected from DSPS (L-isomer) and DPPS.

[0485] 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).

[0486] In some embodiments, the LNP comprises a nucleic acid; an ionizable cationic lipid in a total amount of 50 mol% of the total lipid content in the LNP composition; cholesterol in a total amount of 38.5 mol% of the total lipid content in the LNP composition; one or more phospholipids in a total amount of 7 to 15 mol% of the total lipid content in the LNP composition, and including phosphatidylserine (PS) lipids in a total amount of 3 to 9 mol% of the total lipid content in the LNP composition; and PEG-containing lipids in a total amount of 0.5 to 2.0 mol% of the total lipid content in the LNP composition.

[0487] In some embodiments, the phospholipids consist of one or more phospholipids selected from DSPC, DOPC, DPPC, HSPC, and SM.

[0488] In some embodiments, the PS lipid is one or more L-serine lipids selected from DPPS and DSPS.

[0489] In some embodiments, the one or more phospholipids comprise at least two (L-serine) PS lipids with mismatched acyl chain lengths.

[0490] 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% each based on the total lipid content in the LNP composition.

[0491] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) compositions comprising: a nucleic acid, an ionizable cationic lipid KC3-PA or KC3-OA, and a total amount of (L-serine) PS lipid in an amount of 2.5 to 10 mol % of the total lipid content in the LNP composition.

[0492] 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 DSPC, DPPC, HSPC, and SM.

[0493] In some embodiments, the LNP composition further comprises 0.5 to 2.0 mol% PEG-DMG or PEG-DSG based on the total lipid content in the LNP composition.

[0494] In some embodiments, the ionizable cationic lipid is KC3-PA. In some embodiments, the ionizable cationic lipid is KC3-OA.

[0495] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) compositions comprising: a nucleic acid, a KC3-C17 (C8:1) ionizable cationic lipid; and (L-serine) PS lipid in a total amount of 2.5 to 10 mol % of the total lipid content in the LNP composition.

[0496] In some embodiments, the LNP composition has an N / P ratio of 4 to 7. In some embodiments, the composition has an N / P ratio of 5 to 6. In some embodiments, the composition has an N / P ratio of 5.3.

[0497] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) compositions comprising: a nucleic acid, an ionizable cationic lipid KC3-PA, and a total amount of (L-serine) PS lipids in an amount of 2.5 to 10 mol % of the total lipid content in the LNP composition.

[0498] 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 DSPC, DOPC, DPPC, HSPC, and SM.

[0499] In some embodiments, the LNP composition further comprises 0.5 to 2.0 mol% PEG-DMG or PEG-DSG based on the total lipid content in the LNP composition.

[0500] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) compositions comprising: a nucleic acid, an ionizable cationic lipid selected from KC3-C17 (C8:1); and (L-serine) PS lipid in a total amount of 2.5 to 10 mol % of the total lipid content in the LNP composition.

[0501] In some embodiments, the N / P ratio is 4 to 7. In some embodiments, the N / P ratio is 5 to 6. In some embodiments, the N / P ratio is 3. In some embodiments, the N / P ratio is 7.

[0502] In some embodiments, the nucleic acid is mRNA encoding the SARS-CoV-2 spike protein.

[0503] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) vaccine compositions comprising: mRNA nucleic acid at an N / P ratio of 4 to 7; KC3-PA ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content in the LNP composition; cholesterol in a total amount of 25 to 40 mol% of the total lipid content in the LNP composition; (L-serine) PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition; DSPC phospholipid in a total amount of 5 to 25 mol% of the total lipid content in the LNP composition; and PEG-DMG in a total amount of 0 to 2.5 mol% of the total lipid content in the LNP composition.

[0504] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) vaccine compositions comprising: mRNA nucleic acid with an N / P ratio of 3 to 8; KC3-C17 (C8:1) ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content in the LNP composition; cholesterol in a total amount of 25 to 40 mol% of the total lipid content in the LNP composition; (L-serine) PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition; DSPC phospholipid in a total amount of 5 to 25 mol% of the total lipid content in the LNP composition; and PEG-DMG in a total amount of 0 to 2.5 mol% of the total lipid content in the LNP composition.

[0505] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) vaccine compositions comprising: mRNA nucleic acid with an N / P ratio of 4 to 7; KC3-C15 (C8:1) ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content in the LNP composition; cholesterol in a total amount of 25 to 40 mol% of the total lipid content in the LNP composition; (L-serine) PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition; DSPC phospholipid in a total amount of 5 to 25 mol% of the total lipid content in the LNP composition; and PEG-DMG in a total amount of 0 to 2.5 mol% of the total lipid content in the LNP composition.

[0506] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) vaccine compositions comprising: mRNA nucleic acid with an N / P ratio of 3 to 8; KC3-C18 ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content in the LNP composition; cholesterol in a total amount of 25 to 40 mol% of the total lipid content in the LNP composition; (L-serine) PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition; DSPC phospholipid in a total amount of 5 to 25 mol% of the total lipid content in the LNP composition; and PEG-DMG in a total amount of 0 to 2.5 mol% of the total lipid content in the LNP composition.

[0507] In some embodiments, the nucleic acid is the mRNA of SEQ ID NO:2.

[0508] Some aspects of the present disclosure relate to the use of (L-serine) PS lipids in combination with ionizable cationic lipids as described herein for targeting LNP to dendritic cells. In some embodiments, LNP comprises mRNA. In some embodiments, LNP also comprises cholesterol. In some embodiments, the total amount of (L-serine) PS lipids in LNP is 2.5 to 10 mol% of the total lipid content in the LNP composition. In some embodiments, LNP also comprises one or more other phospholipids, and the other phospholipids include DSPC. In some embodiments, LNP also comprises conjugated lipids. In some embodiments, the LNP comprises: an mRNA nucleic acid having an N / P ratio of 3 to 8; KC3-PA or KC3-C17 (C8: 1) ionizable cationic lipid (ICL) in an amount of 40 to 65 mol% of the total lipid content in the LNP composition; cholesterol in an amount of 25 to 40 mol% of the total lipid content in the LNP composition; (L-serine) PS lipid in an amount of 2.5 to 10 mol% of the total lipid content in the LNP composition; DSPC phospholipid in an amount of 5 to 25 mol% of the total lipid content in the LNP composition; and conjugated lipid in an amount of 0 to 2.5 mol% of the total lipid content in the LNP composition. In some embodiments, the ICL is KC3-PA. In some embodiments, the ICL is KC3-C17 (C8: 1).

[0509] Some aspects of the present disclosure relate to lipid nanoparticle (LNP) compositions comprising ionizable lipids having the following chemical structure:

[0510]

[0511] Where R1 is

[0512]

[0513] 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;

[0514] R2 and R3 are each independently methyl; and

[0515] n is an integer equal to 2 or 3.

[0516] In some embodiments, a is 0. In some embodiments, b is 1. In some embodiments, b is 3.

[0517] In some embodiments, a is 1. In some embodiments, b is 1. In some embodiments, b is 3.

[0518] In some embodiments, n is 2. In some embodiments, n is 3.

[0519] 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. Distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), N-succinyl-distearoylphosphatidylethanolamine (N-Suc-DSPE), N-glutaryl-distearoylphosphatidylethanolamine (N-glu-DSPE), distearoylphosphatidic acid (DSPA), and cardiolipin.

[0520] In some embodiments, the composition comprises an anionic targeting phospholipid other than phosphatidyl-L-serine.

[0521] In some embodiments, the composition comprises an anionic phospholipid selected from DSPG and DPPG.

[0522] In some embodiments, the composition comprises an anionic phospholipid selected from N-Glu-DSPE and N-Suc-DSPE.

[0523] In some embodiments, the composition comprises DSPA anionic phospholipid.

[0524] In some embodiments, the composition comprises cardiolipin anionic phospholipid.

[0525] In some embodiments, the ionizable lipid has the following chemical structure:

[0526]

[0527] In some embodiments, the ionizable lipid has the following chemical structure:

[0528]

[0529] Some aspects of the present disclosure relate to sodium or ammonium salts of compositions of anionic phospholipids of formula (VA-1) having the following chemical structure:

[0530]

[0531] where X + is an ammonium cation or sodium (Na + ) cation; and a is 14, 15 or 16.

[0532] In some embodiments, a is 14 or 16. In some embodiments, X + It is an ammonium cation (NH4 + ).

[0533] In some embodiments, X + It is a sodium cation (Na + )

[0534] In some embodiments, X is an ammonium cation selected from the group consisting of: ammonium (NH4 + ), 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).

[0535] In some embodiments, the anionic phospholipid of formula (VA-1) is the sodium salt of distearoylphosphatidyl-L-serine (DSPS L-isomer). In some embodiments, the anionic phospholipid of formula (VA-1) is the ammonium salt of distearoylphosphatidyl-L-serine (DSPS L-isomer). In some embodiments, the anionic phospholipid of formula (VA-1) is the sodium salt of DPPS (L-isomer). In some embodiments, the anionic phospholipid of formula (VA-1) is the ammonium salt of DPPS (L-isomer). Some embodiments relate to the use of the salt form composition in the preparation of a liposomal nanoparticle (LNP) composition. In some embodiments, the use is combined with one or more of the following LNP components during the preparation of the LNP composition: mRNA nucleic acid; ionizable cationic lipid (ICL); cholesterol; (L-serine) PS lipid; one or more phospholipids; and conjugated lipids. In some embodiments, the use includes the step of combining the ammonium or salt form of the compound of formula (VA-1) with one or more of the following LNP components during the preparation of the LNP composition: mRNA nucleic acid; ionizable cationic lipid (ICL); cholesterol; (L-serine) PS lipid; one or more phospholipids; and conjugated lipids.

[0536] In some embodiments, the LNP is a nucleic acid lipid nanoparticle vaccine composition comprising: an mRNA nucleic acid having an N / P ratio of 4 to 7; an ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content in the LNP composition; cholesterol in a total amount of 25 to 40 mol% of the total lipid content in the LNP composition; (L-serine) PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition; DSPC phospholipid in a total amount of 5 to 25 mol% of the total lipid content in the LNP composition; and PEG-DMG in a total amount of 0 to 2.5 mol% of the total lipid content in the LNP composition.

[0537] In some embodiments, the nucleic acid is the mRNA of SEQ ID NO:2.

[0538] In some embodiments, the LNP composition comprises an ionizable cationic lipid in an amount of 46 to 65 mol% of the total lipid content in the LNP composition. In some embodiments, the LNP composition comprises PS in an amount of about 5 mol% of the total lipid content in the composition. In some embodiments, the LNP composition comprises conjugated lipid in an amount of about 1.5 mol% of the total lipid content in the LNP composition.

[0539] In some embodiments, the conjugated lipid is PEG-DMG; and the PS lipid is selected from DSPS (L-isomer) and DPPS.

[0540] 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).

[0541] Some embodiments relate to the use of (L-serine) PS lipids in combination with the ionizable cationic lipids described herein in LNPs for targeting the LNPs to dendritic cells.

[0542] In some embodiments, LNP comprises mRNA.In some embodiments, LNP also comprises cholesterol.In some embodiments, the total amount of (L-serine) PS lipid in LNP is 2.5 to 10mol% of total lipid content in LNP compositions. In some embodiments, LNP also comprises one or more other phospholipids, and described other phospholipids comprises DSPC. In some embodiments, LNP also comprises the lipid of conjugation.

[0543] In some embodiments, the LNP comprises: an mRNA nucleic acid having an N / P ratio of 3 to 8; 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 in the LNP composition; cholesterol in a total amount of 25 to 40 mol% of the total lipid content in the LNP composition; (L-serine) PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition; DSPC phospholipid in a total amount of 5 to 25 mol% of the total lipid content in the LNP composition; and conjugated lipid in a total amount of 0 to 2.5 mol% of the total lipid content in the LNP composition.

[0544] In some embodiments, the ICL is KC3-PA. In some embodiments, the ICL is KC3-C17(C8:1).

[0545] In some embodiments, the composition comprises an anionic phospholipid selected from DSPG and DPPG in an amount ranging from 2.5% to 7.5% of the total lipid content in the LNP composition. In some embodiments, the composition comprises an anionic phospholipid selected from DSPG and DPPG in an amount ranging from 2.5% to 7.5% of the total lipid content in the LNP composition. In some embodiments, the composition comprises an anionic phospholipid selected from DSPG and DPPG in an amount ranging from 2.5% to 7.5% of the total lipid content in the LNP composition.

[0546] In some embodiments, the LNP further comprises one or more additional phospholipids including DSPC.

[0547] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) compositions comprising: a nucleic acid; a KC3 ionizable cationic lipid in a total amount of 40 to 65 mol % of the total lipid content in the LNP composition; a cholesterol in a total amount of 23.5 to 43.5 mol % of the total lipid content in the LNP composition; a (L-serine) PS lipid in a total amount of 2.5 to 10 mol % of the total lipid content in the LNP composition; a DSPC or HSPC phospholipid in a total amount of 5 to 25 mol % of the total lipid content in the LNP composition; and a PEG-containing conjugated lipid in a total amount of 0.5 to 2.5 mol % of the total lipid content in the LNP composition.

[0548] In some embodiments, the nucleic acid is mRNA.In some embodiments, the N / P ratio is 3 to 8.

[0549] 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).

[0550] In some embodiments, the conjugated lipid is PEG-DMG or PEG-DSG.

[0551] In some embodiments, the composition comprises PEG-containing conjugated lipids in a total amount of 0.5 to 2.0 mol % of the total lipid content in the LNP composition.

[0552] In some embodiments, the composition comprises KC3 ionizable cationic lipids in a total amount of 48 mol % of the total lipid content in the LNP composition.

[0553] In some embodiments, the composition comprises a total amount of DSPC and DSPS that is 10 mol% of the total lipid content in the LNP composition.

[0554] In some embodiments, the composition comprises 5% DSPC or HSPC in a total amount of 5 mol% of the total lipid content in the LNP composition.

[0555] In some embodiments, the composition comprises PEG-DMG in a total amount of 1.5 mol % of the total lipid content in the LNP composition.

[0556] In some embodiments, the composition comprises cholesterol in a total amount that is 40.5 mol% of cholesterol based on the total lipid content in the LNP composition.

[0557] In some embodiments, the composition comprises DSPC phospholipids in a total amount of 10 mol % of the total lipid content in the LNP composition.

[0558] In some embodiments, the PEG-containing conjugated lipid is PEG 2000 -DMG.

[0559] In some embodiments, the composition comprises cholesterol in an amount of 23.5 mol% of the total lipid content in the LNP composition. In some embodiments, the composition comprises cholesterol in an amount of 33.5 mol% of the total lipid content in the LNP composition. In some embodiments, the composition comprises cholesterol in an amount of 38.5 mol% of the total lipid content in the LNP composition. In some embodiments, the composition comprises cholesterol in an amount of 40.5 mol% of the total lipid content in the LNP composition. In some embodiments, the composition comprises cholesterol in an amount of 42.7 mol% of the total lipid content in the LNP composition. In some embodiments, the composition comprises cholesterol in an amount of 43.5 mol% of the total lipid content in 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 in 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 in the LNP composition.

[0560] Some aspects of the present disclosure relate to nucleic acid lipid nanoparticle (LNP) compositions comprising: mRNA nucleic acid; a KC3 ionizable cationic lipid selected from the following: KC3-OA, KC3-PA, KC3-C17 (8:1), and KC3-C15 (C8:1), in a total amount of 45 to 55 mol% of the total lipid content in the LNP composition; cholesterol in a total amount of 33.5 to 43.5 mol% of the total lipid content in the LNP composition; (L-serine) DPPS lipid in a total amount of 5 mol% of the total lipid content in the LNP composition; DSPC or HSPC phospholipids in a total amount of 5 mol% of the total lipid content in the LNP composition; and PEG-DMG conjugated lipid in a total amount of 1.5 mol% of the total lipid content in the LNP composition.

[0561] Some aspects of the present disclosure relate to lipid nanoparticle (LNP) compositions comprising a KC3 ionizable cationic lipid, a (L-serine) PS lipid, cholesterol, one or more phospholipids comprising at least one anionic phospholipid, and a conjugated lipid, wherein the LNP is obtained by a process comprising the step of solubilizing a sodium or ammonium salt of the anionic phospholipid.

[0562] In some embodiments, the composition comprises a nucleic acid. In some embodiments, the nucleic acid is mRNA.

[0563] In some embodiments, the composition is a vaccine.

[0564] In some embodiments, the total amount of phospholipids in the composition is 5 to 25 mol% of the total lipid content in the LNP composition, and the total amount of phosphatidylserine (PS) is 2.5 to 10 mol% of the total lipid content in the LNP composition; and the total amount of conjugated lipids in the composition is 0.5 to 2.5 mol% of the total lipid content in the LNP composition.

[0565] In some embodiments, the composition comprises 48 mol% KC3 ionizable cationic lipid, 40.5 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, wherein each mol% refers to a mol% of the total lipid content in the LNP composition.

[0566] In some embodiments, the composition comprises 48 mol% KC3 ionizable cationic lipid, 38.5 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, wherein each mol% refers to a mol% of the total lipid content in the LNP composition.

[0567] In some embodiments, the composition comprises 46 to 54 mol% KC3 ionizable cationic lipid and 5 mol% (L-serine) DPPS lipid, wherein each mol% refers to a mol% of the total lipid content in the LNP composition.

[0568] In some embodiments, the composition comprises 45 mol% KC3 ionizable cationic lipid, 42.7 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, wherein each mol% refers to a mol% of the total lipid content in the LNP composition.

[0569] In some embodiments, the composition comprises 50 mol% KC3 ionizable cationic lipid, 38.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, and a total 10 mol% phospholipid concentration; wherein each mol% refers to the mol% of the total lipid content in the LNP composition.

[0570] In some embodiments, the composition comprises 48 mol% KC3 ionizable cationic lipid, 40.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, and a total 10 mol% phospholipid concentration; wherein each mol% refers to the mol% of the total lipid content in the LNP composition.

[0571] In some embodiments, the composition comprises 48 mol% KC3 ionizable cationic lipid, 40.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, 5 mol% DSPC or DPPC; and a total 10 mol% phospholipid concentration; wherein each mol% refers to the mol% of the total lipid content in the LNP composition.

[0572] In some embodiments, the composition comprises 46.5 mol% KC3 ionizable cationic lipid, 42 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, wherein each mol% refers to the mol% of the total lipid content in the LNP composition.

[0573] In some embodiments, the composition further comprises 5 mol% DSPC or HSPC based on the total lipid content in the LNP composition.

[0574] In some embodiments, the composition further comprises 1.5 mol% PEG-DMG based on the total lipid content in the LNP composition.

[0575] In some embodiments, the composition comprises 10 mol% of DSPC / DPPC phospholipids based on the total lipid content in the total LNP composition.

[0576] Some aspects of the present disclosure relate to a phosphatidylserine salt selected from the group consisting of sodium DSPS, sodium DPPS, ammonium DSPS, and ammonium DPPS.

[0577] Some aspects of the present disclosure relate to DSPS-Na salt or DPPS-NH4 + Use of a salt in the preparation of LNPs comprising (L-serine)PS lipid, sterol, conjugated lipid, phospholipid for targeting the LNPs to dendritic cells.

[0578] Some aspects of the present disclosure relate to a solution comprising ethanol and DSPS or DPPS, the solution being obtained by a process comprising the step of dissolving a phosphatidylserine salt in ethanol, wherein the phosphatidylserine salt is selected from sodium DSPS, sodium DPPS, ammonium DSPS, and ammonium DPPS.

[0579] Implementation plan:

[0580] Several non-limiting embodiments are described below, each of which is considered to be within the scope of this disclosure.

[0581] Embodiment 1: A lipid nanoparticle (LNP) composition comprising an ionizable lipid having the following chemical structure:

[0582]

[0583] Where R1 is

[0584]

[0585] 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;

[0586] R2 and R3 are each independently (C1-C4)alkyl optionally substituted with hydroxy; and

[0587] n is an integer equal to 2, 3 or 4.

[0588] Embodiment 2: The composition of Embodiment 1, wherein n is 2 or 3.

[0589] Embodiment 3: The composition of any one of Embodiments 1 to 2, wherein a is 0.

[0590] Embodiment 4: The composition of any of Embodiments 1 to 3, wherein b is 1, 2, or 3.

[0591] Embodiment 5: The composition of any one of Embodiments 1 to 2, wherein a is 1.

[0592] Embodiment 6: The composition of any one of Embodiments 1 to 3, wherein b is 1, 2, or 3.

[0593] Embodiment 7: The composition of any of Embodiments 1 to 6, wherein R2 and R3 are each methyl.

[0594] Embodiment 8: The composition of claim 1, wherein R1 is

[0595]

[0596] a is 0 or 1 and b is 1 or 3;

[0597] R2 and R3 are each methyl; and

[0598] n is 2 or 3.

[0599] Embodiment 9: The composition of any one of Embodiments 1 to 8, wherein n is 3.

[0600] Embodiment 10: The composition of any one of Embodiments 1 to 9, comprising:

[0601] a. Nucleic acid;

[0602] b. an ionizable lipid according to any one of embodiments 1 to 9;

[0603] c. sterols;

[0604] d. one or more phospholipids comprising phosphatidylserine (PS) lipids; and

[0605] e. Optionally further comprises a conjugated lipid.

[0606] Embodiment 11: The composition of Embodiment 10, wherein the nucleic acid is mRNA.

[0607] Embodiment 12: The composition of Embodiment 11, wherein the sterol is cholesterol.

[0608] Embodiment 13: The composition of Embodiment 12, wherein the one or more phospholipids consists of:

[0609] a. one or more phospholipids selected from SM, DSPC, HSPC, DPPC and DOPC; and

[0610] b. PS lipids selected from DPPS and DSPS.

[0611] Embodiment 14: The composition of Embodiment 13, wherein the one or more phospholipids consist of:

[0612] a. DSPC; and

[0613] b. One or more PS lipids selected from (L-serine)DPPS and (L-serine)DSPS.

[0614] Embodiment 15: The composition of Embodiment 13, wherein the composition comprises the PS lipids in a total amount of 2.5 to 10 mol% of the total lipids in the composition.

[0615] Embodiment 16: The composition of any one of Embodiments 10 to 15, wherein the conjugated lipid comprises PEG.

[0616] Embodiment 17: A nucleic acid lipid nanoparticle (LNP) composition comprising:

[0617] a. Nucleic acid;

[0618] b. ionizable cationic lipids, wherein the total amount is 40 to 65 mol% of the total lipid content in the LNP composition;

[0619] c. sterols in an amount of 25 to 45 mol% of the total lipid content in the LNP composition; and

[0620] d. one or more phospholipids, the total amount of phospholipids of which is 5 to 25 mol% of the total lipid content in the LNP composition, and comprising a total amount of phosphatidylserine (PS) of 2.5 to 10 mol% of the total lipid content in the LNP composition; and

[0621] e. Optionally further comprising conjugated lipids, the total amount of which is 0.5 to 2.5 mol% of the total lipid content in the LNP composition.

[0622] Embodiment 18: The composition of Embodiment 17, wherein the nucleic acid is mRNA.

[0623] Embodiment 19: The composition of Embodiment 18, wherein the sterol is cholesterol.

[0624] Embodiment 20: The composition of Embodiment 19, wherein the one or more phospholipids consist of DSPC and L-serine PS.

[0625] Embodiment 21: The composition of Embodiment 20, wherein the composition comprises the PS in a total amount of 2.5 to 7.5 mol% of the total lipids in the composition.

[0626] Embodiment 22: The composition of any one of Embodiments 17 to 21, wherein the conjugated lipid comprises PEG.

[0627] Embodiment 23: The composition of Embodiment 22, wherein the conjugated lipid is PEG-DMG.

[0628] Embodiment 24: The composition of Embodiment 23, wherein the LNP comprises the conjugated lipid in a total amount of 0.5 to 2.0 mol% of the total lipid content in the LNP composition.

[0629] Embodiment 25: The composition of Embodiment 24, wherein the LNP comprises the conjugated lipid in a total amount less than 2 mol% of the total lipid content in the LNP composition.

[0630] Embodiment 26: The composition of claim 17, wherein:

[0631] a. the nucleic acid is mRNA,

[0632] b. The total amount of the ionizable cationic lipid is 45 to 55 mol% of the total lipid content in the LNP composition;

[0633] c. sterol is cholesterol in an amount of 35 to 45 mol% of the total lipid content in the LNP composition;

[0634] d. The total amount of phospholipids is 7 to 15 mol% of the total lipid content in the LNP composition;

[0635] e. the one or more phospholipids are composed of DSPC and PS lipids, wherein the PS lipids are one or more lipids selected from DPPS and DSPS in an L-serine configuration; and

[0636] f. The total amount of the PS lipids is about 5 mol% of the total lipid content in the LNP composition.

[0637] Embodiment 27: The composition of Embodiment 26, wherein the composition comprises the PS lipid in a total amount selected from the group consisting of 1.25 mol%, 2.5 mol%, 5 mol%, 7.5 mol% and 10 mol% of the total lipid content in the LNP composition.

[0638] Embodiment 28: A nucleic acid lipid nanoparticle (LNP) composition comprising:

[0639] a. nucleic acid, wherein the nucleic acid is mRNA;

[0640] b. an ionizable cationic lipid, wherein the total amount of the ionizable cationic lipid is 45 to 55 mol% of the total lipid content in the LNP composition;

[0641] c. sterol, wherein the sterol is cholesterol in an amount of 35 to 45 mol% of the total lipid content in the LNP composition;

[0642] d. one or more phospholipids, wherein the total amount of phospholipids of the one or more phospholipids is 10 mol % of the total lipid content in the LNP composition and comprises phosphatidylserine (PS) in an amount of 3 to 9 mol % of the total lipid content in the LNP composition; and

[0643] e. Conjugated lipids, the total amount of the conjugated lipids being 0.5 to 2.0 mol % of the total lipid content in the LNP composition.

[0644] Embodiment 29: The composition of any one of Embodiments 17 to 28, wherein 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).

[0645] Embodiment 30: The composition of claim 29, wherein:

[0646] a. the conjugated lipid is PEG-DMG; and

[0647] b. The PS lipid is selected from DSPS (L-isomer) and DPPS.

[0648] Embodiment 31: The composition of any 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).

[0649] Embodiment 32: The composition of any one of Embodiments 17 to 28, wherein the ionizable cationic lipid is KC3-PA.

[0650] Embodiment 33: The composition of any one of Embodiments 17 to 28, wherein the ionizable cationic lipid is KC3-OA.

[0651] Embodiment 34: The composition of any one of Embodiments 17 to 28, wherein the ionizable cationic lipid is KC3-C17(C8:1).

[0652] Embodiment 35: The nucleic acid lipid nanoparticle (LNP) composition of claim 17, comprising:

[0653] a. Nucleic acid;

[0654] b. ionizable cationic lipids, the total amount of which is 50 mol% of the total lipid content in the LNP composition;

[0655] c. cholesterol, the total amount of which is 38.5 mol% of the total lipid content in the LNP composition;

[0656] d. one or more phospholipids in an amount of 7 to 15 mol% of the total lipid content in the LNP composition, and comprising phosphatidylserine (PS) lipids in an amount of 3 to 9 mol% of the total lipid content in the LNP composition; and

[0657] e. PEG-containing lipids, the total amount of which is 0.5 to 2.0 mol% of the total lipid content in the LNP composition.

[0658] Embodiment 36: The composition of Embodiment 34, wherein the phospholipids consist of one or more phospholipids selected from DSPC, DOPC, DPPC, HSPC, and SM.

[0659] Embodiment 37: The composition of Embodiment 36, wherein the PS lipid is one or more L-serine lipids selected from DPPS and DSPS.

[0660] Embodiment 38: The composition of any of Embodiments 17 to 28, wherein the one or more phospholipids comprise at least two (L-serine) PS lipids with mismatched acyl chain lengths.

[0661] Embodiment 39: The composition of Embodiment 38, wherein the phospholipids are DSPC and DPPS.

[0662] Embodiment 40: The composition of Embodiment 39, wherein the DSPC and DPPS are each present in the LNP in a total amount of 5 mol% each based on the total lipid content in the LNP composition.

[0663] Embodiment 41: A nucleic acid lipid nanoparticle (LNP) composition comprising: a nucleic acid, an ionizable cationic lipid KC3-PA or KC3-OA, and a total amount of (L-serine) PS lipid of 2.5 to 10 mol% of the total lipid content in the LNP composition.

[0664] Embodiment 42: The composition of 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 DSPC, DPPC, HSPC, and SM.

[0665] Embodiment 43: The composition of Embodiment 42, wherein the LNP composition further comprises 0.5 to 2.0 mol% of PEG-DMG or PEG-DSG based on the total lipid content in the LNP composition.

[0666] Embodiment 44: The composition of any one of Embodiments 41 to 43, wherein the ionizable cationic lipid is KC3-PA.

[0667] Embodiment 45: The composition of any one of Embodiments 41 to 43, wherein the ionizable cationic lipid is KC3-OA.

[0668] Embodiment 46: A nucleic acid lipid nanoparticle (LNP) composition comprising: a nucleic acid, a KC3-C17 (C8:1) ionizable cationic lipid; and a total amount of (L-serine) PS lipids in an amount of 2.5 to 10 mol% of the total lipid content in the LNP composition.

[0669] Embodiment 47: The composition of any of Embodiments 41 to 46, wherein the LNP composition has an N / P ratio of 4 to 7.

[0670] Embodiment 48: The composition of Embodiment 47, wherein the LNP composition has an N / P ratio of 5 to 6.

[0671] Embodiment 49: The composition of Embodiment 48, wherein the LNP composition has an N / P ratio of 5.3.

[0672] Embodiment 50: A nucleic acid lipid nanoparticle (LNP) composition comprising: a nucleic acid, an ionizable cationic lipid KC3-PA, and a total amount of (L-serine) PS lipids in an amount of 2.5 to 10 mol% of the total lipid content in the LNP composition.

[0673] Embodiment 51: The composition of 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 DSPC, DOPC, DPPC, HSPC, and SM.

[0674] Embodiment 52: The composition of 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 in the LNP composition.

[0675] Embodiment 53: A nucleic acid lipid nanoparticle (LNP) composition comprising: a nucleic acid, an ionizable cationic lipid selected from KC3-C17 (C8:1); and a (L-serine) PS lipid in an amount of 2.5 to 10 mol% of the total lipid content in the LNP composition.

[0676] Embodiment 54: The composition of any of Embodiments 17-21, 26-28, 35-44, or 53, wherein the N / P ratio is 4 to 7.

[0677] Embodiment 55: The composition of Embodiment 54, wherein the N / P ratio is 5 to 6.

[0678] Embodiment 56: The composition of Embodiment 55, wherein the N / P ratio is 3.

[0679] Embodiment 57: The composition of Embodiment 55, wherein the N / P ratio is 7.

[0680] Embodiment 58: The composition of any one of claims 17-21, 26-28, 35-47, or 53, wherein the nucleic acid is mRNA encoding the SARS-CoV-2 spike protein.

[0681] Embodiment 59: A nucleic acid lipid nanoparticle (LNP) vaccine composition comprising:

[0682] mRNA nucleic acid with a N / P ratio of 4 to 7;

[0683] b. a total amount of 40 to 65 mol% of the total lipid content of the LNP composition of KC3-PA ionizable cationic lipids;

[0684] c. a total amount of 25 to 40 mol% cholesterol based on the total lipid content of the LNP composition;

[0685] d. a total amount of 2.5 to 10 mol% (L-serine) PS lipids based on the total lipid content of the LNP composition;

[0686] e. a total amount of 5 to 25 mol% of DSPC phospholipids based on the total lipid content of the LNP composition; and

[0687] f. PEG-DMG in a total amount of 0 to 2.5 mol% based on the total lipid content in the LNP composition.

[0688] Embodiment 60: A nucleic acid lipid nanoparticle (LNP) vaccine composition comprising:

[0689] mRNA nucleic acid with a N / P ratio of 3 to 8;

[0690] b. a total amount of 40 to 65 mol% of KC3-C17 (C8: 1) ionizable cationic lipids based on the total lipid content of the LNP composition;

[0691] c. a total amount of 25 to 40 mol% cholesterol based on the total lipid content of the LNP composition;

[0692] d. a total amount of 2.5 to 10 mol% (L-serine) PS lipids based on the total lipid content of the LNP composition;

[0693] e. a total amount of 5 to 25 mol% of DSPC phospholipids based on the total lipid content of the LNP composition; and

[0694] f. PEG-DMG in a total amount of 0 to 2.5 mol% based on the total lipid content in the LNP composition.

[0695] Embodiment 61: A nucleic acid lipid nanoparticle (LNP) vaccine composition comprising:

[0696] mRNA nucleic acid with a N / P ratio of 4 to 7;

[0697] b. a total amount of 40 to 65 mol% of KC3-C15 (C8: 1) ionizable cationic lipids based on the total lipid content of the LNP composition;

[0698] c. a total amount of 25 to 40 mol% cholesterol based on the total lipid content of the LNP composition;

[0699] d. a total amount of 2.5 to 10 mol% (L-serine) PS lipids based on the total lipid content of the LNP composition;

[0700] e. a total amount of 5 to 25 mol% of DSPC phospholipids based on the total lipid content of the LNP composition; and

[0701] f. PEG-DMG in a total amount of 0 to 2.5 mol% based on the total lipid content in the LNP composition.

[0702] Embodiment 62: A nucleic acid lipid nanoparticle (LNP) vaccine composition comprising:

[0703] mRNA nucleic acid with a N / P ratio of 3 to 8;

[0704] b. a total amount of 40 to 65 mol% of KC3-C18 ionizable cationic lipids based on the total lipid content of the LNP composition;

[0705] c. a total amount of 25 to 40 mol% cholesterol based on the total lipid content of the LNP composition;

[0706] d. a total amount of 2.5 to 10 mol% (L-serine) PS lipids based on the total lipid content of the LNP composition;

[0707] e. a total amount of 5 to 25 mol% of DSPC phospholipids based on the total lipid content of the LNP composition; and

[0708] f. PEG-DMG in a total amount of 0 to 2.5 mol% based on the total lipid content in the LNP composition.

[0709] Embodiment 63: The composition of any one of Embodiments 59 to 62, wherein the nucleic acid is the mRNA of SEQ ID NO: 2.

[0710] Embodiment 64: Use of a (L-serine) PS lipid in combination with the ionizable cationic lipid of any one of embodiments 1 to 9 in the LNP for targeting the LNP to dendritic cells.

[0711] Embodiment 65: The use of Embodiment 64, wherein the LNP comprises mRNA.

[0712] Embodiment 66: The use of any one of Embodiments 64 to 65, wherein the LNP further comprises cholesterol.

[0713] Embodiment 67: The use of 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 in the LNP composition.

[0714] Embodiment 68: The use of Embodiment 67, wherein the LNP further comprises one or more additional phospholipids, the additional phospholipids comprising DSPC.

[0715] Embodiment 69: The use of Embodiment 68, wherein the LNP further comprises a conjugated lipid.

[0716] Embodiment 70: The use of Embodiment 64, wherein the LNP comprises:

[0717] mRNA nucleic acid with a N / P ratio of 3 to 8;

[0718] b. a total amount of 40 to 65 mol% of KC3-PA or KC3-C17 (C8: 1) ionizable cationic lipid (ICL) based on the total lipid content of the LNP composition;

[0719] c. a total amount of 25 to 40 mol% cholesterol based on the total lipid content of the LNP composition;

[0720] d. a total amount of 2.5 to 10 mol% (L-serine) PS lipids based on the total lipid content of the LNP composition;

[0721] e. a total amount of 5 to 25 mol% of DSPC phospholipids based on the total lipid content of the LNP composition; and

[0722] f. The total amount of conjugated lipids is 0 to 2.5 mol % of the total lipid content in the LNP composition.

[0723] Embodiment 71: The use of Embodiment 70, wherein the ICL is KC3-PA.

[0724] Embodiment 72: The use of Embodiment 70, wherein the ICL is KC3-C17(C8:1).

[0725] Embodiment 73: A lipid nanoparticle (LNP) composition comprising an ionizable lipid having the following chemical structure:

[0726]

[0727] Where R1 is

[0728]

[0729] 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;

[0730] R2 and R3 are each independently methyl; and

[0731] n is an integer equal to 2 or 3.

[0732] Embodiment 74: The composition of Embodiment 73, wherein a is 0.

[0733] Embodiment 75: The composition of Embodiment 74, wherein b is 1.

[0734] Embodiment 76: The composition of Embodiment 74, wherein b is 3.

[0735] Embodiment 77: The composition of Embodiment 73, wherein a is 1.

[0736] Embodiment 78: The composition of Embodiment 77, wherein b is 1.

[0737] Embodiment 79: The composition of Embodiment 77, wherein b is 3.

[0738] Embodiment 80: The composition of any one of Embodiments 73 to 79, wherein n is 2.

[0739] Embodiment 81: The composition of any one of Embodiments 73 to 79, wherein n is 3.

[0740] Embodiment 82: The composition of any one of Embodiments 17 to 28, wherein 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. Distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), N-succinyl-distearoylphosphatidylethanolamine (N-Suc-DSPE), N-glutaryl-distearoylphosphatidylethanolamine (N-glu-DSPE), distearoylphosphatidic acid (DSPA), and cardiolipin.

[0741] Embodiment 83: The composition of any of Embodiments 17 to 28 or 82, wherein the composition comprises an anionic targeting phospholipid other than phosphatidyl-L-serine.

[0742] Embodiment 84: The composition of any one of Embodiments 17 to 28 or 82 to 83, wherein the composition comprises an anionic phospholipid selected from DSPG and DPPG.

[0743] Embodiment 85: The composition of any one of Embodiments 17 to 28 or 82 to 83, wherein the composition comprises an anionic phospholipid selected from N-Glu-DSPE and N-Suc-DSPE.

[0744] Embodiment 86: The composition of any one of Embodiments 17 to 28 or 82 to 83, wherein the composition comprises DSPA anionic phospholipid.

[0745] Embodiment 87: The composition of any one of Embodiments 17 to 28 or 82 to 83, wherein the composition comprises cardiolipin anionic phospholipid.

[0746] Embodiment 88: The composition of any one of Embodiments 1 to 63 or 73 to 87, wherein the ionizable lipid has the following chemical structure:

[0747]

[0748] Embodiment 89: The use of any one of Embodiments 64 to 72, wherein the ionizable lipid has the following chemical structure:

[0749]

[0750] Embodiment 90: A sodium salt or ammonium salt of an anionic phospholipid composition of formula (VA-1), wherein formula (VA-1) has the following chemical structure:

[0751]

[0752] in:

[0753] X + is an ammonium cation or sodium (Na + ) cations; and

[0754] a is 14, 15 or 16.

[0755] Embodiment 91: The composition of Embodiment 90, wherein a is 14 or 16.

[0756] Embodiment 92: The composition of any one of Embodiments 90 or 91, wherein X + It is an ammonium cation (NH4 + ).

[0757] Embodiment 93: The composition of any one of Embodiments 90 or 91, wherein X + It is a sodium cation (Na + ).

[0758] Embodiment 94: The composition of any one of Embodiments 90 to 93, wherein the anionic phospholipid of formula (VA-1) is the sodium salt of distearoylphosphatidyl-L-serine (DSPS L-isomer).

[0759] Embodiment 95: The composition of any one of Embodiments 90 to 93, wherein the anionic phospholipid of formula (VA-1) is the ammonium salt of distearoylphosphatidyl-L-serine (DSPS L-isomer).

[0760] Embodiment 96: The composition of any one of Embodiments 90 to 93, wherein the anionic phospholipid of formula (VA-1) is the sodium salt of DPPS (L-isomer).

[0761] Embodiment 97: The composition of any one of Embodiments 90 to 93, wherein the anionic phospholipid of formula (VA-1) is the ammonium salt of DPPS (L-isomer).

[0762] Embodiment 98: Use of the salt form composition of any one of Embodiments 90 to 97 in the preparation of a liposomal nanoparticle (LNP) composition.

[0763] Embodiment 99: The use of Embodiment 98, in combination with one or more of the following LNP components during the preparation of the LNP composition:

[0764] mRNA nucleic acid;

[0765] ionizable cationic lipids (ICLs);

[0766] cholesterol;

[0767] (L-serine)PS lipid;

[0768] one or more phospholipids; and

[0769] Conjugated lipids.

[0770] Embodiment 100: The use of Embodiment 98, comprising the step of combining the ammonium or salt form of the compound of formula (VA-1) with one or more of the following LNP components during the preparation of the LNP composition:

[0771] mRNA nucleic acid;

[0772] The ionizable cationic lipid (ICL) of any one of claims 1 to 9 or 73 to 88;

[0773] cholesterol;

[0774] (L-serine)PS lipid;

[0775] one or more phospholipids; and

[0776] Conjugated lipids.

[0777] Embodiment 101: The use of any one of Embodiments 98 to 100, wherein the LNP is a nucleic acid lipid nanoparticle vaccine composition comprising:

[0778] mRNA nucleic acid with a N / P ratio of 4 to 7;

[0779] b. an ionizable cationic lipid according to any one of embodiments 1 to 9 or 73 to 88, wherein the total amount is 40 to 65 mol% of the total lipid content in the LNP composition;

[0780] c. a total amount of 25 to 40 mol% cholesterol based on the total lipid content of the LNP composition;

[0781] d. a total amount of 2.5 to 10 mol% (L-serine) PS lipids based on the total lipid content of the LNP composition;

[0782] e. a total amount of 5 to 25 mol% of DSPC phospholipids based on the total lipid content of the LNP composition; and

[0783] f. PEG-DMG in a total amount of 0 to 2.5 mol% based on the total lipid content in the LNP composition.

[0784] Embodiment 102: The use of Embodiment 101, wherein the nucleic acid is the mRNA of SEQ ID NO: 2.

[0785] Embodiment 103: The composition of any one of Embodiments 17 to 63 or 73 to 89, wherein the LNP composition comprises the ionizable cationic lipid in a total amount of 46 to 65 mol% of the total lipid content in the LNP composition.

[0786] Embodiment 104: The composition of any one of Embodiments 17 to 63, 73 to 89, or 103, wherein the LNP composition comprises the PS in a total amount of about 5 mol% of the total lipids in the composition.

[0787] Embodiment 105: The composition of any one of Embodiments 17 to 63, 73 to 89, or 103 to 104, wherein the LNP composition comprises the conjugated lipid in a total amount of about 1.5 mol% of the total lipid content in the LNP composition.

[0788] Embodiment 106: The composition of any one of Embodiments 82 to 87, wherein:

[0789] The conjugated lipid is PEG-DMG; and

[0790] The PS lipid is selected from DSPS (L-isomer) and DPPS.

[0791] Embodiment 107: The composition of any 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).

[0792] Embodiment 108: The composition of any one of Embodiments 82 to 87, wherein the ionizable cationic lipid is KC3-PA.

[0793] Embodiment 109: The composition of any one of Embodiments 82 to 87, wherein the ionizable cationic lipid is KC3-OA.

[0794] Embodiment 110: The composition of any one of Embodiments 82 to 87, wherein the ionizable cationic lipid is KC3-C17(C8:1).

[0795] Embodiment 111: Use of a (L-serine) PS lipid in combination with the ionizable cationic lipid of any one of embodiments 73 to 87 in the LNP for targeting the LNP to dendritic cells.

[0796] Embodiment 112: The use of Embodiment 111, wherein the LNP comprises mRNA.

[0797] Embodiment 113: The use of any one of Embodiments 111 to 112, wherein the LNP further comprises cholesterol.

[0798] Embodiment 114: The use of 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 in the LNP composition.

[0799] Embodiment 115: The use of Embodiment 114, wherein the LNP further comprises one or more additional phospholipids, wherein the additional phospholipids include DSPC.

[0800] Embodiment 116: The use of Embodiment 115, wherein the LNP further comprises a conjugated lipid.

[0801] Embodiment 117: The use of Embodiment 111, wherein the LNP comprises:

[0802] mRNA nucleic acid with a N / P ratio of 3 to 8;

[0803] b. a total amount of 40 to 65 mol% of KC3-PA or KC3-C17 (C8: 1) ionizable cationic lipid (ICL) based on the total lipid content of the LNP composition;

[0804] c. a total amount of 25 to 40 mol% cholesterol based on the total lipid content of the LNP composition;

[0805] d. a total amount of 2.5 to 10 mol% (L-serine) PS lipids based on the total lipid content of the LNP composition;

[0806] e. a total amount of 5 to 25 mol% of DSPC phospholipids based on the total lipid content of the LNP composition; and

[0807] f. The total amount of conjugated lipids is 0 to 2.5 mol % of the total lipid content in the LNP composition.

[0808] Embodiment 118: The use of Embodiment 117, wherein the ICL is KC3-PA.

[0809] Embodiment 119: The use of Embodiment 117, wherein the ICL is KC3-C17(C8:1).

[0810] Embodiment 120: The composition of any one of Embodiments 90 to 97, wherein X is an ammonium cation selected from the group consisting of: ammonium (NH4 + ), alkylammonium, dialkylammonium and trialkylammonium salts.

[0811] Embodiment 121: The composition of 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).

[0812] Embodiment 122: The composition of any one of Embodiments 17 to 28 or 82 to 83, wherein the composition comprises anionic phospholipids selected from DSPG and DPPG in a total amount of 2.5% to 7.5% of the total lipid content in the LNP composition.

[0813] Embodiment 123: The composition of Embodiment 122, wherein the composition comprises DSPG anionic phospholipids in a total amount of 2.5% to 7.5% of the total lipid content in the LNP composition.

[0814] Embodiment 124: The composition of Embodiment 122, wherein the composition comprises DPPG anionic phospholipids in a total amount of 2.5% to 7.5% of the total lipid content in the LNP composition.

[0815] Embodiment 125: The use of Embodiment 114, wherein the LNP further comprises one or more additional phospholipids, wherein the additional phospholipids include DSPC.

[0816] Embodiment 126: A nucleic acid lipid nanoparticle (LNP) composition comprising:

[0817] a. Nucleic acid;

[0818] b. a total amount of 40 to 65 mol% of the total lipid content of the LNP composition of KC3 ionizable cationic lipids;

[0819] c. a total amount of 23.5 to 43.5 mol% cholesterol based on the total lipid content of the LNP composition;

[0820] d. a total amount of 2.5 to 10 mol% (L-serine) PS lipids based on the total lipid content of the LNP composition;

[0821] e. a total amount of 5 to 25 mol% of DSPC or HSPC phospholipids based on the total lipid content of the LNP composition; and

[0822] f. PEG-containing conjugated lipids in a total amount of 0.5 mol% to 2.5 mol% of the total lipid content in the LNP composition.

[0823] Embodiment 127: The composition of Embodiment 126, wherein the nucleic acid is mRNA.

[0824] Embodiment 128: The composition of any of Embodiments 126 to 127, wherein the N / P ratio is 3 to 8.

[0825] Embodiment 129: The composition of any of Embodiments 126 to 127, wherein the KC3 ionizable cationic lipid is selected from: KC3-OA, KC3-PA, KC3-C17(8:1), and KC3-C15(C8:1).

[0826] Embodiment 130: The composition of Embodiment 129, wherein the KC3 ionizable cationic lipid is KC3-OA.

[0827] Embodiment 131: The composition of Embodiment 129, wherein the KC3 ionizable cationic lipid is KC3-PA.

[0828] Embodiment 132: The composition of Embodiment 129, wherein the KC3 ionizable cationic lipid is KC3-C17(C8:1).

[0829] Embodiment 133: The composition of Embodiment 129, wherein the KC3 ionizable cationic lipid is KC3-C15(C8:1).

[0830] Embodiment 134: The composition of any one of Embodiments 126 to 133, wherein the conjugated lipid is PEG-DMG or PEG-DSG.

[0831] Embodiment 135: The composition of Embodiment 134, wherein the composition comprises the PEG-containing conjugated lipid in a total amount of 0.5 to 2.0 mol% of the total lipid content in the LNP composition.

[0832] Embodiment 136: The composition of any one of Embodiments 126 to 135, wherein the composition comprises the KC3 ionizable cationic lipid in a total amount that is 48 mol% of the total lipid content in the LNP composition.

[0833] Embodiment 137: The composition of any one of Embodiments 126 to 136, wherein the composition comprises a total amount of DSPC and DSPS that is 10 mol% of the total lipid content in the LNP composition.

[0834] Embodiment 138: The composition of any one of Embodiments 126 to 137, wherein the composition comprises 5% DSPC or HSPC in a total amount of 5 mol% of the total lipid content in the LNP composition.

[0835] Embodiment 139: The composition of any one of Embodiments 126 to 137, wherein the composition comprises PEG-DMG in an amount totaling 1.5 mol% of the total lipid content in the LNP composition.

[0836] Embodiment 140: The composition of any one of Embodiments 126 to 137, wherein the composition comprises cholesterol in a total amount that is 40.5 mol% of cholesterol based on the total lipid content in the LNP composition.

[0837] Embodiment 141: The composition of any one of Embodiments 126 to 137, wherein the composition comprises the DSPC phospholipid in a total amount of 10 mol% of the total lipid content in the LNP composition.

[0838] Embodiment 142: The composition of any one of Embodiments 126 to 141, wherein the PEG-containing conjugated lipid is PEG2000 -DMG.

[0839] Embodiment 143: The composition of any one of Embodiments 126 to 139, wherein the composition comprises a total amount of said cholesterol that is 23.5 mol% of the total lipid content in the LNP composition.

[0840] Embodiment 144: The composition of any one of Embodiments 126 to 139, wherein the composition comprises a total amount of said cholesterol that is 33.5 mol% of the total lipid content in the LNP composition.

[0841] Embodiment 145: The composition of any one of Embodiments 126 to 139, wherein the composition comprises a total amount of said cholesterol that is 38.5 mol% of the total lipid content in the LNP composition.

[0842] Embodiment 146: The composition of any one of Embodiments 126 to 139, wherein the composition comprises a total amount of said cholesterol that is 40.5 mol% of the total lipid content in the LNP composition.

[0843] Embodiment 147: The composition of any one of Embodiments 126 to 139, wherein the composition comprises a total amount of said cholesterol that is 42.7 mol% of the total lipid content in the LNP composition.

[0844] Embodiment 148: The composition of any one of Embodiments 126 to 139, wherein the composition comprises a total amount of said cholesterol that is 43.5 mol% of the total lipid content in the LNP composition.

[0845] Embodiment 149: The composition of any one of Embodiments 126 to 139, wherein the composition comprises a total amount of said cholesterol of 33.5 to 43.5 mol% of the total lipid content in the LNP composition.

[0846] Embodiment 150: The composition of any one of Embodiments 126 to 149, wherein the composition comprises the KC3 ionizable cationic lipid in a total amount of 45 to 55 mol% of the total lipid content in the LNP composition.

[0847] Embodiment 151: A nucleic acid lipid nanoparticle (LNP) composition comprising:

[0848] a.mRNA nucleic acid;

[0849] b. KC3 ionizable cationic lipids selected from the group consisting of KC3-OA, KC3-PA, KC3-C17 (8: 1), and KC3-C15 (C8: 1), wherein the total amount is 45 to 55 mol% of the total lipid content in the LNP composition;

[0850] c. a total amount of 33.5 to 43.5 mol% of cholesterol based on the total lipid content of the LNP composition;

[0851] d. a total amount of 5 mol% (L-serine) DPPS lipids based on the total lipid content of the LNP composition;

[0852] e. a total amount of 5 mol% of DSPC or HSPC phospholipids based on the total lipid content of the LNP composition; and

[0853] f. PEG-DMG conjugated lipids in an amount of 1.5 mol% of the total lipid content in the LNP composition.

[0854] Embodiment 152: 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 conjugated lipid, wherein the LNP is obtained by a method comprising the step of dissolving a sodium salt or an ammonium salt of the anionic phospholipid.

[0855] Embodiment 153: The composition of Embodiment 152, wherein the anionic phospholipid is a salt of any one of claims 90 to 97.

[0856] Embodiment 154: The composition of any of Embodiments 152 to 153, wherein the composition comprises a nucleic acid.

[0857] Embodiment 155: The composition of Embodiment 154, wherein the nucleic acid is mRNA.

[0858] Embodiment 156: The composition of Embodiment 155, wherein the composition is a vaccine.

[0859] Embodiment 157: The composition of any of Embodiments 152 to 156, wherein the total amount of phospholipids in the composition is 5 to 25 mol% of the total lipid content in the LNP composition, and the total amount of phosphatidylserine (PS) is 2.5 to 10 mol% of the total lipid content in the LNP composition; and the total amount of conjugated lipids in the composition is 0.5 to 2.5 mol% of the total lipid content in the LNP composition.

[0860] Embodiment 158: The composition of any one of Embodiments 152 to 156, wherein the composition comprises

[0861] 48 mol% of the KC3 ionizable cationic lipids,

[0862] 40.5 mol% cholesterol, and

[0863] 5 mol% (L-serine) DPPS lipid,

[0864] Each mol% refers to the mol% of the total lipid content in the LNP composition.

[0865] Embodiment 159: The composition of any one of Embodiments 152 to 156, wherein the composition comprises

[0866] 48 mol% of the KC3 ionizable cationic lipids,

[0867] 38.5 mol% cholesterol, and

[0868] 5 mol% (L-serine) DPPS lipid,

[0869] Each mol% refers to the mol% of the total lipid content in the LNP composition.

[0870] Embodiment 160: The composition of any one of Embodiments 152 to 156, wherein the composition comprises

[0871] 46 to 54 mol% of said KC3 ionizable cationic lipid, and

[0872] 5 mol% (L-serine) DPPS lipid,

[0873] Each mol% refers to the mol% of the total lipid content in the LNP composition.

[0874] Embodiment 161: The composition of any one of Embodiments 152 to 156, wherein the composition comprises

[0875] 45 mol% of the KC3 ionizable cationic lipid,

[0876] 42.7 mol% cholesterol, and

[0877] 5 mol% (L-serine) DPPS lipid,

[0878] Each mol% refers to the mol% of the total lipid content in the LNP composition.

[0879] Embodiment 162: The composition of any one of Embodiments 152 to 156, wherein the composition comprises

[0880] 50 mol% of the KC3 ionizable cationic lipid,

[0881] 38.5 mol% cholesterol,

[0882] 5 mol% (L-serine) DPPS lipid, and

[0883] total 10 mol% phospholipid concentration;

[0884] Wherein each mol% refers to the mol% of the total lipid content in the LNP composition.

[0885] Embodiment 163: The composition of any one of Embodiments 152 to 156, wherein the composition comprises

[0886] 48 mol% of the KC3 ionizable cationic lipids,

[0887] 40.5 mol% cholesterol,

[0888] 5 mol% (L-serine) DPPS lipid, and

[0889] total 10 mol% phospholipid concentration;

[0890] Each mol% refers to the mol% of the total lipid content in the LNP composition.

[0891] Embodiment 164: The composition of any one of Embodiments 152 to 156, wherein the composition comprises

[0892] 48 mol% of the KC3 ionizable cationic lipids,

[0893] 40.5 mol% cholesterol,

[0894] 5 mol% (L-serine) DPPS lipid,

[0895] 5 mol% DSPC or DPPC; and

[0896] total 10 mol% phospholipid concentration;

[0897] Each mol% refers to the mol% of the total lipid content in the LNP composition.

[0898] Embodiment 165: The composition of any one of Embodiments 152 to 156, wherein the composition comprises

[0899] 46.5 mol% of the KC3 ionizable cationic lipids,

[0900] 42 mol% cholesterol,

[0901] 5 mol% (L-serine) DPPS lipid,

[0902] Each mol% refers to the mol% of the total lipid content in the LNP composition.

[0903] Embodiment 166: The composition of any one of Embodiments 158 to 165, wherein the composition further comprises 5 mol% DSPC or HSPC totaling to the total lipid content in the LNP composition.

[0904] Embodiment 167: The composition of any one of Embodiments 158 to 166, wherein the composition further comprises 1.5 mol% PEG-DMG totaling to the total lipid content in the LNP composition.

[0905] Embodiment 168: The composition of any one of Embodiments 158 to 163, wherein the composition comprises a total of 10 mol% DSPC / DPPC phospholipids based on the total lipid content in the LNP composition.

[0906] Embodiment 169: A phosphatidylserine salt selected from sodium DSPS, sodium DPPS, ammonium DSPS and ammonium DPPS.

[0907] Embodiment 170: DSPS-Na salt or DPPS-NH4 + Use of a salt in the preparation of LNPs comprising (L-serine)PS lipid, a sterol, a conjugated lipid, a phospholipid, for targeting the LNPs to dendritic cells.

[0908] Embodiment 171: A solution comprising ethanol and DSPS or DPPS, said solution being obtained by a process comprising the step of dissolving a phosphatidylserine salt in ethanol, wherein said phosphatidylserine salt is selected from the group consisting of sodium DSPS, sodium DPPS, ammonium DSPS and ammonium DPPS.

[0909] Example

[0910] Although the present disclosure has been described in conjunction with certain embodiments and many details have been set forth for illustrative purposes, 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 vary considerably 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 features, terms, or elements of the various illustrative components and examples.

[0911] Unless otherwise specifically stated, the isomeric form of the phosphatidylserine lipid used in the examples is phosphatidyl-L-serine.

[0912] Certain examples are provided below to illustrate various embodiments of the embodiments disclosed herein. Those of ordinary skill in the art will recognize that the various embodiments disclosed herein are not limited to these specific illustrative examples.

[0913] Example 1A: Synthesis of ionizable lipids

[0914] Scheme 1 Synthesis of acid intermediates from AKG-UO-1 to AKG-UO-3. Figures 47A to 47B .

[0915]

[0916] The acid intermediates (6Z,12Z)-6,12-octadecadienoic acid and (6Z,12Z)-6,12-hexadecadienoic acid were prepared by a general synthesis, as shown in Error! Reference source not found, involving i) the preparation of a triphenylene glycol from 5-bromopentanol and the corresponding aldehyde. Initial Witting reaction of the ylide, ii) conversion of the terminal alcohol to the bromide via mesylation and substitution, iii) repetition of the sequence of ylide synthesis and Witting reaction, and finally iv) periodate oxidation of the terminal alcohol. The resulting acid intermediates were used to synthesize AKG-UO-1 to AKG-UO-4, see below.

[0917] Scheme 2 Synthesis of the acid intermediate of AKG-UO-5

[0918]

[0919] 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, involving i) alkylation of silyl-protected 10-hydroxy-1-decyne with (5Z)-1-bromo-5-octene, ii) catalytic hydrogenation of the alkyne to the cis-olefin, iii) removal of the silyl protection on the alcohol, and finally iv) oxidation of the terminal alcohol to the desired acid.

[0920] Scheme 3 Synthesis of acid intermediates of AKG-BDG-01 and AKG-BDG-02

[0921]

[0922] The synthesis of two disulfide acid intermediates used in the synthesis of AKG-BDG-1 and AKG-BDG-2 is shown in Scheme 4.

[0923] The general synthesis of the acid intermediate of AKG-BDG-1 involves i) synthesis of 4-mercaptobutyric acid from 4-bromobutyric acid, ii) reaction of 4-mercaptobutyric acid with DPS to give 4-(2-pyridyldisulfanyl)butyric acid, iii) catalytic hydrogenation of 3-decyn-1-ol to the cis-olefin, iv) tosylation of the primary alcohol, v) displacement of the tosyl group with thiourea to give the terminal thiol, and finally vi) coupling of the terminal thiol with 4-(2-pyridyldisulfanyl)butyric acid prepared in step ii above to give the disulfide-containing acid intermediate. A similar synthetic sequence was followed starting from 3-dodecyn-1-ol to produce the second acid intermediate for the synthesis of AKG-BDG-2.

[0924] Scheme 4 Synthesis of AKG-UO-1, AKG-UO-4, AKG-UO-5, AKG-BDG-1, and AKG-BDG-2

[0925]

[0926] The general synthesis of lipids AKG-UO-1, AKG-UO-4, AKG-UO-5, AKG-BDG-1, and AKG-BDG-2 shown in Scheme 4 involves the following steps: i) tosylation of the primary alcohol of a commercially available chiral dioxolane; ii) displacement of the tosyl group with a dimethylamine group to give a tertiary amine; iii) acid-catalyzed deprotection of the diol; and finally iv) esterification of the diol with the corresponding acid intermediate synthesized according to Schemes 1 to 3. AKG-UO-2 was prepared following a similar synthetic sequence starting from a different dioxolane and the corresponding acid intermediate, as shown in Scheme 5 below.

[0927] Scheme 5 Synthesis of AKG-UO-2

[0928]

[0929] The general synthesis of trialkyl phosphates comprising the lipid AKG-UO-3 shown in Scheme 6 involves the following steps: i) reaction of the primary alcohol of a commercially available chiral dioxolane with methyl dichlorophosphite to give the corresponding dialkyl phosphite; ii) displacement of the chloride in the dialkyl phosphite by treatment with 3-bromopropanol to give the corresponding trialkyl phosphite; iii) acid-catalyzed deprotection of the diol; iv) esterification of the diol with the corresponding acid intermediate synthesized according to Scheme 1; and finally v) displacement of the bromo group with a dimethylamine group to give a tertiary amine.

[0930] Scheme 6 Synthesis of AKG-UO-3

[0931]

[0932] Alternatively, acid intermediates having two methylene groups between the double bond positions in the hydrocarbon chain are synthesized as described in Caballeira et al., Chem. Phys. Lipids, Vol. 100, pp. 33-40, 1999, or as described by D'yakonov et al. (D'yakonov et al., Med. Chem. Res., 2016, Vol. 25, pp. 30-39; D'yakonov et al., Chem. Commun. 2013, Vol. 49, pp. 8401-8403; D'yakonov et al., 2020, Phytochem. Rev.).

[0933] Example 1B: Synthesis of ionizable lipids - see Figure 48

[0934] 1.2-((S)-2,2-bis((6Z,12Z)-octadec-6,12-dien-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylethane-1-amine (AKG-KC2-01, O-12095)

[0935] 2.3-((S)-2,2-bis((6Z,12Z)-octadec-6,12-dien-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-01, O-12096)

[0936] 3. 2-((S)-2,2-Di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethane-1-amine (AKG-KC2-OA, O-11880)

[0937] 4. 2-((S)-2,2-Di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethane-1-amine (AKG-KC2-PA, O-11879)

[0938] 5. 3-((S)-2,2-Di((Z)-octadec-9-en-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-OA, O-11957)

[0939] 6. 3-((S)-2,2-Di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-PA, O-12418)

[0940] 7. 3-((S)-2,2-Di((Z)-heptadeca-8-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-C17(C8:1))

[0941] 8. (S)-3-(2,2-diheptadecan-1,3-dioxolane-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-C17)

[0942] Synthesis of: 2-((S)-2,2-di((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylethane-1-amine (AKG-KC2-01, O-12095)

[0943] 3-((S)-2,2-Di((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-01, O-12096)

[0944] See also Figure 48 .

[0945] Experimental procedures

[0946] Synthesis of (6Z,12Z)-1-bromooctadeca-6,12-diene, 2

[0947]

[0948] To a solution of (6Z, 12Z)-octadec-6,12-diene-1-ol, 1 (3.6 g, 13.7 mmol) in dichloromethane (50 mL) was added methanesulfonyl chloride (1.26 mL, 16.4 mmol) and triethylamine (3.6 mL, 20.5 mmol) at 0 ° C. The resulting solution was warmed to room temperature and stirred for 2 hours. The mixture was quenched with water and extracted with dichloromethane (2 × 100 mL). The combined organics were washed with brine, dried over magnesium sulfate, and then filtered. The filtrate was concentrated under vacuum to give a crude oil. The resulting oil was dissolved in diethyl ether (50 mL) and added to a stirred slurry of magnesium bromide etherate (7 g, 27.4 mmol) in diethyl ether (50 mL) at 0 ° C. The mixture was warmed to room temperature and stirred for 2 hours. The reaction mixture was quenched with water and extracted with ethyl acetate (2 × 100 mL). 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 crude oil was purified by chromatography on silica using 5% to 10% ethyl acetate in n-hexane as eluent to give (6Z,12Z)-1-bromooctadec-6,12-diene, 3 (2.9 g, 8.89 mmol, 65%) as a yellow oil.

[0949] 1 H NMR (300MHz, CDCl3): 5.36-5.33 (m, 4H), 3.42-3.37 (t, J=7.5Hz, 2H), 2.04-1. 97 (m, 8H), 1.83-1.83 (m, 2H), 1.37-1.28 (m, 14H), 0.90-0.86 (t, J=6.6Hz, 3H).

[0950] Synthesis of (6Z,12Z,25Z,31Z)-heptatriacontac-6,12,25,31-tetraen-19-ol, 3

[0951]

[0952] At room temperature under argon, a solution of (6Z, 12Z) -1- bromooctadecane -6,12-diene, 2 (2 g, 6.08 mmol) in ether (10 mL) was added to a mixture of magnesium chips (162 mg, 6.69 mmol) and iodine in ether (2 mL). The mixture was stirred at room temperature for 90 minutes (consuming magnesium chips), followed by the addition of ethyl formate (0.24 mL, 3.04 mmol). After stirring at room temperature for one hour, the reaction was quenched with a 1N HCl solution. The mixture was extracted with ethyl acetate (2 × 100 mL), and the combined organics were washed with water and then with brine. The organics were dried over magnesium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude oil. The resulting oil was dissolved in ethanol (10 mL) and added to a solution of potassium hydroxide (260 mg) in water (3 mL). After stirring for 12 hours, the pH of the mixture was adjusted to 4 with 2N HCl. The aqueous solution was extracted with dichloromethane (2×) and combined. The organic matter was washed with brine, then dried under magnesium sulfate and filtered. The filtrate was concentrated under vacuum to obtain a crude oil. The crude oil was purified on silica using 10% to 30% ethyl acetate in n-hexane as eluent to obtain (6Z, 12Z, 25Z, 31Z)-heptatriacontac-6, 12, 25, 31-tetraene-19-ol, 3 (0.29 g, 0.55 mmol, 18%) as a clear oil.

[0953] 1 H NMR (300MHz, CDCl3): 5.36-5.32 (m, 8H), 3.57 (bs, 1H), 3.33-3.32, (m, 2H), 2.13-1.97 (m, 16H), 1.36-1.29 (m, 34H), 0.90-0.86 (t, J=6.6Hz, 6H).

[0954] Synthesis of (6Z,12Z,25Z,31Z)-heptatriacontac-6,12,25,31-tetraen-19-one, 4

[0955]

[0956] To a mixture of (6Z,12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-ol, 3 (0.29 g, 0.55 mmol) and sodium carbonate (3 mg, 0.03 mmol) in dichloromethane at 0°C was added pyridinium chlorochromate. The mixture was heated to room temperature and stirred for one hour. After one hour, silica gel (10 g) was added to the reactant and the mixture was filtered. The filtrate was concentrated and the resulting oil was purified on silica using 10% to 20% ethyl acetate in n-hexane as eluent to obtain (6Z, 12Z, 25Z, 31Z)-heptatriacontac-6, 12, 25, 31-tetraene-19-one, 4 (0.12 g, 0.23 mmol, 42%) as a clear oil.

[0957] 1 H NMR (300MHz, CDCl3): 5.36-5.32 (m, 8H), 3.36-3.32, (m, 1H), 2.40-2.35 (t, J=6.6Hz, 3H) , 2.14-2.00 (m, 16H), 1.58-1.54 (m, 4H), 1.34-1.29 (m, 28H), 0.90-0.86 (t, J=6.6Hz, 6H).

[0958] 2-((S)-2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)-1,3-dioxolane-4-yl)ethane- Synthesis of 1-ol, 7

[0959]

[0960] (6Z,12Z,25Z,31Z)-heptatriacontriacontria-6,12,25,31-tetraen-19-one, 4 (0.12 g, 0.23 mmol), (4S)-(+)-4-(2-hydroxyethyl)-2,2-dimethyl-1,3-dioxolane 5 (0.20 g, 1.38 mmol) and pyridinium p-toluenesulfonate were added. The mixture of (9mg) in toluene (10mL) is heated under reflux under nitrogen positive pressure.After 12 hours, mixture is concentrated under vacuum to obtain thick oil.Gained thick oil is purified by chromatography on silica using 20% ​​to 40% ethyl acetate in n-hexane as eluent, to obtain 2-((S)-2,2-bis((9Z, 12Z)-octadecane-9,12-diene-1-yl)-1,3-dioxolane-4-yl) ethane-1-ol as clear oil, 7 (0.11g, 0.17mmol, 77%).

[0961] 1H NMR (300MHz, CDCl3): 5.36-5.32 (m, 8H), 4.25-4.20 (m, 1H), 4.10-4.06 (m, 1H), 3.82-3.77 (m, 1H), 3.54-3.49 (m, 1H), 2.23-2.1 9 (t, J=6.6Hz, 3H), 2.14-2.00 (m, 16H), 1.84-1.78 (m, 2H), 1.62-1.51 (m, 6H), 1.34-1.29 (m, 28H), 0.90-0.86 (t, J=6.6Hz, 6H).

[0962] 3-((S)-2,2-bis((9Z,12Z)-octadec-9,12-dien-1-yl)-1,3-dioxolane-4-yl)propane- Synthesis of 1-ol, 8

[0963]

[0964] (6Z,12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-one, 4 (0.50 g, 0.95 mmol), (S)-(3)-(2,2-dimethyl-1,3-dioxolan-4-yl)propanol 6 (0.76 g, 4.75 mmol) and pyridinium p-toluenesulfonate were added. The mixture of (36mg) in toluene (10mL) is heated under reflux under nitrogen positive pressure.After 12 hours, mixture is concentrated under vacuum to obtain thick oil.Gained thick oil is purified by chromatography on silica using 20% ​​to 40% ethyl acetate in n-hexane as eluent, to obtain 3-((S)-2,2-bis((9Z, 12Z)-octadecane-9,12-diene-1-yl)-1,3-dioxolane-4-yl) propane-1-ol as clear oil, 8 (0.48g, 0.76mmol, 80%).

[0965] 1 H NMR (300MHz, CDCl3): 5.34-5.29 (m, 8H), 4.06-4.02 (m, 2H), 3.67-3.47 (m, 2H), 3.45-3.43 (m, 1H), 2.12-2.01 (m, 16H), 1.65-1.62 (m, 8H), 1.34-1.29 (m, 32H), 0.89-0.85 (t, J=6.6Hz, 6H).

[0966] 2-((S)-2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)-1,3-dioxolane-4-yl)-N,N- Synthesis of dimethylethane-1-amine (AKG-KC2-01, O-12095)

[0967]

[0968] To a solution of 2-((S)-2,2-bis((9Z,12Z)-octadec-9,12-dien-1-yl)-1,3-dioxolan-4-yl)ethane-1-ol, 7 (0.49 g, 0.79 mmol) in dichloromethane (10 mL) was added methanesulfonyl chloride (73 μL, 0.95 mmol) and triethylamine (0.26 mL, 1.2 mmol) at 0°C. The solution was warmed to room temperature and stirred for another hour. The reaction was quenched with water and extracted with dichloromethane (2×100 mL). The organics were washed with brine, then dried over magnesium sulfate and filtered. The filtrate was concentrated under vacuum to give a crude oil. 2M dimethylamine solution (10 mL) was added to the resulting crude oil and allowed to stir for 24 hours. The mixture was then quenched with water and extracted with dichloromethane (2×100 mL). The combined organic matter was washed with brine, then dried over magnesium sulfate and then filtered. The filtrate was concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 5% to 100% ethyl acetate in n-hexane as eluent to give 2-((S)-2,2-bis((9Z,12Z)-octadec-9,12-diene-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylethane-1-amine, (AKG-KC2-01, O-12095), (206 mg, 0.32 mmol, 41%) as a clear oil.

[0969] 1 H NMR (300MHz, CDCl3): 5.35-5.32 (m, 8H), 4.08-4.03 (m, 2H), 3.47 (t, J=68Hz, 1H), 2.36-2.27 (m, 2H), 2.21 (s, 6 H), 2.01-1.99 (m, 16H), 1.88-1.77 (m, 2H), 1.68-1.53 ​​(m, 6H), 1.42-1.19 (m, 34H), 0.96-0.86 (t, J=3.7Hz, 6H).

[0970] C 43 H 79 MS (APCI) of NO2: 642.6

[0971] 3-((S)-2,2-di((6Z,12Z)-octadec-6-12-dien-4-yl)-1,3-dioxolane-4-yl)-N,N- Synthesis of dimethylpropane-1-amine (AKG-KC3-01, O-12096)

[0972]

[0973] The procedure has been described previously.

[0974] 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), as a clear oil (255 mg, 0.39 mmol, 51%).

[0975] 1 H NMR (300MHz, CDCl3): 5.39-5.32 (m, 8H), 4.06-4.02 (m, 2H), 3.48-3.44 (m, 1H), 2.35-2.30 (m, 2H), 2. 25 (s, 6H), 2.01-1.98 (m, 16H), 1.70-1.51 (m, 12H), 1.35-1.25 (m, 32H), 0.90-0.85 (t, J=6.6Hz, 6H). C 44 H 81 MS (APCI) of NO2: 656.6

[0976] The following synthesis: 2-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethane-1-amine (AKG-KC2-OA, O-11880)

[0977] 2-((S)-2,2-Di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethane-1-amine (AKG-KC2-PA, O-11879)

[0978] 3-((S)-2,2-Di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-OA, O-11957)

[0979] 3-((S)-2,2-Di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-PA, O-12418)

[0980] See also Figure 49 .

[0981] Experimental procedures (refer to the previously described synthesis of AKG-KC2-01)

[0982] Synthesis of (Z)-1-bromooctadec-9-ene 3

[0983]

[0984] The procedure has been described previously.

[0985] (Z)-1-bromooctadec-9-ene (6.4 g, 19.33 mmol) as a clear oil.

[0986] 1 H NMR (300MHz, CDCl3): 5.36-5.32 (m, 2H), 3.41 (t, J=7.5Hz, 2H), 2.01-1.99 (m, 4H), 1.87-1.82 (m, 2H), 1.44-1.26 (m, 22H), 0.87 (t, J=6.6Hz, 3H).

[0987] (Z)-16-Bromohexadec-7-ene 4

[0988]

[0989] 1 H NMR (300MHz, CDCl3): 5.36-5.32 (m, 2H), 3.42 (t, J=7.5Hz, 2H), 2.01-1.99 (m, 4H), 1.87-1.82 (m, 2H), 1.44-1.26 (m, 18H), 0.89 (t, J=6.6Hz, 3H).

[0990] Synthesis of (9Z,28Z)-heptatriaconta-9,28-dien-19-ol 5

[0991]

[0992] The procedure has been described previously.

[0993] (9Z,28Z)-heptatriaconta-9,28-dien-19-ol as a solid (1.2 g, 2.25 mmol, 47%). H NMR (300 MHz, CDCl3): 5.36-5.29 (m, 4H), 357 (bs, 1H), 2.01-1.97 (m, 8H), 1.42-1.26 (m, 53H), 0.89 (t, J = 6.6 Hz, 6H).

[0994] (7Z, 26Z)-tricara-7,26-dien-17-ol

[0995]

[0996] 1 H NMR (300MHz, CDCl3): 5.36-5.29 (m, 4H), 3.57 (bs, 1H), 2.01-1.97 (m, 8H), 1.42-1.26 (m, 45H), 0.89 (t, J=6.6Hz, 6H).

[0997] Synthesis of (9Z,28Z)-heptatriaconta-9,28-dien-19-one 7

[0998]

[0999] The procedure has been described previously.

[1000] (9Z,28Z)-heptatriaconta-9,28-dien-19-one (0.89 g, 1.67 mmol, 74%) as a clear oil.

[1001] 1 H NMR (300MHz, CDCl3): 5.36-5.29 (m, 4H), 2.03-1.98 (m, 8H), 1.42-1.26 (m, 52H), 0.90-0.89 (t, J=6.6Hz, 6H).

[1002] (7Z, 26Z)-tricara-7,26-dien-17-one 8

[1003]

[1004] HNMR (300MHz, CDCl3): 5.36-5.29 (m, 4H), 2.03-1.98 (m, 8H), 1.42-1.26 (m, 44H), 0.90-0.89 (t, J=6.6Hz, 6H)

[1005] Synthesis of 2-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol become

[1006]

[1007] The procedure has been described previously.

[1008] 2-((S)-2,2-Di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol (0.39 g, 0.63 mmol, 74%) as a clear oil.

[1009] H NMR (300MHz, CDCl3): 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=6.8Hz, 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=6.3Hz, 6H).

[1010] 2-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol, 10 synthesis

[1011]

[1012] The procedure has been described previously.

[1013] 2-((S)-2,2-Di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol (1.02 g, 1.65 mmol, 51%) was obtained as a clear oil.

[1014] 1 H NMR (300MHz, 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-6.6Hz, 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=6.3Hz, 6H).

[1015] 3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol, 11 synthesis

[1016]

[1017] The procedure has been described previously.

[1018] 3-((S)-2,2-Di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol as a clear oil (0.41 g, 0.65 mmol, 76%)

[1019] 1 H NMR (300MHz, CDCl3): 5.39-5.32 (m, 4H), 4.06-4.03 (m, 2H), 3.71-3.67 (m, 2H), 3.47-3.46 (m, 1H), 2 .01-1.99 (m, 10H), 1.66-1.59 (m, 4H), 1.56-1.54 (m, 6H), 1.34-1.26 (m, 44H), 0.87 (t, J=6.3Hz, 6H).

[1020] Synthesis of 3-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol

[1021]

[1022] The procedure has been described previously.

[1023] 3-((S)-2,2-Di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol (0.9 g, 1.56 mmol, 80%) as a clear oil.

[1024] 1 H NMR (300MHz, CDCl3): 5.39-5.28 (m, 4H), 4.06-4.01 (m, 2H), 3.71-3.67 (m, 2H), 3.47-3.46 (m, 1H), 2 .01-1.99 (m, 10H), 1.66-1.59 (m, 4H), 1.56-1.54 (m, 6H), 1.34-1.26 (m, 37H), 0.87 (t, J=6.3Hz, 6H).

[1025] 2-((S)-2,2-Di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethyl Synthesis of Alkyl-1-amine (AKG-KC2-OA, O-11880)

[1026]

[1027] The procedure has been described previously.

[1028] 2-((S)-2,2-Di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethane-1-amine, (AKG-KC2-OA, O-11880) (200 mg, 0.31 mmol, 49%) as a clear oil.

[1029] 1 H NMR (300MHz, CDCl3): 5.38-5.28 (m, 4H), 4.08-4.01 (m, 2H), 3.48 (t, J=6.8Hz, 1H), 2.39-2.24 (m, 2H), 2.21 (s, 6H), 2.01-1.97 (m, 8H), 1.82-1.77 (m, 2H), 1.68-1.52 (m, 6H), 1.34-1.26 (m, 46H), 0.87 (t, = 6.3Hz, 6H).

[1030] C 43 H 83 MS (APCI) of NO2: 646.7

[1031] Synthesis of 2-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethane-1-amine (AKG-KC2-PA, O-11879)

[1032]

[1033] The procedure has been described previously.

[1034] 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), (195 mg, 0.33 mmol, 18%) was obtained as a clear oil.

[1035] 1 H NMR (300MHz, CDCl3): 5.35-5.28(m, 4H), 4.08-4.02(m, 2H), 3.48(t, J=6.6Hz, 1H), 2.38-2.27(m, 2H), 2.20( s, 6H), 2.01-1.99 (m, 8H), 1.97-1.80 (m, 2H), 1.77-1.52 (m, 6H), 1.34-1.29 (m, 38H), 0.87 (t, J=6.3Hz, 6H).

[1036] C 39 H 75 MS (APCI) of NO2: 590.6

[1037] Synthesis of 3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-OA, O-11957)

[1038]

[1039] The procedure has been described previously.

[1040] 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), (160 mg, 0.24 mmol, 37%) as a clear oil.

[1041] 1 H NMR (300MHz, CDCl3): 5.39-5.28 (m, 4H), 4.06-4.01 (m, 2H), 3.44 (t, J=6.8Hz, 1H), 2.26 (t, J=6.8Hz, 2H), 2.2 0 (s, 6H), 2.01-1.97 (m, 8H), 1.82-1.77 (m, 2H), 1.60-1.43 (m, 8H), 1.34-1.26 (m, 46H), 0.87 (t, J=6.3Hz, 6H).

[1042] C 44 H 85 MS (APCI) of NO2: 660.6

[1043] Synthesis of 3-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-PA, O-12418)

[1044]

[1045] The procedure has been described previously.

[1046] 3-((S)-2,2-Di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine, (AKG-KC3-PA, O-12418), (300 mg, 0.49 mmol, 32%) as a clear oil.

[1047] 1 H NMR (300MHz, CDCl3): 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=6.6Hz, 6H).

[1048] C 44 H 85 MS (APCI) of NO2: 604.6

[1049] Synthesis of 3-((S)-2,2-di((Z)-heptadeca-8-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine, AKG-KC3-C17(C8:1)

[1050] Synthesis of (S)-3-(2,2-diheptadecan-1,3-dioxolane-4-yl)-N,N-dimethylpropane-1-amine, AKG-KC3-C17

[1051] See also Figure 50 .

[1052] Experimental procedures

[1053] Synthesis of (9Z,26Z)-pentatriacontac-9,26-dien-18-one, 2

[1054]

[1055] At 0 ℃, triethylamine (5.8mL, 33.3mmol) is added to a stirred solution of oleoyl chloride (10g, 33.3mmol) in toluene (50mL). A large amount of precipitates are formed, and the mixture is allowed to stir at room temperature for 8 hours. The mixture is quenched with 2% sulfuric acid solution and subsequently extracted with ethyl acetate. The organic matter is washed with salt water, then dried over magnesium sulfate and filtered. The filtrate is concentrated under vacuum to obtain a crude oil. The gained oil is diluted with ethanol (20mL), and [2N NaOH] (30mL) is added. The mixture is heated at 100 ℃ for 12 hours, then cooled. The mixture is diluted with 2N HCl solution until it is 4 to obtain a pH. The mixture is extracted with ethyl acetate. The combined organic matter is washed with salt water, then dried over magnesium sulfate and filtered. The filtrate is concentrated under vacuum to obtain a crude oil. The oil was purified on silica using 10% to 20% ethyl acetate in n-hexane as eluent to afford (9Z,26Z)-pentatriaconta-9,26-dien-18-one, 2 (3.8 g, 44%) as a yellow oil.

[1056] 1 H NMR (300MHz, CDCl3): δppm 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=6.6Hz, 3H).

[1057] 3-((S)-2,2-di((Z)-heptadeca-8-en-1-yl)-1,3-dioxolane-4-yl)propan-1-ol, 3 become

[1058]

[1059] The procedure for synthesizing AKG-KC2-01 was previously described.

[1060] 3-((S)-2,2-Di((Z)-heptadeca-8-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol, 3 (0.7 g, 1.15 mmol, 65%) was obtained as a clear oil.

[1061] 1H NMR (300MHz, CDCl3): δppm5.38-5.31(m, 4H), 4.08-4.02(m, 2H), 3.67-3.66(m, 2H), 3.48-3.43(m, 1H ), 215-2.13 (m, 1H), 2.00-1.98 (m, 8H), 1.65-1.56 (m, 8H), 1.27-1.25 (m, 44H), 0.88 (t, J=6.6Hz, 6H).

[1062] Synthesis of (S)-3-(2,2-diheptadecan-1,3-dioxolane-4-yl)propan-1-ol, 4

[1063]

[1064] By 3-((S)-2,2-bis((Z)-heptadecan-8-ene-1-yl)-1,3-dioxolane-4-yl)propane-1-ol (1.3g, 2.15mmol) in methanol / ethyl acetate (20mL, 1:1 / v:v) solution under 1atm (hydrogen balloon) through 10% palladium carbon (100mg) hydrogenation 2 hours.The mixture is evacuated hydrogen and flowed with nitrogen.The mixture is filtered through diatomaceous earth, and the filtrate is concentrated under vacuum to obtain (S)-3-(2,2-diheptadecan-1,3-dioxolane-4-yl)propane-1-ol as clear oil, 4 (1.3g, quantitative).

[1065] 1 H NMR (300MHz, CDCl3): δppm 4.10-4.01 (m, 2H), 3.71-3.64 (m, 1H), 3.47-3.43 (m, 2H), 2.05-2.01 (m, 1H), 1.63-1.51 (m, 8H), 1.42-1.22 (m, 58H), 0.87 (t, J=6.6Hz, 6H).

[1066] Synthesis of 3-((S)-2,2-di((Z)-heptadeca-8-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine, AKG-KC3-C17(C8:1)(O-12620)

[1067]

[1068] The procedure for synthesizing AKG-KC2-01 was previously described.

[1069] 3-((S)-2,2-Di((Z)-heptadeca-8-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine, (AKG-KC3-C17(C8:1), O-12620), as a clear oil (290 mg, 0.46 mmol, 40%).

[1070] C 42 H 81 MS (APCI) of NO2 + ):632.6

[1071] 1 H NMR (300MHz, CDCl3): δppm5.38-5.28(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=6.8Hz, 6H).

[1072] Synthesis of (S)-3-(2,2-diheptadecan-1,3-dioxolane-4-yl)-N,N-dimethylpropane-1-amine, AKG-KC3-C17 (O-12637)

[1073]

[1074] The procedure for synthesizing AKG-KC2-01 was previously described.

[1075] (S)-3-(2,2-diheptadecan-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine, (AKG-KC3-C17, O-12637), as a solid (275 mg, 0.43 mmol, 22%).

[1076] C 42 H 85 MS (APCI) of NO2 + ):636.6

[1077] 1 H NMR (300MHz, CDCl3): δppm4.06-4.00(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=6.6Hz, 6H).

[1078] Example 1C. Synthesis of Dilauroyl-(S)-Glycerol-mPEG2000 (PEG(2000)-DL)

[1079] Experimental procedures

[1080] Synthesis of mPEG2000 tosylate 2

[1081]

[1082] To a solution of poly(ethylene glycol) methyl ether 1 (7 g, 3.5 mmol) in dichloromethane (30 mL) at 0°C was added p-toluenesulfonyl chloride (0.9 g, 7 mmol) and triethylamine (1.8 mL, 10.5 mmol). The resulting mixture was stirred at room temperature for 12 hours and then quenched with water. The mixture was extracted with dichloromethane and the 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 10% methanol in dichloromethane as eluent to give mPEG2000 tosylate (5.2 g, 70%) as a white solid.

[1083] 1H NMR (300MHz, CDCl3): δppm7.79-7.76 (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).

[1084] Synthesis of (S)-(+)-1,2-isopropylideneglycerol mPEG2000 4

[1085]

[1086] At 0 ° C, 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). 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, dried over 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 eluent to obtain (S)-(+)-1,2-isopropylidene glycerol mPEG 2000 (3.3 g, 56%) as a clear oil.

[1087] 1H NMR (300MHz, CDCl3): δppm4.32 (dd, J=11.8, 6.3Hz, 1H), 3.99 (dd, J=8.2, 6.3Hz, 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).

[1088] Synthesis of (R)-3-(mPEG2000)-propane-1,2-diol 5

[1089]

[1090] By (S)-(+)-1,2-isopropylidene glycerol mPEG 2000 (3.3g, 1.56mmol) and 15mL [1N HCl] in THF (10mL) mixture at room temperature stirred one hour.After one hour, the mixture was concentrated under vacuum to obtain (R)-3-(mPEG2000)-propane-1,2-diol (3.4g, quantitative) as a white solid.Proceed in the absence of further purification.

[1091] 1H NMR (300MHz, CDCl3): 8ppm4.1-4.12(m, 1H), 3.68-3.51(m, 180H), 3.39-3.36(m, 4H).

[1092] Synthesis of Dilauroyl-(S)-Glycerol-mPEG2000

[1093]

[1094] 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 and the 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 5% to 100% diethyl ether in hexane as eluent to give dilauroyl-(S)-glycerol-mPEG2000 (0.66 g, 18%) as a white solid.

[1095] MS (MALDI): CHCA matrix: 2431.57

[1096] 1H NMR (300MHz, CDCl3): δppm 5.23-5.18 (m, 1H), 4.32 (dd, J=11.8, 3.6Hz, 1H), 4.13 (dd, J=11.8, 6.3Hz, 1H), 3.85-3.74 (m, 1H), 3.68-3.5 1 (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).

[1097] Example 1D. Synthesis of mPEG2000-DLPE

[1098]

[1099] 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-tetradecanoic acid tritriacont-133-yl) carbonate, 2

[1100] To a solution of poly(ethylene glycol) methyl ether (4 g, 2 mmol) in dichloromethane (20 mL) was added 4-nitrophenyl chloroformate (603 mg, 3 mmol) and pyridine (0.5 mL, 6 mmol) at 0°C. The resulting mixture was stirred at room temperature for 3 hours and then quenched with water. The mixture was extracted with dichloromethane and the 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 5% to 10% methanol in dichloromethane as eluent to give 4-nitrophenyl chloroformate as a white solid.

[1101] (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

[1102] Tetradecanoic acid tritriacont-133-yl) carbonate (3.4 g, 82%).

[1103] 1H NMR (300MHz, CDCl3): δppm8.28-8.25(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).

[1104] (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-tetrapentacarbonyloxa-136-azatriacont-138-yl)oxy)phosphoryl)oxy)propane-1,2-diyldidodecanoate, mPEG2000-DLPE

[1105] 4-Nitrophenyl

[1106] A mixture of (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-tetradecanoic acid tritriacont-alk-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) was 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% to 5% methanol in dichloromethane as eluent to give (2R)-3-((hydroxy((1,35-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-pentadecanoyl-136-azaoctatriacont-138-yl)oxy)phosphoryl)oxy)propane-1,2-diyldidodecanoate mPEG2000-DLPE (2 g, 46%).

[1110] 1H NMR (300MHz, CDCl3): δppm5.17-5.15(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=1.3Hz, 6H).

[1111] Example 1E. Synthesis of Distearoylphosphatidyl-D-serine (O-12153) - see Figure 51 ((Benzyloxy)carbonyl)-D-serine benzyl ester, 2

[1112] To a solution of Z-(D)-serine-OH (2.4 g, 10 mmol) in DMF (10 mL) was added benzyl bromide (1.2 mL, 10 mmol) and cesium carbonate (3.2 g, 10 mmol). The resulting mixture was stirred at room temperature for 12 hours and then quenched with water. The mixture was extracted with dichloromethane and the 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 20% ​​to 40% ethyl acetate in hexanes as eluent to give ((benzyloxy)carbonyl)-D-serine benzyl ester (2.5 g, 76%) as a white solid.

[1113] O-((Benzyloxy)(diisopropylamino)phosphino)-N-((Benzyloxy)carbonyl)-D-serine benzyl ester, 4

[1114] A mixture of 1-(benzyloxy)-N,N,N',N'-tetraisopropylphosphodiamidate, 3 (2.8 g, 8.4 mmol), ((benzyloxy)carbonyl)-D-serine ester 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 0.1% Et3N / n-hexane solution) using 10% ethyl acetate in n-hexane as eluent to give O-((benzyloxy)(diisopropylamino)phosphino)-N-((benzyloxy)carbonyl)-D-serine benzyl ester 4 (2.0 g, 47%) as a clear oil.

[1115] MS(APCI+):567.2(M+1);1H NMR (300MHz, CDCl3): δppm7.33-7.30 (m, 15H), 5.81-5.61 (dd, J=11.8, 3.6Hz, 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).

[1116] (2R)-3-(((Benzyloxy)((R)-3-(Benzyloxy)-2-(((Benzyloxy)carbonyl)amino)-3-oxopropoxy)phosphoryl)oxy)propane-1,2-diyl distearate, 6

[1117] A mixture of 1.2 distearoyl-sn-glycerol, 5 (1.0 g, 1.6 mmol), O-((benzyloxy)(diisopropylamino)phosphino)-N-((benzyloxy)carbonyl)-D-serine benzyl ester 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 hours. After 6 hours, 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 hour and then quenched with saturated sodium thiosulfate. The mixture was extracted with ethyl acetate and the 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 10% to 20% ethyl acetate in hexanes as 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.

[1118] 1H NMR (300MHz, CDCl3): δppm 7.33-7.25 (m, 15H), 5.91-5.79 (dd, J=11.8, 3.6Hz, 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). .

[1119] O-(((R)-2,3-bis(stearoyloxy)propoxy)(hydroxy)phosphoryl)-D-serine bistriethylamine salt,

[1120] 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, THF (10:2:4, v:v:v, 10 mL) was hydrogenated over 10% palladium on carbon (40 mg) at 1 atmosphere at room temperature. After 3 hours, the mixture was degassed, flushed with nitrogen, and filtered through celite. Triethylamine (0.5 mL) was added to the filtrate, and then concentrated under vacuum to give an oil. The oil was purified on a C18 column using methanol (0.5% triethylamine) as eluent to give O-(((R)-2,3-bis(stearoyloxy)propoxy)(hydroxy)phosphoryl)-D-serine bistriethylamine salt (360 mg, 50%) as a white solid.

[1121] MS, (APCI+):567.2(M+1), 1H NMR (300MHz, CDCl3): δppm5.30-5.20(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=0.6Hz, 6H).

[1122] Example 2. Determination of in vitro cytotoxicity in human hepatocytes or cancer cells

[1123] The LNPs were tested in vitro in a series of 10 dilutions to determine the IC50 in human hepatocytes / liver (HepG2; ATCC #HB8065) ​​cells. As these formulations are generally expected to be non-toxic, a positive control, Lipofectamine, was included in all studies. TM 3000 (ThermoFisher #L3000015) complexed with mRNA (2 μl reagent / 1 μg mRNA). The mRNA used was CleanCap FLuc, EGFP, or MCherry reporter mRNA (5 moU; Trilink #L-7202, #L-7201, or #L-7203). Data are reported as complete cell viability curves and calculated actual IC50 values ​​for each compound.

[1124] Adherent cells were grown to approximately 80% confluence. The cells were trypsinized by adding 0.25% trypsin-EDTA (Gibco #25200-072), and the cells were subsequently centrifuged and 5 ml of growth medium (MEM medium; Corning #10010CM) was added to disperse the cells. Cell density was determined using a hemocytometer. Growth medium (MEM medium comprising 10% FBS; Corning #35015CV) was added to the cells to adjust to a suitable cell concentration. 200 μl of cells (5,000 cells / well) were then added to a 96-well transparent flat-bottom plate (Costar #9804) and incubated for 24 hours at 37°C in a humidified incubator with 5% CO .

[1125] Serial dilutions of LNP formulations were prepared using growth medium as a solvent. These compounds were provided in sterile aqueous solutions at a concentration of 1 mg / ml mRNA. To perform the dilutions, each LNP stock solution was warmed to room temperature. These were further diluted 4× in growth medium to achieve a maximum mRNA concentration of 250 μg / ml.

[1126] By aspirating the old culture medium and replacing the old culture medium with 200 μ l of culture medium containing LNP, LNP is added to the wells with a series of 1:3 dilutions starting from the initial 250 μ g / ml concentration of each LNP. The plate is incubated for 72 hours at 37 ° C in a humidified incubator with 5% CO . At the end of the LNP incubation period, the culture medium in each well is replaced with 100 μ l 1 × PrestoBlue cell viability reagent (ThermoFisher catalog number A13261). The plate is incubated for 30 minutes to 2 hours at 37 ° C in a humidified incubator with 5% CO . At 30, 60 and 120 minutes, readings are read. SpectraMax M5 plate reader (Molecular Devices) is used to read the fluorescence under 560 nm excitation and 590 nm emission. Background is corrected by subtracting the RFU of the control (background control well) comprising only culture medium from all sample readings. Cytotoxicity percentage is calculated using the following formula:

[1127] % cytotoxicity = [(RFU. 培养基 -RFU 处理 ) / RFU. 培养基 ]×100%

[1128] IC50 was determined using GraphPad Prism using the following formula:

[1129] Y=100 / (1+10^((LogIC50-X)*Hill Slope)))

[1130] In some embodiments, Lipofectamine TM The cytotoxicity of the positive control for the 3000 (ThermoFisher # L3000015) complexed mRNA (2 μl reagent / 1 μg mRNA) can be 5 to 100 times more toxic than the compounds disclosed herein. This indicates that in an in vitro hepatotoxicity assay, the disclosed compounds are less toxic than commercial transfection reagents. In some embodiments, the compounds described herein form less toxic LNPs in vivo than commercially available transfection reagents.

[1131] Example 3. Determination of pKa of ionizable lipids

[1132] The pKa of ionizable cationic lipids can be calculated in several ways. For lipids, this is sometimes difficult because the membrane structure and adjacent lipids in the membrane can affect the dissociation characteristics of the amino group, thereby potentially giving inaccurate values. In situ measurements are ideal, where the apparent pKa of an ionizable lipid is measured when the lipid is in its intended environment, in this case as part of an LNP (Jayaraman 2012, Sabins 2018).

[1133] For each LNP preparation, amino lipid pKa value is by measuring the fluorescence of 2-(p-toluidinyl)-6-naphthalenesulfonic acid (TNS) during the titration from pH 3 to 12 to determine.TNS is an anionic molecule, it is not fluorescent in solution, but can improve fluorescence when associated with positive lipid membrane, and this characteristic has been used to detect membrane surface charge in the past.Prepared main buffer stock solution (10mM sodium phosphate, 10mM sodium borate, 10mM sodium citrate, 150mM sodium chloride), described main buffer stock solution is used to prepare the buffer solution under multiple pH value for determining apparent pKa.Use 1M sodium hydroxide and 1M hydrochloric acid, prepare about 20 kinds of unique buffer solutions under about 3 to 12 different pH values ​​from main buffer stock solution.300mM6-(p-toluidinyl)-2-naphthalenesulfonic acid sodium salt (TNS reagent) dissolved in dimethyl sulfoxide (DMSO) is used as stock solution. Prepare LNP and purify it into the desired pH buffer solution with a final mRNA concentration of 0.04mg / mL. Using a 96-well plate preloaded with a desired buffer solution, add LNP containing mRNA so that the final concentration of mRNA is 0.7 μg / mL. Add TNS to each well so that the DMSO concentration is 1% (v / v). After mixing, measure the fluorescence (Ex / Em 331nm / 445nm) of TNS in each well, and apply S-shaped best fit analysis to the fluorescence data. pKa is determined to be the pH that causes half-maximum fluorescence intensity. The apparent pKa measured for compounds 1 to 36 is in the pH range of 6.0 to 7.0.

[1134] Example 4. Measurement of LNP cellular uptake.

[1135] The measurement of LNP cellular uptake is achieved by fluorescence imaging and / or fluorescence quantification. There are many suitable fluorescent tracers available, such as 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), 3,3'-dilinoleyloxacarbocyanine perchlorate (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 are weakly fluorescent in water, but when incorporated into lipid membranes (such as lipid membranes present in LNPs), they show high fluorescence. It is important that the selected lipid is photostable and has a high extinction coefficient.

[1136] Under a fluorescence microscope, the LNP comprising these types of lipids is manifested. In one method, the LNP lipid formulation comprises a fluorescent lipid tracer, such as 1,1'-dioctadecyl-3,3,3', 3'-tetramethyl indole dicarbocyanine-5,5'-disulfonic acid (DiI5-DS), of 0.1 to 0.5mol% total lipid. In a suitable cell culture dish, for example, cells of interest are cultured in a 24-well plate (Corning). The day before the uptake study, cells are inoculated with 50% confluence and cultured overnight under suitable conditions (such as 37°C, 5% CO2, 90% to 100% humidity). LNP is added to cell culture medium with 0.1 to 100 μg / mL mRNA, allowed to interact with cells for a period of time (4 to 24 hours), then cells are washed three times with culture medium to remove the LNP not internalized, subsequently observed. Cells are observed under a microscope with fluorescence detection capability. Untreated cells are used as background controls, and the relative extent of LNP cellular uptake is determined according to the fluorescence intensity signal from cells. Alternatively, quantitative measurement of cellular fluorescent lipids can be achieved by pelleting the cells, lysing them with a detergent such as Triton-X100, and quantifying fluorescence by spectrofluorometry or quantifying fluorescent lipid tracers by HPLC.

[1137] In a similar manner, quantification of fluorescently labeled mRNA has been achieved. For example, dye-labeled enhanced green fluorescent protein (EGFP) and firefly luciferase (FLuc) mRNA (both transcribed with a 1:3 ratio of cyanine 5-UTP:5-methoxy-UTP) are currently available from Trilink Biotechnologies. The excitation maximum of cyanine 5 is 650 nm and the emission maximum is 670 nm. Replacement at this ratio results in mRNA that is easily visualized and can still be translated in cell culture. By capturing fluorescently labeled mRNA, the intracellular delivery of mRNA can be visualized using the above method.

[1138] LNP uptake in cells can be achieved through endogenous methods (e.g., ApoE-mediated) or through exogenous methods (e.g., active targeting). LNP systems containing ionizable cationic lipids were found to utilize a "natural" targeting process, in which they adsorb apolipoprotein E (ApoE) in the blood (Cullis et al 2017) and are subsequently actively taken up in hepatocytes by a number of receptors containing ApoE-binding ligands (Williams et al. 2010). By using non-overlapping fluorophores, the intracellular distribution and organelle accumulation dynamics of mRNA and LNPs can be tracked independently.

[1139] The mRNA cell expression level can be quantified by using a reporter system 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 with 0.1 to 100 μg / mL mRNA. By replacing the culture medium after 4 to 24 hours, the cells can be washed to make the LNP without internalization. At 24 hours, the GFP signal is quantified by fluorescence microscopy or flow cytometry. In this way, it is possible to distinguish between LNP formulation groups based on the reporter protein expression level.

[1140] Example 5. Transfection selectivity index

[1141] The Transfection Selectivity Index (TSI) was calculated to determine the relative transfection efficiency in mammalian cells compared to the relative toxicity in those same cells. The selectivity index was calculated using the following formula:

[1142] TSI=EF 哺乳动物 / IC 50，哺乳动物

[1143] Among them EF 哺乳动物 is the transfection efficiency expressed in ng protein / million cells, and IC 50，哺乳动物 It is related to the cell viability of the same preparations and is expressed as half-maximal inhibitory concentration.

[1144] LNPs using compounds (1 to 36) described herein had 50% higher TSI compared to LNPs prepared using otherwise identical LNPs prepared with the control molecule DLin-MC3-DMA as the ICL.

[1145] Example 6. Determination of lipid peroxidation.

[1146] The extent of oxidation can be determined using a forced degradation assay in which LNP samples are treated with 3% H2O2 at 25°C and sampled for lipid oxidation products at days 0, 1, 3, and 5 (Blessy et al. (2014) Journal of Pharmaceutical Analysis 4, 159-165). The oxidation reactants can be quenched 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) reactivity assay for the lipid peroxidation end product malondialdehyde (MDA) (Gutteridge (1982) FEBS Letters 150, 454-458), or by detection using an HPLC assay with evaporative light scattering detection (ELSD) or charged aerosol detection (CAD). Lipid oxidation and isomerization impurity structures can be assigned based on known literature precedent and are expected to be mixtures of isomers.

[1147] Generally, it is known in the art that lipids with multiple unsaturations in the acyl chain are more susceptible to oxidation (see Reis and Spickett (2012) Biochim Biophys Acta 1818, 2374-2387).

[1148] It is expected that compounds 1 to 36 described herein have a lower susceptibility to oxidative damage or degradation when compared to a control LNP comprising DLin-KC2-DMA lipid or when compared to a control LNP comprising DLin-MC3-DMA. In some embodiments, the compounds provided herein have a reduction of greater than 30%, greater than 50%, greater than 75%, greater than 90%, and greater than 95% in oxidative byproducts when compared to a control LNP.

[1149] Example 7. Preparation of ligand-targeted LNPs

[1150] Antibody ligands in the form of antibody Fab' fragments or single-chain Fv fragments are prepared by any method known in the art (e.g., as described in U.S. Patent Application No. 20180271998 to Drummond et al.; U.S. Patent No. 10,406,225 to Zhou et al.; U.S. Patent No. 8,974,792 to Marks et al., which are incorporated herein by reference in their entireties), and are provided for specific uptake of LNPs into target cells (e.g., immune cells). To provide a conjugate 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 then is conjugated according to Figure 2 Conjugated to a maleimide-terminated PEG-DSPE anchor. The ligand is expressed in bacteria or eukaryotic cells and isolated from the cell mass or growth medium using standard methods such as protein affinity chromatography or metal chelate chromatography. To activate the thiol group of the terminal cysteine ​​residue, the ligand is incubated for 1 hour in the...

Claims

1. A lipid nanoparticle (LNP) vaccine composition comprising: a. Nucleic acid; b. an ionizable cationic lipid having an N / P ratio of 3 to 8 relative to the nucleic acid, wherein the ionizable lipid has a chemical structure: Where R1 is a C containing one or two olefins 15 -C 19 of an alkyl group, and wherein the ionizable cationic lipid is present in the LNP vaccine composition in a total amount of 40 to 65 mol% of the total lipid content in the LNP composition, and the ionizable cationic lipid is 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); c. sterols, the total amount of which is 25 to 45 mol% of the total lipid content in the LNP composition; d. one or more phospholipids, the total amount of phospholipids being 5 to 25 mol % of the total lipid content in the LNP composition; and e. Conjugated lipids, the total amount of which is 0.5 to 2.5 mol % of the total lipid content in the LNP composition.

2. The composition of claim 1, wherein the one or more phospholipids comprise phosphatidylserine (PS) lipids in a total amount of 2.5 to 10 mol % of the total lipid content in the LNP composition.

3. The composition of claim 2, wherein the PS-lipid is DSPS or DPPS.

4. The composition of claim 1, wherein the second phospholipid is selected from the group consisting of DSPC, HSPC, DPPC and sphingomyelin.

5. The LNP vaccine composition of claim 1, wherein the ionizable cationic lipid comprises a monounsaturated alkyl chain or a diunsaturated alkyl chain, wherein the olefins are separated by at least two methylene groups.

6. The composition of claim 1, wherein 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).

7. The composition of any one of claims 1 to 6, which has an N / P ratio of 5 to 6 relative to the nucleic acid.

8. The composition of claim 7, wherein the nucleic acid is RNA.

9. The composition of claim 8, wherein the nucleic acid is mRNA, optionally chemically modified RNA, and optionally further modified with N-methylpseudouridine.

10. A lipid nanoparticle (LNP) composition comprising: a. Nucleic acid; b. an ionizable cationic lipid having an N / P ratio of 3 to 8 relative to the nucleic acid, wherein the ionizable lipid has a chemical structure: Where R1 is a C containing one or two olefins 15 -C 19 of an alkyl group, and wherein the ionizable cationic lipid is present in the LNP composition in a total amount of 40 to 65 mol % of the total lipid content in the LNP composition, and the ionizable cationic lipid is optionally selected from the group consisting of Compound 3 (Table 1A), Compound 8 (Table 1A), DLin-KC3-DMA, KC3-01, KC3-OA, KC3-PA, KC3-C17 (8: 1), and KC3-C15 (C8: 1); c. sterols, the total amount of which is 25 to 45 mol% of the total lipid content in the LNP composition; d. one or more phospholipids, the total amount of phospholipids of which is 5 to 25 mol% of the total lipid content in the LNP composition; and comprising a total amount of dipalmitoylphosphatidylserine (DPPS) lipids of 2.5 to 10 mol% of the total lipid content in the LNP composition; and e. Conjugated lipids, the total amount of which is 0.5 to 2.5 mol % of the total lipid content in the LNP composition.

11. The composition of claim 10, wherein the second phospholipid is selected from the group consisting of DSPC, HSPC and sphingomyelin.

12. The composition of any one of claims 10 to 11, wherein the N / P ratio of the ionizable cationic lipid relative to the nucleic acid is 5 to 6.

13. The LNP composition of claim 12, wherein the ionizable cationic lipid comprises two monounsaturated alkyl chains or two diunsaturated alkyl chains, and wherein the olefins are separated by at least two methylene groups.

14. The LNP composition of claim 13, wherein 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).

15. The composition of claim 14, wherein 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).

16. The composition of any one of claims 10 to 15, wherein the nucleic acid is mRNA.

17. The composition of claim 16, wherein the mRNA is chemically modified with N-methyl pseudouridine.

18. A nucleic acid lipid nanoparticle (LNP) composition comprising: a. Nucleic acid; b. an ionizable cationic lipid having an N / P ratio of 3 to 8 relative to the nucleic acid, the total amount of which is 40 to 65 mol% of the total lipid content in the LNP composition; c. sterols, the total amount of which is 25 to 45 mol% of the total lipid content in the LNP composition; d. one or more phospholipids, the total amount of phospholipids is 5 to 25 mol% of the total lipid content in the LNP composition; and e. A conjugated lipid, the total amount of which is 0.5 to 2.5 mol% of the total lipid content in the LNP composition, the conjugated lipid comprising a PEG-lipid having a poly(ethylene glycol) chain terminally connected to a linker moiety and two hydrocarbon chains terminally connected to the same linker moiety, wherein the hydrocarbon chains are saturated C independently selected from n-dodecyl (lauryl) and n-dodecanoyl (lauroyl). 12 chain.

19. The composition of claim 18, wherein the linker moiety is glyceryl, N-oxycarbonylglycerophosphorylethanolaminocarbonyl, oxycarbonylamide, or oxyacetamide.

20. The composition of claim 19, wherein the poly(ethylene glycol) chain is a methoxy-poly(ethylene glycol) having an average molecular weight of 2000.

21. The composition of claim 20, 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-dilaurylamide, or mPEG-N,N-dilaurylacetamide.

22. The composition of any one of claims 18 to 21, wherein the LNPs have a z-average particle size of 60 to 150 nm and are freeze-thaw stable.

23. The composition of any one of claims 18 to 22, wherein one or more phospholipids comprises phosphatidylserine (PS) lipids in a total amount of 2.5 to 10 mol % of the total lipid content in the LNP composition, and wherein the LNP composition comprises phosphatidylserine (PS) lipids in a total amount of 2.5 to 10 mol % of the total lipid content in the LNP composition.

24. A nucleic acid lipid nanoparticle (LNP) human vaccine composition comprising: a. Nucleic acid; b. an ionizable cationic lipid having an N / P ratio of 3 to 8 relative to the nucleic acid, the total amount of which is 40 to 65 mol% of the total lipid content in the LNP composition; c. sterols, the total amount of which is 25 to 45 mol% of the total lipid content in the LNP composition; d. one or more phospholipids, the total amount of phospholipids of which is 5 to 25 mol% of the total lipid content in the LNP composition, and comprising a total amount of phosphatidylglycerol (PG) of 1.0 to 10 mol% of the total lipid content in the LNP composition; and e. Conjugated lipids, the total amount of which is 0.5 to 2.5 mol % of the total lipid content in the LNP composition.

25. The composition of claim 24, wherein 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).

26. The composition of claim 24, wherein: a. the sterol is cholesterol; and b. The phosphatidylglycerol (PG) is an anionic phospholipid selected from distearoylphosphatidylglycerol (DSPG) and dipalmitoylphosphatidylglycerol (DPPG).

27. The composition of claim 24, wherein the nucleic acid is mRNA and the ionizable cationic lipid is present at an N / P ratio of 4 to 7 relative to the nucleic acid.

28. The composition of claim 24, wherein the conjugated lipid is selected from the group consisting of PEG-DMG, PEG-DLG, and PEG-DLPE.

29. The composition of claim 26, wherein the one or more phospholipids comprises a phospholipid selected from distearoylphosphatidylcholine (DSPC) and hydrogenated soy phosphatidylcholine (HSPC).

30. A method for preparing a nucleic acid delivery composition comprising a lipid, wherein the lipid comprises phosphatidylserine, the method comprising the step of dissolving the phosphatidylserine in ethanol, wherein the phosphatidylserine is in the form of an ammonium salt of the phosphatidylserine.

31. The method of claim 30, wherein the phosphatidylserine is DPPS.

32. The method of any one of claims 30 to 31, wherein the phosphatidylserine is dissolved in ethanol to a concentration exceeding 0.2 mM.

33. The method of any one of claims 30 to 32, wherein the ammonium salt is a salt comprising ammonium selected from the group consisting of ammonium, alkylammonium, dialkylammonium, trialkylammonium, and tetraalkylammonium.

34. The method of any one of claims 30 to 32, wherein the ammonium is selected from an ammonium form 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, or tromethamine (tris(hydroxymethyl)aminomethane).

35. The method of any one of claims 30 to 32, wherein the nucleic acid delivery composition is the composition of any one of claims 1 to 29.

36. A method for delivering a nucleic acid into a cell, comprising the steps of: The lipid nanoparticle (LNP) is contacted with the cell, wherein the cell is a human dendritic cell, wherein the LNP composition is obtained by a method comprising one or more steps of any one of claims 30 to 34.

37. The method of claim 36, wherein the LNP comprises the ionizable cationic lipid of any one of claims 1 to 29.

38. The method of any one of claims 36 or 37, wherein the cell is in a human or animal subject.

39. The method of claim 38, wherein the LNP is administered to the subject intramuscularly, subcutaneously, intradermally, or topically.

40. A LNP composition comprising: a. nucleic acid, wherein the nucleic acid is mRNA; b. cholesterol sterols, the total amount of which is 25 to 40 mol% of the total lipid content in the LNP composition; c. an ionizable cationic lipid having an N / P ratio of 3 to 8 relative to the nucleic acid, the total amount of which is 40 to 65 mol% of the total lipid content in the LNP composition; d. one or more phospholipids, the total amount of which is 5 to 25 mol% of the total lipid content in the LNP composition, the one or more phospholipids being selected from: 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 in the LNP composition; and ii. distearoylphosphatidylcholine (DSPC) phospholipids, the total amount of which is 5 to 25 mol% of the total lipid content in the LNP composition; and e. PEG-containing conjugated lipids, the total amount of which is 0.5 to 2.5 mol % of the total lipid content in the LNP composition.

41. A method for administering a nucleic acid to a subject in need thereof, comprising administering to the human subject a nucleic acid lipid nanoparticle (LNP) composition comprising: a. Nucleic acid; b. an ionizable cationic lipid having an N / P ratio of 3 to 8 relative to the nucleic acid, the total amount of which is 40 to 65 mol% of the total lipid content in the LNP composition; c. sterols, the total amount of which is 25 to 45 mol% of the total lipid content in the LNP composition; d. one or more phospholipids, the total amount of phospholipids of which is 5 to 25 mol% of the total lipid content in the LNP composition, and comprising a total amount of phosphatidylglycerol (PG) of 1.0 to 10 mol% of the total lipid content in the LNP composition; and e. Conjugated lipids, the total amount of which is 0.5 to 2.5 mol % of the total lipid content in the LNP composition.

42. A nucleic acid lipid nanoparticle (LNP) composition comprising: a. Nucleic acid; b. an ionizable cationic lipid having an N / P ratio of 3 to 8 relative to the nucleic acid, the total amount of which is 40 to 65 mol% of the total lipid content in the LNP composition; c. sterols, the total amount of which is 25 to 45 mol% of the total lipid content in the LNP composition; d. one or more phospholipids, the total amount of phospholipids of which is 5 to 25 mol% of the total lipid content in the LNP composition, and comprising a total amount of phosphatidylserine (PS) lipids of 2.5 to 10 mol% of the total lipid content in the LNP composition; and e. Conjugated lipids, the total amount of which is 0.5 to 2.5 mol % of the total lipid content in the LNP composition.

43. The composition of claim 42, wherein: a. The nucleic acid is mRNA b. the ionizable cationic lipid is present in the LNP composition at an N / P ratio of 4 to 7 relative to the nucleic acid; c. the sterol is cholesterol; and d. The conjugated lipid is a PEG-containing conjugated lipid.

44. The composition of claim 43, wherein the one or more phospholipids comprise at least two phospholipids having mismatched acyl chain lengths.

45. The composition of claim 44, wherein the phosphatidylserine (PS) lipid is dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS).

46. ​​The composition of claim 45, wherein the one or more phospholipids comprises a phospholipid selected from distearoylphosphatidylcholine (DSPC) and hydrogenated soy phosphatidylcholine (HSPC).

47. The composition of claim 46, wherein the one or more phospholipids consist of distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidyl-L-serine ((L-serine) DPPS).

48. The composition of claim 47, wherein the PEG-containing conjugated lipid is PEG(2000)-dimyristoylglycerol (PEG-DMG).

49. LNP composition, wherein the ionizable lipid has the following chemical structure: Where R1 is 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 hydroxy; and n is an integer equal to 2, 3 or 4.

50. The composition of claim 49, wherein: a. R2 and R3 are each methyl; and bn is 3 or 4.

51. The composition of any one of claims 49, 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).

52. The composition of any one of claims 49 to 51, wherein the ionizable cationic lipid is KC3-PA.

53. The composition of any one of claims 49 to 51, wherein the ionizable cationic lipid is KC3-OA.

54. The composition of any one of claims 49 to 51, wherein the ionizable cationic lipid is KC3-C17(C8:1).

55. An ionizable cationic lipid selected from KC3-PA, KC3-C17(8:1), and KC3-C15(C8:1).

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