Lipid nanoparticles for preventing tuberculosis or other mycobacterial infections

Abstract

Some aspects of the present disclosure provide improved Mycobacterium tuberculosis vaccine compositions for the delivery of immunogenic nucleic acids to ionizable lipid nanoparticles of cells. Anionic phospholipids, including phosphatidylserine and phosphatidylglycerol, are included in lipid nanoparticles to improve transfection efficiency in dendritic cells. In some embodiments, the incorporation of monounsaturated alkyl chain analogs in dimethylaminopropyl-dioxolane or heterocyclic ketal ionizable lipids in formulations provides high levels of transfection in human dendritic cells and exhibits good stability against oxidative damage as compared to other ionizable lipids in the same family. Further aspects of the present disclosure provide an mRNA encoding a tandem peptide and optionally comprise a second mRNA encoding a tandem MHC-I tuberculosis epitope, the mRNA encoding the tandem peptide encoding a plurality of MHC-II tuberculosis epitopes.

Description

[0001] Related Applications

[0002] This patent application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 476,916, filed December 22, 2022, which is incorporated by reference in its entirety.

[0003] Incorporation by Reference of Sequence Listing

[0004] The specification includes a sequence listing submitted concurrently herewith, which includes the file 191016-010702_PCT_SL.xml of size 324,933 bytes, which was created on December 21, 2023, the content of which is incorporated by reference herein. TECHNICAL FIELD

[0005] Some aspects of the present disclosure relate to lipid nanoparticles (LNP) that target dendritic cells incorporating mRNA that encodes a combination of specific CD8 and CD4 T cell epitopes present in mycobacterium tuberculosis. In some embodiments, LNP comprising one or more cationic ionizable lipids are useful for delivery of mRNA, for dendritic cell targeting, or methods using these LNP compositions as vaccines for the prevention of tuberculosis or other mycobacterial infections. BACKGROUND

[0006] Lipid nanoparticles (LNP) 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 comprise ionizable cationic lipids (ICLs). However, certain ICL compounds are known in the art to be undesirably sensitive to oxidation during storage. Thus, there is a need for improved ICL compounds that have improved stability to oxidative degradation upon storage, while also providing desirable transfection activity or potency in cells when incorporated into LNP with a therapeutic agent, such as a nucleic acid.

[0007] LNP compositions, including stable nucleic acid lipid particle (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 significant challenge to human health. For example, Mycobacteria is a genus of bacteria that causes tuberculosis (TB). According to the World Health Organization, TB is among the top 10 causes of death and the leading cause of death from a single infectious agent worldwide. Despite the best efforts currently, there remains a significant challenge in developing effective vaccines for the prevention of many infectious diseases. New efforts to identify individual antigenic peptides or combinations of antigenic peptides help to improve the efficiency of vaccines. Nonetheless, there remains important opportunities in engineering adjuvants to help effectively deliver and present these antigenic sequences to professional antigen presenting cells such as dendritic cells. mRNA encoding antigenic peptides or proteins in combination with ionizable cationic lipid nanoparticles represent a particularly promising strategy in vaccine development. There is a need for safe and effective therapeutics comprising LNP pharmaceutical compositions for the delivery of mRNA for the treatment and prevention of a variety of diseases, including vaccine compositions. SUMMARY

[0008] Provided herein are lipid nanoparticle (LNP) compositions and methods of making and using the same. In some embodiments, the LNP compositions comprise a nucleic acid, such as a messenger ribonucleic acid (mRNA). In some embodiments, the LNP compositions are vaccines comprising an LNP formulation comprising an mRNA encoding an immune system epitope or an antigen recognized by the immune system.

[0009] In some aspects, the LNP comprises a nucleic acid comprising a chemically modified mRNA, wherein the chemically modified mRNA comprises N1-methyl pseudouridine. In some aspects, the LNP comprises a nucleic acid comprising a 5’ untranslated region (UTR) and a 3’ UTR, a polyA tail of about 80 to about 140 nucleotides in length, and (i) a 5’ enzymatic cap or (ii) a 5’ clean cap. In some aspects, the LNP comprises a chemically modified mRNA, wherein the chemically modified mRNA comprises N1-methyl pseudouridine.

[0010] In some embodiments, the LNP composition comprises: (a) a nucleic acid; (b) an ionizable cationic lipid comprising a KC3 ionizable cationic lipid at an N / P ratio of 3 to 7 relative to the nucleic acid, the ionizable cationic lipid being present in the LNP composition in a total amount of 46 to 54 mol% of the total lipid content in the LNP composition; (c) one or more phospholipids in a total amount of 5 to 20 mol% of the total lipid content in the LNP composition; (d) one or more anionic phospholipids in a total amount of 2 to 8 mol% of the total lipid content in the LNP composition; (e) a conjugated lipid in a total amount of 1 to 3.5 mol% of the total lipid content in the LNP composition; and (f) a sterol, e.g., cholesterol (e.g., in an amount to provide the remaining portion of the LNP composition). In some aspects, the one or more anionic phospholipids is phosphatidylserine (PS) or phosphatidylglycerol (PG). In some aspects, the one or more anionic phospholipids is selected from the group consisting of: dipalmitoylphosphatidyl-L-serine (DPPS), or distearoylphosphatidyl-L-serine (DSPS), distearoylphosphatidylglycerol (DSPG), and dipalmitoyphosphatidylglycerol (DPPG). In some aspects, the one or more phospholipids comprises distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC), or a combination thereof. In some aspects, the conjugated lipid is PEG(2000)-dimyristoyl glycerol (PEG-DMG). In some aspects, the sterol is cholesterol. In some aspects, the ionizable cationic lipid comprises 3-((S)-2,2-dio((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine (KC3-OA). In some aspects, the ionizable cationic lipid further comprises a KC4 ionizable cationic lipid, e.g., 4- exo-rac-2,2-dio((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylbutane-1-amine (AKG-KC4-OA).In some aspects, the LNP composition consists of 48 mol% KC3-OA, 5 mol% DPPS or DSPG, 5 to 10 mol% DSPC or HSPC, 1.5 mol% PEG-DMG, and cholesterol. In some aspects, the LNP composition consists of 48 mol% KC3-OA, 5 mol% DPPS or DSPG, 5 mol% DSPC or HSPC, 1.5 mol% PEG-DMG, and 40.5 mol% cholesterol. In some aspects, the LNP composition consists of 48 mol% KC3-OA, 5 mol% DPPS or DSPG, 10 mol% DSPC or HSPC, 1.5 mol% PEG-DMG, and 35.5 mol% cholesterol.

[0011] In some embodiments, a method of eliciting a T cell response in a host is provided, the method comprising administering to the host a nucleic acid sequence disclosed herein or a nucleic acid having at least 90% sequence identity or complementarity to a sequence disclosed herein and / or a sequence encoding a T cell epitope from Mycobacterium tuberculosis (Mtb), or a polynucleotide sequence having at least 90% identity or complementarity to a sequence disclosed herein and / or a polynucleotide sequence of a Mtb antigen recognized by a T cell.

[0012] A lipid nanoparticle (LNP) composition consists of: a messenger ribonucleic acid (mRNA) encoding one or more Mycobacterium tuberculosis (Mtb) proteins selected from the group consisting of: CFP10 / Rv3874, ESAT-6 / Rv3875, Mtb32A / Rv0125, Mtb39A / Rv1196, Ag85B / Rv1886c, EsxW / Rv3620c, EsxV / Rv3619c, PE13 / Rv1195, PPE30 / Rv1802, PPE40 / Rv2356c, and TB10.4 / Rv0288; an ionizable cationic lipid comprising KC3 ionizable cationic lipid at an N / P ratio of 4 to 6 relative to the mRNA, the ionizable cationic lipid being present in the LNP composition in a total amount of 46 to 54 mol% of the total lipid content in the LNP composition; one or more phospholipids selected from the group consisting of: distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), and dipalmitoylphosphatidylcholine (DPPC), the total amount of the one or more phospholipids being 10 to 18 mol% of the total lipid content in the LNP composition; one or more anionic phospholipids selected from the group consisting of: dipalmitoylphosphatidyl-L-serine (DPPS), or distearoylphosphatidyl-L-serine (DSPS), distearoylphosphatidylglycerol (DSPG), and dipalmitoylphosphatidylglycerol (DPPG), the total amount of the one or more anionic phospholipids being 2 to 8 mol% of the total lipid content in the LNP composition; PEG(2000)-dimyristoylglycerol (PEG-DMG) in a total amount of 1 to 3.5 mol% of the total lipid content in the LNP composition; and cholesterol (e.g., 35.5 to 40.5 mol% cholesterol).

[0013] Some aspects of the disclosure relate to lipid nanoparticle (LNP) compositions comprising KC3 ionizable cationic lipids, cholesterol, one or more phospholipids containing at least one anionic phospholipid, conjugated lipids, and one or more nucleic acid sequences encoding T cell epitopes from Mycobacterium tuberculosis (Mtb).

[0014] In some embodiments, the LNP composition comprises 48 mol% KC3 ionizable cationic lipids, 40.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipids, 5 mol% DSPC or DPPC, and a total phospholipid concentration of 10 mol%, wherein each mol% refers to mol% of the total lipid content in the LNP composition.

[0015] In some embodiments, the LNP composition comprises 48 mol% KC3 ionizable cationic lipid, 40.5 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP composition comprises 48 mol% KC3 ionizable cationic lipid, 38.5 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP composition comprises 45 mol% KC3 ionizable cationic lipid, 42.7 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP composition comprises 50 mol% KC3 ionizable cationic lipid, 38.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, and a total phospholipid concentration of 10 mol%, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP composition comprises 48 mol% KC3 ionizable cationic lipid, 40.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, and a total phospholipid concentration of 10 mol%, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP 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 phospholipid concentration of 10 mol%, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP composition comprises 46.5 mol% KC3 ionizable cationic lipid, 42 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP composition comprises 15 mol% total phospholipid and 35.5 mol% cholesterol. In some embodiments, the LNP composition comprises 10 mol% total phospholipid and 40.5 mol% cholesterol. In some embodiments, the LNP composition comprises 40.5 mol% cholesterol, 5% anionic lipid (DPPS), and 5% PC (DSPC or DPPC) and a total phospholipid concentration of 10 mol%. In some embodiments, the LNP composition comprises 48 mol% cationic ionizable lipid, 5 mol% PC (DPPC), 5 mol% anionic lipid (DPPS), 40.5 mol% cholesterol, 1.5 mol% conjugated lipid (PEG-DMG).

[0016] In some aspects, the LNP comprises a nucleic acid sequence (e.g., mRNA) encoding a T cell epitope or Mtb antigen recognized by T cells from Mycobacterium tuberculosis (Mtb). In some aspects, the LNP comprises a nucleic acid sequence that is mRNA encoding a concatenated sequence of T cell epitopes or Mtb antigens recognized by T cells present in Mtb. In some aspects, the LNP comprises a nucleic acid sequence that is mRNA encoding one or more Mtb proteins selected from CFP10 / Rv3874, ESAT-6 / Rv3875, Mtb32A / Rv0125, Mtb39A / Rv1196, and Ag85B / Rv1886c. In some aspects, the LNP comprises a nucleic acid sequence that is mRNA comprising one or more nucleic acid sequences selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 220. In some aspects, the LNP comprises a nucleic acid sequence that is mRNA encoding one or more Mtb proteins selected from EsxW / Rv3620c, EsxV / Rv3619c, PE13 / Rv1195, PPE30 / Rv1802, PPE40 / Rv2356c, and TB10.4 / Rv0288. In some aspects, the LNP comprises a nucleic acid sequence that is mRNA comprising one or more nucleic acid sequences selected from SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO: 221, and SEQ ID NO: 222. In some aspects, the LNP comprises a nucleic acid sequence comprising a concatenated nucleic acid coding sequence comprising N-terminal and C-terminal signal peptides selected from Sec / MITD, Lamp1, HLA-Dra, or tPA. In some aspects, the LNP comprises a nucleic acid sequence that is mRNA having a sequence selected from SEQ ID NO: 34, 36, 38, 40, 42, 44, 224, and 226. In some aspects, the LNP comprises a nucleic acid that is mRNA encoding an amino acid sequence selected from SEQ ID NO: 33, 35, 37, 39, 41, 43, 86-105, 207-210, 223, and 225.

[0017] In some embodiments, the one or more nucleic acids are mRNA. In some embodiments, the mRNA encodes a concatenated sequence of T cell epitopes present in Mtb. In some embodiments, the concatenated sequence of T cell epitopes comprises the amino acid sequences set forth in SEQ ID NOs: 1-17, 106-137, 138-203. In some embodiments, the concatenated sequence of T cell epitopes comprises an amino acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequences set forth in SEQ ID NOs: 1-17, 45-85, 106-137, 138-203. In some embodiments, the concatenated nucleotide sequence comprises two or more sequences encoding peptides or proteins that can elicit MHC class II restricted CD4 T cell responses.

[0018] In some embodiments, the two or more MHC class II epitopes are selected from the group of EsxV (Rv3619), EsxW (Rv3620c), EsxB / CFP10 (Rv3874), EsxA / ESAT-6 (Rv3875), AMtb39A (Rv1196), Ag85B (Rv1886c), and EsxH / TB10.4 (Rv0288). In some embodiments, the two or more MHC class II epitopes comprise a peptide or protein from EsxV (Rv3619), EsxW (Rv3620c), EsxB / CFP10 (Rv3874), EsxA / ESAT-6 (Rv3875), AMtb39A (Rv1196), Ag85B (Rv1886c), and EsxH / TB10.4 (Rv0288) (SEQ ID NOs. 1-7).

[0019] In some embodiments, the concatenated nucleic acid coding sequence comprises, in order from N- to C-terminus, seven proteins selected from the group of EsxB / CFP10 (Rv3874),

[0020] EsxB / CFP10 (Rv3874), EsxA / ESAT-6 (Rv3875), EsxW (Rv3620c), EsxV (Rv3619), EsxH / TB10.4 (Rv0288), ΔAg85B (Rv1886c), and ΔMtb39A (Rv1196), or EsxB / CFP10 (Rv3874), EsxA / ESAT-6 (Rv3875), EsxW (Rv3620c), EsxH / TB10.4 (Rv0288), EsxV (Rv3619), and ΔAg85B (Rv1886c), or EsxB / CFP10 (Rv3874), ΔMtb39A (Rv1196), EsxA / ESAT-6 (Rv3875), EsxW (Rv3620c), EsxH / TB10.4 (Rv0288), EsxV (Rv3619), and ΔAg85B (Rv1886c). (SEQ ID NO 18, 19, and 20).

[0021] In some embodiments, the composition comprises nucleic acids encoding 5 or more non-overlapping CD4 T cell epitopes in the form of peptides, wherein optionally the peptides are 12 to 50 amino acids long.

[0022] In some embodiments, the tandem nucleic acid coding sequence optionally comprises 10 selected MHC-II epitopes comprising:

[0023] AQIYQAVSAQAAAIH (SEQ ID NO. 9), PSPSMGRDIKVQFQS (SEQ ID NO. 10), GINTIPIAINEAEYV (SEQ ID NO. 11), AAFQGAHARFVAAAA (SEQ ID NO. 12), AGWLAFFRDLVARGL (SEQ ID NO 13), ASIIRLVGAVLAEQH (SEQ ID NO. 14), MSFVTTQPEALAAAA (SEQ ID NO. 8), MHVSFVMAYPEMLAA (SEQ ID NO. 15), AYGSFVRTVSLPVGA (SEQ ID NO. 16), and LENDNQLLYNYPGAL (SEQ ID NO. 17) o

[0024] In some embodiments, the tandem nucleic acid coding sequence comprises a GPGPG (SEQ ID NO. 228) linker sequence between each tandem epitope.

[0025] In some embodiments, the one or more nucleic acids comprise the nucleic acid sequence set forth in SEQ ID NO: 34, 36, 38, 40, 42, and 44.

[0026] In some embodiments, the one or more nucleic acids comprise a nucleic acid sequence having at least 90% identity, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 34, 36, 38, 40, 42, and 44.

[0027] In some embodiments, the tandem nucleic acid coding sequence comprises N-terminal and C-terminal signal peptides selected from Sec / MITD, Lamp1, HLA-Dr alpha, or tPA.

[0028] In some embodiments, the one or more nucleic acids comprise chemically modified mRNA, wherein the chemically modified mRNA comprises N1-methylpseudouridine.

[0029] In some embodiments, the one or more nucleic acids comprise a 5’ untranslated region (UTR) and a 3’ UTR, a polyA tail of about 80 to about 140 nucleotides in length, and (i) a 5’ enzymatic cap or (ii) a 5’ clean cap.

[0030] In some embodiments, the one or more nucleic acids are mRNA having a sequence selected from SEQ ID NO: 34, 36, 38, 40, 42, 44, 224, and 226.

[0031] In some embodiments, the one or more nucleic acids are mRNA, and wherein the amino acid sequence encoded by the mRNA is selected from SEQ ID NO: 33, 35, 37, 39, 41, 43, 86 to 105, 207 to 210, 223, and 225.

[0032] In some embodiments, the nucleic acid encoded tandem sequence comprises two or more MHC class I epitopes selected from SEQ ID NO: 106 to 137 and 138 to 203.

[0033] In some embodiments, the nucleic acid encoded tandem sequence comprises two or more MHC class I epitopes present in Mycobacterium tuberculosis that removes epitopes present in BCG and is selected from SEQ ID NO: 86 to 95.

[0034] In some embodiments, the nucleic acid encoded tandem sequence comprises two or more MHC class I epitopes that are ordered to minimize production of linkage neoepitopes and are selected from SEQ ID NOs: 86-105.

[0035] In some embodiments, the cationic lipid is KC3-OA, KC3-PA, KC3-01, KC3-C17(8:1), or KC3-C15(C8:1).

[0036] In some embodiments, the LNP comprises a conjugated lipid in a total amount of less than 2 mol% of the total lipid content in the LNP composition.

[0037] In some embodiments, the total amount of ionizable cationic lipid is 45 to 55 mol% of the total lipid content in the LNP composition; the total amount of cholesterol is 35 to 45 mol% of the total lipid content in the LNP composition; the total amount of one or more phospholipids is 7 to 15 mol% of the total lipid content in the LNP composition; the one or more phospholipids consists of DSPC, and the PS lipid is one or more lipids selected from DPPS and DSPS in the L-serine configuration; and the total amount of PS lipid is about 5 mol% of the total lipid content in the LNP composition.

[0038] In some embodiments, the conjugated lipid is PEG-DMG; and the PS lipid is selected from DSPS (L-isomer) and DPPS.

[0039] In some embodiments, the ionizable cationic lipid is KC3-OA.

[0040] In some embodiments, the N / P ratio of the LNP composition is 4 to 7. In some embodiments, the N / P ratio of the LNP composition is 5 to 6.

[0041] Provided in some aspects of the disclosure are nucleic acid lipid nanoparticle (LNP) compositions comprising: an mRNA that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the nucleic acid sequence set forth in SEQ ID NO: 34, 36, 38, 40, 42, and 44; an ionizable cationic lipid KC3-PA; and a (L-serine) PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition.

[0042] In some embodiments, the PS lipid is (L-serine)DSPS, (L-serine)DPPS, or a mixture thereof, and the LNP composition further comprises cholesterol and a second phospholipid selected from the group consisting of: DSPC, DOPC, DPPC, HSPC, and SM.

[0043] In some aspects of the present disclosure, a nucleic acid lipid nanoparticle (LNP) composition is provided, comprising: an mRNA having the sequence of SEQ ID The nucleic acid sequences shown in NOs:34, 36, 38, 40, 42 and 44 are at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more); a KC3 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 23.5 to 43.5 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 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.

[0044] In some embodiments, the ionizable lipid has the following chemical structure:

[0045]

[0046] in:

[0047] R1 is

[0048] 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;

[0049] R2 and R3 are each independently methyl; and

[0050] n is an integer equal to 2 or 3. In some embodiments, n is 3.

[0051] In some embodiments, the composition is a vaccine.

[0052] In some aspects of the present disclosure, provided are pharmaceutical compositions comprising the lipid nanoparticles described herein, and a pharmaceutically acceptable carrier.

[0053] Some aspects of the present disclosure relate to nucleic acids encoding tandem amino acid sequences of T cell epitopes present in Mycobacterium tuberculosis, said nucleic acids being at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the nucleic acid sequences shown in SEQ ID NOs: 34, 36, 38, 40, 42, and 44.

[0054] 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. Some 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.

[0055] In some embodiments, LNP compositions include targeting ligands directly for cell surface receptors, thereby with high specificity, lipid nanoparticles are targeted to including dendritic cells. 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 contain dipalmitoylphosphatidylcholine (DPPC).

[0056] 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 lipids were more active than KC2 or diacyl ionizable lipids (UO series) in transfecting human dendritic cells. 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.

[0057] In some embodiments, certain salts of phosphatidylserine targeting lipids are provided. For example, in some embodiments, the phosphatidylserine targeting lipids can be provided as an ammonium salt of DPPS, which has improved biophysical properties and higher solubility in the presence of ethanol, which is the preferred solvent for the preparation of LNPs. DSPS or the sodium salt of DSPS is not soluble in ethanol and requires both the presence of methanol and heating for it to form, as does the ammonium salt of DSPS. It is expected that other ammonium salts of phosphatidylserine will yield the same advantages in terms of solubility and biophysical properties.

[0058] In some embodiments, ionizable cationic lipid compositions useful for the preparation of liposomal nanoparticle (LNP) compositions are provided. In some embodiments, liposomal compositions comprising ionizable cationic lipids having (a) a straight chain C 16 or C 18 hydrocarbon chains each comprising a single unsaturated alkenyl double bond within each polyolefin chain, covalently bound to a headgroup comprising a dialkylaminoalkyl group. In some embodiments, the headgroup of the ionizable cationic lipid has a dialkylamino group having a pKa of about 6.3 to 7.5. In some embodiments, the headgroup of the ionizable cationic lipid comprises a heterocyclyl or alkyl moiety covalently bound to the dialkylamino group. In some embodiments, the headgroup of the ionizable cationic lipid optionally further comprises a phospho group. In some embodiments, each lipid tail of the ionizable cationic lipid compound is identical and each lipid tail collectively has an olefin having a total length of 15, 16, 17, or 18 carbons.

[0059] In some embodiments, the LNP compositions comprise a KC3 ionizable cationic lipid. Unless otherwise indicated, the term “KC3 ionizable cationic lipid” as used herein refers to an ionizable cationic lipid having the following chemical formula:

[0060]

[0061] wherein each R1 is the same or different and is a straight chain C 15 to C 19 hydrocarbon chain each comprising one or more unsaturated alkenyl double bonds within each polyolefin chain; R2 and R3 are each independently methyl; and n is 3. In some aspects, each R1 in the KC3 ionizable cationic lipid is the same and is a straight chain C 16 or C 18 hydrocarbon chain each comprising a single unsaturated alkenyl double bond within each polyolefin chain.

[0062] In some embodiments, the LNP composition comprises a KC4 ionizable cationic lipid. The term "KC4 ionizable cationic lipid" as used herein, unless otherwise indicated, refers to an ionizable cationic lipid having the following chemical formula:

[0063]

[0064] wherein each R1is the same or different, and is a linear C 15 to C 19 hydrocarbon chain, each comprising one or more unsaturated alkenyl double bonds within each polyene chain; R2and R3are each independently methyl; and n is 4. In some aspects, each R1in the KC4 ionizable cationic lipid is the same, and is a linear C 16 or C 18 hydrocarbon chain, each comprising a single unsaturated alkenyl double bond within each polyene chain.

[0065] In some embodiments, the LNP composition comprises a mixture of a KC3 ionizable cationic lipid and a KC4 ionizable cationic lipid.

[0066] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I):

[0067]

[0068] wherein:

[0069] R1is

[0070] 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;

[0071] R2and R3are each independently (C1-C4)alkyl optionally substituted with hydroxyl; and

[0072] n is an integer equal to 2, 3, or 4.

[0073] In some embodiments, an ionizable cationic lipid composition is provided. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein a is 0 or 1; b is 1, 2, 3, or 4, provided that the sum of a+b is 1, 2, 3, or 4; R2and R3are each methyl; and n is an integer equal to 3.

[0074] In some embodiments, an ionizable cationic lipid composition is provided. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I-A):

[0075]

[0076] wherein:

[0077] R1is

[0078] 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;

[0079] R2and R3are each methyl; and

[0080] n is an integer equal to 3.

[0081] In some embodiments, the LNP composition containing an ionizable cationic lipid comprises the same pair of lipid hydrocarbon tails having a total of 15, 16, 17, or 18 carbons and comprising a single alkene group or pair of alkene groups. In some embodiments, the LNP composition comprises an ionizable cationic lipid selected from the group consisting of:

[0082]

[0083] 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).

[0084] 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.

[0085] The salt form of a targeting lipid can affect its solubility in alcohol-containing solvents used in the preparation of lipid nanoparticles. In some embodiments, an ionizable cationic lipid composition is provided. In some embodiments, a 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 comprising a conjugated lipid. In some embodiments, a lipid nanoparticle (LNP) composition comprises: an mRNA nucleic acid; an ionizable lipid disclosed herein; cholesterol; one or more phospholipids selected from the group consisting of DSPC, DPPC, and DOPC; and a PS lipid selected from the group consisting of DPPS, DSPS, and DOPS; and optionally further comprising a conjugated lipid comprising PEG.

[0086] In some embodiments, the LNP composition can comprise an anionic phospholipid. In some embodiments, the LNP composition is prepared using a sodium salt or an ammonium salt of an anionic phospholipid. In some embodiments, the anionic phospholipid salt is a compound of Formula (V-A-l), which has the following chemical structure:

[0087]

[0088] wherein:

[0089] X + is an ammonium (NH4 + ) or sodium (Na + ) cation; and

[0090] a is 14, 15, or 16.

[0091] In some embodiments, the anionic phospholipid salt is selected from:

[0092]

[0093] In some embodiments, the anionic phospholipid salt is a DSPS (L-isomer) sodium salt. In some embodiments, the anionic phospholipid salt is a DSPS (L-isomer) ammonium salt. In some embodiments, the anionic phospholipid salt is a DPPS (L-isomer) sodium salt. In some embodiments, the anionic phospholipid salt is a DPPS (L-isomer) ammonium salt. In some embodiments, the targeting lipid is a sodium salt or an ammonium salt of dipalmitoyl phosphatidyl-L-serine (DPPS) or distearoyl phosphatidyl-L-serine (DSPS). In some embodiments, the targeting lipid is a sodium salt or an ammonium salt of dipalmitoyl phosphatidyl-L-serine (DPPS) or distearoyl phosphatidyl-L-serine (DSPS).

[0094] In some embodiments, the LNP composition can comprise an anionic phospholipid selected from the following, or an ammonium salt or a sodium salt thereof:

[0095]

[0096] In some embodiments, the salt form of phosphatidylserine is highly soluble in ethanol. In some embodiments, it is soluble at greater than 0.5 mg / ml, greater than 1 mg / mL, greater than 5 mg / mL, greater than 10 mg / mL, or greater than 20 mg / mL. In some embodiments, the salt is an ammonium salt. In some embodiments, the salt is an ammonium salt itself, an alkylammonium, a dialkylammonium, or a trialkylammonium salt. In some embodiments, the amine is selected from the group consisting of ammonia, dimethylamine, diethylamine, triethylamine, trimethylamine, 2-(dimethylamino)ethanol, diethanolamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methyl-glucamine, imidazole, histidine, lysine, arginine, 4-(2-hydroxyethyl)-morpholine, piperazine, 1-(2-hydroxyethyl)-pyrrolidine, triethanolamine, and tromethamine (tris(hydroxymethyl)aminomethane), and in some embodiments, the targeting lipid is an ammonium salt of DPPS.

[0097] Anionic phospholipids isolated from phosphatidyl-L-serine are also considered targeting lipids for LNPs. These include phosphatidylglycerol (PG), phosphatidic acid (PA), N- glutaryl-phosphatidylethanolamine (N-Glu-PE), N-succinyl-phosphatidylethanolamine (N-Suc-PE), and cardiolipin. In some embodiments, the LNP comprises an anionic phospholipid isolated from phosphatidyl-L-serine, which can be used as a targeting lipid for the LNP. In some embodiments, the LNP comprises an anionic phospholipid selected from the group consisting of phosphatidylglycerol (PG), phosphatidic acid (PA), N-glutaryl- phosphatidylethanolamine (N-Glu-PE), N-succinyl-phosphatidylethanolamine (N-Suc-PE), and cardiolipin. Distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), N-succinyl-distearoylphosphatidylethanolamine (N-Suc-DSPE), N-glutaryl-distearoylphosphatidylethanolamine (N-glu-DSPE), distearoylphosphatidic acid (DSPA), and cardiolipin are also provided as anionic phospholipids.

[0098] In some embodiments, there is provided a lipid nanoparticle (LNP) composition comprising a composition of ionizable cationic lipids. In some embodiments, there is provided a lipid nanoparticle (LNP) composition comprising an ionizable cationic lipid. In some embodiments, the LNP composition comprises an mRNA nucleic acid. In some embodiments, the lipid nanoparticle (LNP) composition further comprises a PS lipid in a total amount of 2.5 to 10 mol% of the total lipids in the LNP composition. In some embodiments, the lipid nanoparticle (LNP) composition further comprises a PS lipid selected from DSPS (L-isomer) and DPPS. In some embodiments, the lipid nanoparticle (LNP) composition comprises a conjugated lipid in 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 in a total amount less than 2 mol% of the total lipid content in the LNP composition, and the conjugated lipid is PEG-DMG.

[0099] 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 comprising 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 the group consisting of SM, DSPC, HSPC, DPPC, and DOPC; and a PS lipid selected from the group consisting of DPPS and DSPS; and optionally further comprising a conjugated lipid comprising 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 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 comprising a conjugated lipid in a total amount of 0.5 to 2.5 mol% of the total lipid content in the LNP composition.

[0100] 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).

[0101] Some aspects of the disclosure relate to a lipid nanoparticle (LNP) composition comprising an ionizable lipid having the following chemical structure:

[0102]

[0103] wherein:

[0104] R1is

[0105] 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;

[0106] R2and R3are each independently (C1-C4)alkyl optionally substituted with hydroxyl; and

[0107] n is an integer equal to 2, 3, or 4.

[0108] 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, R2and R3are each methyl.

[0109] In some embodiments, R1is

[0110]

[0111] a is 0 or 1, and b is 1 or 3; R2and R3are each methyl; and n is 2 or 3. In some embodiments, n is 3.

[0112] In some embodiments, it comprises: a nucleic acid; an ionizable lipid described herein, a sterol; one or more phospholipids comprising phosphatidylserine (PS) lipids; and optionally a conjugated lipid.

[0113] In some embodiments, the nucleic acid is mRNA.

[0114] In some embodiments, the sterol is cholesterol.

[0115] 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.

[0116] In some embodiments, the one or more phospholipids consist of: DSPC; and one or more PS lipids selected from (L-serine)DPPS and (L-serine)DSPS.

[0117] 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.

[0118] In some embodiments, the conjugated lipid comprises PEG.

[0119] Some aspects of the disclosure relate to a nucleic acid lipid nanoparticle (LNP) composition comprising: a nucleic acid; ionizable cationic lipids 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 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 a conjugated lipid in a total amount of 0.5 to 2.5 mol% of the total lipid content in the LNP composition.

[0120] In some embodiments, the nucleic acid is mRNA.

[0121] In some embodiments, the sterol is cholesterol.

[0122] In some embodiments, the one or more phospholipids consists of: DSPC and L-serine PS.

[0123] In some embodiments, the composition comprises PS in a total amount of 2.5 to 7.5 mol% of the total lipid in the composition.

[0124] In some embodiments, the conjugated lipid comprises PEG. In some embodiments, the conjugated lipid is PEG-DMG.

[0125] In some embodiments, 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. In some embodiments, the total amount of conjugated lipid is less than 2 mol% of the total lipid content in the LNP composition.

[0126] 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 consists of DSPC, and the PS lipid is one or more lipids selected from DPPS and DSPS in L-serine configuration; and the total amount of PS lipid is about 5 mol% of the total lipid content in the LNP composition.

[0127] In some embodiments, the composition comprises PS lipid in a total amount selected from 1.25 mol%, 2.5 mol%, 5 mol%, 7.5 mol%, and 10 mol% of the total lipid content in the LNP composition.

[0128] Some aspects of the disclosure relate to a nucleic acid lipid nanoparticle (LNP) composition 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 of 35 to 45 mol% of the total lipid content in the LNP composition; one or more phospholipids, wherein the total amount of the one or more phospholipids is 10 mol% of the total lipid content in the LNP composition, and comprises phosphatidylserine (PS) in a total amount of 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.

[0129] 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).

[0130] In some embodiments, the conjugated lipid is PEG-DMG; and the PS lipid is selected from the group consisting of: DSPS (L-isomer) and DPPS.

[0131] 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).

[0132] 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 comprising phosphatidylserine (PS) lipids in a total amount of 3 to 9 mol% of the total lipid content in the LNP composition; and a PEG-containing lipid in a total amount of 0.5 to 2.0 mol% of the total lipid content in the LNP composition.

[0133] In some embodiments, the phospholipid consists of: one or more phospholipids selected from the group consisting of DSPC, DOPC, DPPC, HSPC, and SM.

[0134] In some embodiments, the PS lipid is one or more L-serine lipids selected from the group consisting of DPPS and DSPS.

[0135] In some embodiments, the one or more phospholipids comprise at least two (L-serine)PS lipids having mismatched acyl chain lengths.

[0136] 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.

[0137] Some aspects of the disclosure relate to a nucleic acid liposome nanoparticle (LNP) composition comprising: a nucleic acid, an ionizable cationic lipid KC3-PA or KC3-OA, and a (L-serine)PS lipid in a total amount of 2.5 to 10 mol% based on the total lipid content in the LNP composition.

[0138] In some embodiments, the nucleic acid is an mRNA, the PS lipid is (L-serine)DSPS, (L-serine)DPPS, or a mixture thereof, and the LNP composition further comprises cholesterol and a second phospholipid selected from the group consisting of DSPC, DPPC, HSPC, and SM.

[0139] 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.

[0140] In some embodiments, the ionizable cationic lipid is KC3-PA. In some embodiments, the ionizable cationic lipid is KC3-OA.

[0141] Some aspects of the disclosure relate to a nucleic acid liposome nanoparticle (LNP) composition comprising: a nucleic acid, a KC3-C17(C8:1) ionizable cationic lipid, and a (L-serine)PS lipid in a total amount of 2.5 to 10 mol% based on the total lipid content in the LNP composition.

[0142] 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.

[0143] Some aspects of the disclosure relate to a nucleic acid liposome nanoparticle (LNP) composition comprising: a nucleic acid, an ionizable cationic lipid KC3-PA, and a (L-serine)PS lipid in a total amount of 2.5 to 10 mol% based on the total lipid content in the LNP composition.

[0144] In some embodiments, the nucleic acid is mRNA, the PS lipid is (L-serine)DSPS, (L-serine)DPPS, or a mixture thereof, and the LNP composition further comprises cholesterol and a second phospholipid selected from the group consisting of DSPC, DOPC, DPPC, HSPC, and SM.

[0145] 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.

[0146] Some aspects of the disclosure relate to a nucleic acid lipid nanoparticle (LNP) composition comprising: a nucleic acid, an ionizable cationic lipid selected from KC3-C17(C8:1), and a (L-serine)PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition.

[0147] 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.

[0148] In some embodiments, the nucleic acid is an mRNA encoding a SARS-CoV-2 spike protein.

[0149] Some aspects of the disclosure relate to a nucleic acid lipid nanoparticle (LNP) vaccine composition comprising: an mRNA nucleic acid having an N / P ratio of 4 to 7; a 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; 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 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.

[0150] Some aspects of the disclosure relate to a nucleic acid lipid nanoparticle (LNP) vaccine composition comprising: an mRNA nucleic acid having an N / P ratio of 3 to 8; a 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; 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 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.

[0151] Some aspects of the disclosure relate to a nucleic acid lipid nanoparticle (LNP) vaccine composition comprising: an mRNA nucleic acid having an N / P ratio of 4 to 7; 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.

[0152] Some aspects of the disclosure relate to a nucleic acid lipid nanoparticle (LNP) vaccine composition comprising: an mRNA nucleic acid having an N / P ratio of 3 to 8; 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.

[0153] In some embodiments, the nucleic acid is an mRNA encoding a tandem sequence of T cell epitopes. In some embodiments, the mRNA encodes a tandem sequence of MHC-II epitopes. In some embodiments, the mRNA encodes a tandem sequence of MHC-I epitopes.

[0154] Some aspects of the disclosure relate to the use of a combination of a (L-serine) PS lipid in a LNP with an ionizable cationic lipid described herein for targeting the LNP to dendritic cells. In some embodiments, the LNP comprises mRNA. In some embodiments, the LNP further comprises cholesterol. In some embodiments, the total amount of (L-serine) PS lipid in the LNP is 2.5 to 10 mol% of the total lipid content in the LNP composition. In some embodiments, the LNP further comprises one or more additional phospholipids, including DSPC. In some embodiments, the LNP further comprises a conjugated lipid. In some embodiments, the LNP comprises: mRNA nucleic acid at an N / P ratio of 3 to 8; KC3-PA or KC3-C17(C8:1) ionizable cationic lipid (ICL) at a total amount of 40 to 65 mol% of the total lipid content in the LNP composition; cholesterol at a total amount of 25 to 40 mol% of the total lipid content in the LNP composition; (L-serine) PS lipid at a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition; DSPC phospholipid at a total amount of 5 to 25 mol% of the total lipid content in the LNP composition; and conjugated lipid at a total 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).

[0155] Some aspects of the disclosure relate to a lipid nanoparticle (LNP) composition comprising an ionizable lipid having the following chemical structure:

[0156]

[0157] wherein:

[0158] R1is 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;

[0159] R2and R3are each independently methyl; and

[0160] n is an integer equal to 2 or 3.

[0161] In some embodiments, a is 0. In some embodiments, b is 1. In some embodiments, b is 3.

[0162] In some embodiments, a is 1. In some embodiments, b is 1. In some embodiments, b is 3.

[0163] In some embodiments, n is 2. In some embodiments, n is 3.

[0164] 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.

[0165] In some embodiments, the composition comprises an anionic targeting phospholipid other than phosphatidyl-L-serine.

[0166] In some embodiments, the composition comprises an anionic phospholipid selected from the group consisting of DSPG and DPPG.

[0167] In some embodiments, the composition comprises an anionic phospholipid selected from the group consisting of N-Glu-DSPE and N-Suc-DSPE.

[0168] In some embodiments, the composition comprises a DSPA anionic phospholipid.

[0169] In some embodiments, the composition comprises a cardiolipin anionic phospholipid.

[0170] In some embodiments, the ionizable lipid has the following chemical structure:

[0171]

[0172] In some embodiments, the ionizable lipid has the following chemical structure:

[0173]

[0174] Some aspects of the present disclosure relate to a sodium salt or an ammonium salt of a composition of an anionic phospholipid of Formula (V-A-1) having the following chemical structure:

[0175]

[0176] Formula (V-A-1),

[0177] wherein X + is an ammonium cation or a sodium (Na + ) cation; and a is 14, 15, or 16.

[0178] In some embodiments, a is 14 or 16. In some embodiments, X + is an ammonium cation (NH4 + ).

[0179] In some embodiments, X is a sodium cation (Na + ). + ).

[0180] 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).

[0181] In some embodiments, the anionic phospholipid of formula (V-A-1) is a sodium salt of distearoylphosphatidyl-L-serine (DSPS L-isomer). In some embodiments, the anionic phospholipid of formula (V-A-1) is an ammonium salt of distearoylphosphatidyl-L-serine (DSPS L-isomer). In some embodiments, the anionic phospholipid of formula (V-A-1) is a sodium salt of DPPS (L-isomer). In some embodiments, the anionic phospholipid of formula (V-A-1) is an ammonium salt of DPPS (L-isomer). Some embodiments relate to use of a salt form composition of any one of claims 90-97 in the manufacture of a liposomal nanoparticle (LNP) composition. In some embodiments, the use is in combination with one or more of the following LNP components during manufacture of the LNP composition: an mRNA nucleic acid, an ionizable cationic lipid (ICL), cholesterol, a (L-serine) PS lipid, one or more phospholipids, and a conjugated lipid. In some embodiments, the use includes the step of combining the ammonium or salt form of the compound of formula (V-A-1) with one or more of the following LNP components during manufacture of the LNP composition: an mRNA nucleic acid, an ionizable cationic lipid (ICL) of any one of claims 1-9 or 73-88, cholesterol, a (L-serine) PS lipid, one or more phospholipids, and a conjugated lipid.

[0182] 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; the ionizable cationic lipid of any one of claims 1 to 9 or 73 to 88 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; 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 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.

[0183] In some embodiments, 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. In some embodiments, the LNP composition comprises the PS in a total amount of about 5 mol% of the total lipid in the composition. In some embodiments, 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.

[0184] In some embodiments, the conjugated lipid is PEG-DMG and the PS lipid is selected from the group consisting of DSPS (L-isomer) and DPPS.

[0185] 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).

[0186] Some embodiments relate to the use of a (L-serine) PS lipid in combination with the ionizable cationic lipids described herein in LNP for targeting the LNP to dendritic cells.

[0187] In some embodiments, the LNP comprises an mRNA. In some embodiments, the LNP further comprises cholesterol. In some embodiments, the (L-serine) PS lipid is in a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition. In some embodiments, the LNP further comprises one or more additional phospholipids, the additional phospholipids comprising DSPC. In some embodiments, the LNP further comprises a conjugated lipid.

[0188] In some embodiments, the LNP comprises: an mRNA nucleic acid having an N / P ratio of 3 to 8; a 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; 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 phospholipid in a total amount of 5 to 25 mol% of the total lipid content in the LNP composition; and a conjugated lipid in a total amount of 0 to 2.5 mol% of the total lipid content in the LNP composition.

[0189] In some embodiments, the ICL is KC3-PA. In some embodiments, the ICL is KC3-C17(C8:1).

[0190] In some embodiments, the composition comprises an anionic phospholipid selected from DSPG and DPPG in a total amount of 2.5% to 7.5% of the total lipid content in the LNP composition. In some embodiments, the composition comprises DSPG anionic phospholipid in a total amount of 2.5% to 7.5% of the total lipid content in the LNP composition. In some embodiments, the composition comprises DPPG anionic phospholipid in a total amount of 2.5% to 7.5% of the total lipid content in the LNP composition.

[0191] In some embodiments, the LNP further comprises one or more additional phospholipids, the additional phospholipids comprising DSPC.

[0192] Some aspects of the disclosure relate to a nucleic acid lipid nanoparticle (LNP) composition 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; 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 mol% to 2.5 mol% of the total lipid content in the LNP composition.

[0193] In some embodiments, the nucleic acid is an mRNA. In some embodiments, the N / P ratio is 3 to 8.

[0194] 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).

[0195] In some embodiments, the conjugated lipid is PEG-DMG or PEG-DSG.

[0196] In some embodiments, the composition comprises PEG-containing conjugated lipid in a total amount of 0.5 to 2.0 mol% of the total lipid content in the LNP composition.

[0197] In some embodiments, the composition comprises KC3 ionizable cationic lipid in a total amount of 48 mol% of the total lipid content in the LNP composition.

[0198] In some embodiments, the composition comprises DSPC and DSPS in a total amount of 10 mol% of the total lipid content in the LNP composition.

[0199] 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.

[0200] 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.

[0201] In some embodiments, the composition comprises cholesterol in a total amount of 40.5 mol% of the total lipid content in the LNP composition.

[0202] In some embodiments, the composition comprises DSPC phospholipid in a total amount of 10 mol% of the total lipid content in the LNP composition.

[0203] In some embodiments, the PEG-containing conjugated lipid is PEG 2000 -DMG.

[0204] In some embodiments, the composition comprises cholesterol in a total amount of 23.5 mol% of the total lipid content in the LNP composition. In some embodiments, the composition comprises cholesterol in a total amount of 33.5 mol% of the total lipid content in the LNP composition. In some embodiments, the composition comprises cholesterol in a total amount of 38.5 mol% of the total lipid content in the LNP composition. In some embodiments, the composition comprises cholesterol in a total amount of 40.5 mol% of the total lipid content in the LNP composition. In some embodiments, the composition comprises cholesterol in a total amount of 42.7 mol% of the total lipid content in the LNP composition. In some embodiments, the composition comprises cholesterol in a total amount of 43.5 mol% of the total lipid content in the LNP composition. In some embodiments, the composition comprises cholesterol in a total 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 a total amount of 45 to 55 mol% of the total lipid content in the LNP composition.

[0205] Some aspects of the disclosure relate to a nucleic acid lipid nanoparticle (LNP) composition comprising: an mRNA nucleic acid; a KC3 ionizable cationic lipid selected from 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; a (L-serine)DPPS lipid in a total amount of 5 mol% of the total lipid content in the LNP composition; a DSPC or HSPC phospholipid in a total amount of 5 mol% of the total lipid content in the LNP composition; and a PEG-DMG conjugated lipid in a total amount of 1.5 mol% of the total lipid content in the LNP composition.

[0206] Some aspects of the disclosure relate to a lipid nanoparticle (LNP) composition comprising: a KC3 ionizable cationic lipid, a (L-serine)PS lipid, cholesterol, one or more phospholipids comprising at least one anionic phospholipid, and a conjugated lipid, wherein the LNP is obtained by a method comprising a step of dissolving a sodium or ammonium salt of the anionic phospholipid.

[0207] In some embodiments, the composition comprises a nucleic acid. In some embodiments, the nucleic acid is an mRNA.

[0208] In some embodiments, the composition is a vaccine. In some embodiments, the composition is an injectable vaccine composition.

[0209] In some embodiments, the total amount of phospholipid 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 lipid in the composition is 0.5 to 2.5 mol% of the total lipid content in the LNP composition.

[0210] 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 mol% of the total lipid content in the LNP composition.

[0211] 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 mol% of the total lipid content in the LNP composition.

[0212] 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 mol% of the total lipid content in the LNP composition.

[0213] 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 mol% of the total lipid content in the LNP composition.

[0214] 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 mol% of the total lipid content in the LNP composition.

[0215] 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 mol% of the total lipid content in the LNP composition.

[0216] 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 mol% of the total lipid content in the LNP composition.

[0217] 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 mol% of total lipid content in the LNP composition.

[0218] In some embodiments, the composition further comprises DSPC or HSPC for a total of 5 mol% of total lipid content in the LNP composition.

[0219] In some embodiments, the composition further comprises PEG-DMG for a total of 1.5 mol% of total lipid content in the LNP composition.

[0220] In some embodiments, the composition comprises DSPC / DPPC phospholipid for a total of 10 mol% of total lipid content in the LNP composition.

[0221] Some aspects of the disclosure relate to a phosphatidylserine salt selected from the group consisting of DSPS sodium, DPPS sodium, DSPS ammonium, and DPPS ammonium.

[0222] Some aspects of the disclosure relate to a DSPS-Na salt or DPPS-NH4 + Use of a salt in the manufacture of an LNP comprising a (L-serine)PS lipid, a sterol, a conjugated lipid, a phospholipid, for targeting the LNP to dendritic cells.

[0223] Some aspects of the disclosure relate to a solution comprising ethanol and DSPS or DPPS, the solution being obtained by a method comprising the step of dissolving a phosphatidylserine salt in ethanol, wherein the phosphatidylserine salt is selected from the group consisting of DSPS sodium, DPPS sodium, DSPS ammonium, and DPPS ammonium. BRIEF DESCRIPTION OF DRAWINGS

[0224] Figure 1A Effect of 0 to 2.5 mol% DSPS inclusion on transfection efficiency of dendritic cells (MutuDC1940) using mCherry mRNA LNPs formulated with DLin-KC2-DMA as ionizable cationic lipid. ICL was kept at 50 mol%, cholesterol at 38.5 mol%, PEG-DMG at 1.5 mol%, and the DSPS content was varied. DSPS was included by reducing the DSPC content by the same mol% as the added DSPS. Cells were incubated with each formulation at a concentration of 1 ug mRNA / mL for 24 hours. UT samples correspond to cells without added LNP. Lipofect refers to samples treated with Lipofectamine.

[0225] Figure 1B Effect of 0 to 7.5 mol% DSPS inclusion on transfection efficiency of dendritic cells (MutuDC1940) using mCherry mRNA LNPs formulated with DLin-KC2-DMA as ionizable cationic lipid. ICL was kept at 50 mol%, cholesterol at 38.5 mol%, PEG-DMG at 1.5 mol%, and the DSPS content was varied. DSPS was included by reducing the DSPC content by the same mol% as the DSPS added. Cells were incubated with each formulation at a concentration of 1 ug mRNA / mL for 24 hours. UT samples correspond to cells without LNP added. Lipofectamine transfection refers to samples treated with Lipofectamine.

[0226] Figure 1C Effect of 0 to 7.5 mol% DSPS inclusion on transfection efficiency of dendritic cells (MutuDC1940) using mCherry mRNA LNPs formulated with DLin-KC2-DMA as ionizable cationic lipid. ICL was kept at 50 mol%, cholesterol at 38.5 mol%, PEG-DMG at 1.5 mol%, and the DSPS content was varied. DSPS was included by reducing the DSPC content by the same mol% as the DSPS added. Cells were incubated with each formulation at a concentration of 0.3 ug mRNA / mL for 24 hours. UT samples correspond to cells without LNP added.

[0227] Figure 1D Effect of 0 to 7.5 mol% DSPS inclusion on transfection efficiency of dendritic cells (MutuDC1940) using mCherry mRNA LNPs formulated with DLin-KC2-DMA as ionizable cationic lipid. ICL was kept at 50 mol%, cholesterol at 38.5 mol%, PEG-DMG at 1.5 mol%, and the DSPS content was varied. DSPS was included by reducing the DSPC content by the same mol% as the DSPS added. Cells were incubated with each formulation at a concentration of 0.1 ug mRNA / mL for 24 hours. UT samples correspond to cells without LNP added.

[0228] Figure 2 Transfection of murine dendritic cells (MutuDC1940) using LNPs containing multiple ICLs (KC2, KC2-OA, KC3-OA, and SM-102) and 5 mol% DSPS, and compared to LNPs using Glu-DSPE or Suc-DSPE instead of DSPS. UT samples correspond to cells without LNP added.

[0229] Figure 3DSPS or DPPS improved transfection of mCherry LNPs with ICLs containing KC2, KC2-01, KC2-PA, KC3-01, and KC3-OA. UT samples correspond to cells without added LNPs.

[0230] Figure 4 Effect of PEG-DMG concentration in LNPs containing AUG-UO-1 with and without 5 mol% DSPS on transfection of dendritic cells. Y-axis shows % PEG used in the composition, followed by the concentration of mRNA added to the cells (0.11, 0.33, or 1 pg / mL). UT samples correspond to cells without added LNPs.

[0231] Figure 5A Effect of N / P on mCherry expression by LNPs containing KC2-01 in murine dendritic cells at 1 pg / ml. UT samples correspond to cells without added LNPs.

[0232] Figure 5B Effect of N / P on mCherry expression by LNPs containing KC2-01 in murine dendritic cells at 0.33 pg / ml. UT samples correspond to cells without added LNPs.

[0233] Figure 6 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.

[0234] Figure 7A mRNA sequence for VRN-029, which is a SARS-COV2 Spike protein production sequence.

[0235] Figure 7B 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 UOl, and increasing mol% of PEG-DMG. Middle graph shows endpoint antibody titers at day 34. Right graph shows corresponding CD4 T cell responses.

[0236] Figure 7CEffect 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, along 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.

[0237] Figure 7D Total anti-Spike antibody titers and CD4 responses from mice immunized with mRNA-LNPs using 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.

[0238] Figure 7E Total anti-Spike antibody titers and CD4 responses from mice immunized with mRNA-LNPs using 7.5% DSPS and the 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.

[0239] Figure 7F Effect of phosphatidylserine incorporation on the immunogenicity of mRNA-LNPs. 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.

[0240] Figure 7G Effect of phosphatidylserine tail composition (DPPS vs DSPS) on B cell responses (Panel A) and T cell responses (Panel B) to mRNA-LNP priming. Antibody data were log-transformed prior to analysis. Data were analyzed using one-way ANOVA with Tukey's multiple comparison test.

[0241] Figure 8A Comparison of mCherry expression by KC2-01 LNPs, 7.5 mol% DSPS (D isomer), and DSPS (L isomer) at 1 μg / mL mRNA for 24 hours.

[0242] Figure 8B Comparison of KC2-01 LNP, 7.5 mol% DSPS (D isomer), and DSPS (L isomer) on mCherry expression at 0.33 pg / mL mRNA for 24 hours.

[0243] Figure 9 Comparison of KC2 LNP containing 5 mol% and 7.5 mol% DSPS (L-isomer) with LNP made with SM-102 or ALC-0315 on mCherry expression at 1 pg / mL mRNA for 24 hours. Y-axis is mean fluorescence intensity (MFI). UT sample corresponds to cells with no LNP added.

[0244] Figure 10 Comparison of UOl, SM102, ALC-0315 formulation alone or with DSPS added on mCherry expression at 1 pg / mL mRNA for 24 hours. Lipo refers to Lipofectamine MessengerMax (ThermoFisher) used at the same dose level as LNP according to manufacturer’s instructions. UT sample corresponds to cells with no LNP added.

[0245] Figure 11 Oxidative degradation of liposomes containing KC3 (DLin-KC3-DMA), which is a polyunsaturated ICL with a single methylene between two alkenes, and liposomes containing ICLs with monounsaturated alkyl chains (KC3-OA, KC3-PA, or KC3-C17 (C8: l)) and a fully saturated ICL (KC3-C17). Effect of hydrogen peroxide on the stability of a single ionizable cationic lipid measured by CAD-HPLC.

[0246] Figure 12 Comparison in murine dendritic cells of LNP containing polyunsaturated KC3, monounsaturated KC3-OA, KC3-PA, or KC3 C17 (C8: l), and fully saturated KC3 C17 on mCherry expression at 0.3 or 1 pg / mL mRNA for 24 hours with or without DPPS (NH4 + salt. UT sample corresponds to cells with no LNP added.

[0247] Figure 13Comparison of LNP containing polyunsaturated KC2 or KC3 with a single methylene between the two alkenes, polyunsaturated KC3-01 with four methylenes between the two alkenes, monounsaturated KC3-OA, KC3-PA or KC3C17 (C8:1), and ALC-0315 in human dendritic cells for mCherry expression at 0.1 or 1 pg / mL mRNA for 24 hours. All LNP except ALC-0315 contain DPPS (NH4 + Untreated samples correspond to cells without added LNP.

[0248] Figure 14 Comparison of LNP formulations with 5 mol% DSPS and 46 to 54 mol% KC3-OA to ALC-0315 and SM-102 LNP controls for mCherry expression at 0.1 and 1 pg / mL mRNA for 24 hours in human dendritic cells. Untreated DC samples correspond to human dendritic cells without added LNP.

[0249] Figure 15 Comparison of LNP formulations with 0 or 5 mol% DSPS and 50 mol% KC2-O1 at an N / P ratio of 4 to 7 for mCherry expression at 0.1 pg / mL mRNA for 24 hours in human dendritic cells. These are also compared to LNP containing KC3-OA and 5 mol% DSPS at an N / P ratio of 5. Untreated DC samples correspond to human dendritic cells without added LNP.

[0250] Figure 16A Comparison of LNP formulations containing polyunsaturated KC3 with monounsaturated KC3-OA and KC3-PA for vaccine immunogenicity. Total anti-spike antibody titers from mice immunized with mRNA-LNP using LNP containing KC3, KC3-OA or KC3-PA targeted with 5 mol% DSPS or DPPS. For KC3-OA and KC3-PA LNP, each formulation was also evaluated with a C16 DPPC or C18 DSPC neutral phosphatidylcholine component. All LNP contained 1.5 mol% PEG-DMG. The figure shows endpoint antibody titers on day 21 after an initial priming injection of 1 pg mRNA / mouse.

[0251] Figure 16BComparison of LNP formulations comprising polyunsaturated KC3 with monounsaturated KC3-OA and KC3-PA on vaccine immunogenicity. Total anti-spike antibody titers from mice immunized with mRNA-LNP comprising KC3, KC3-OA or KC3-PA LNP targeted with 5 mol% DSPS or DPPS. Each formulation was also evaluated with a C16 DPPC or C18 DSPC neutral phosphatidylcholine component for KC3-OA and KC3-PA LNP. All LNP contained 1.5 mol% PEG-DMG. The graph shows endpoint antibody titers at day 34 after a prime at 1 pg mRNA / mouse followed by a boost at day 21.

[0252] Figure 17A Comparison of mCherry expression at 1 pg / mL mRNA for 24 hours in human dendritic cells for LNP formulations with 5 mol% DSPS and 43 to 48 mol% KC3-OA versus ALC-0315 and SM-102 LNP controls. KC3-OA LNP prepared with 45 mol% KC3-OA and 5 mol% DSPS at N / P ratios of 5, 5.5, 6.0 and 6.5 were also compared. Finally, LNP with 45 mol% KC3-OA at N / P of 5 and 6 were evaluated with 1 or 3 mol% PEG-SA replacing 1.5 mol% PEG-DMG. Untreated DC samples correspond to human dendritic cells without added LNP.

[0253] Figure 17B Comparison of mCherry expression at 0.1 pg / mL mRNA for 24 hours in human dendritic cells for LNP formulations with 5 mol% DSPS and 43 to 48 mol% KC3-OA versus ALC-0315 and SM-102 LNP controls. KC3-OA LNP prepared with 45 mol% KC3-OA and 5 mol% DSPS at N / P ratios of 5, 5.5, 6.0 and 6.5 were also compared. Finally, LNP with 45 mol% KC3-OA at N / P of 5 and 6 were evaluated with 1 or 3 mol% PEG-SA replacing 1.5 mol% PEG-DMG. Untreated DC samples correspond to human dendritic cells without added LNP.

[0254] Figure 18AComparison of mCherry expression of LNP formulations with 0-10 mol% DSPG containing UO-1 or KC3-01 in human dendritic cells after 24 hours incubation at 1 pg / mL mRNA. ALC-0315 and SM-102 LNP controls at 1 pg / mL mRNA are also included and untreated DC samples correspond to human dendritic cells without added LNP.

[0255] Figure 18B Comparison of mCherry expression of LNP formulations with 0-10 mol% DSPG containing UO-1 or KC3-01 in human dendritic cells after 24 hours incubation at 0.1 pg / mL mRNA. ALC-0315 and SM-102 LNP controls at 0.1 pg / mL mRNA are also included and untreated DC samples correspond to human dendritic cells without added LNP.

[0256] Figure 19 Comparison of LNP formulations containing dilinoleyl KC2, monounsaturated KC3-OA, and four methylene-interrupted polyunsaturated ICLs (KC3-01, AKG-UO1, and AKG-UO9) on vaccine immunogenicity. LNP containing ALC-0315 is included as a control. All LNPs contain 1.5 mol% PEG-DMG. Total anti-spike antibody titers from mice immunized with mRNA-LNP were determined on day 21 after an initial priming injection of 1 pg mRNA / mouse on day 1.

[0257] Figure 20A Comparison of mCherry expression of LNP formulations with 5 mol% various anionic phospholipids containing 48 mol% KC3-OA in human dendritic cells after 24 hours incubation at 1 pg / mL mRNA. All LNPs contain 2.5 mol% DSPC, 50 mol% UO-1, and 1.5 mol% PEG-DMG. Anionic phospholipids include phosphatidylglycerol, DOPG, DSPG, DPPG, and DMPG, and DSPS. In some LNPs, DSPG and DSPS are combined individually, or with DSPC. In this study, two donors were used to generate human dendritic cells and untreated DC samples correspond to human dendritic cells without added LNP.

[0258] Figure 20BComparison of mCherry expression for LNP formulations with 5 mol% of various anionic phospholipids including 48 mol% KC3-OA in human dendritic cells after 24 hours incubation at 0.1 pg / mL mRNA. All LNPs include 2.5 mol% DSPC, 50 mol% UO-1, and 1.5 mol% PEG-DMG. Anionic phospholipids include phosphatidylglycerol, DOPG, DSPG, DPPG, and DMPG, and DSPS. In some LNPs, DSPG and DSPS are combined alone, or with DSPC. In this study, two donors were used to generate human dendritic cells, and untreated DC samples correspond to human dendritic cells without added LNP.

[0259] Figure 21 Comparison of mCherry expression for LNP formulations including KC3-OA LNPs with 5 mol% DSPS (Na + salt) or 5 mol% DPPS (NH4 + salt) in murine dendritic cells after 24 hours incubation at 1 pg / mL mRNA. Also included are ALC-0315 and SM-102 LNP controls at 1 pg / mL mRNA. UT samples correspond to cells without added LNP.

[0260] Figure 22A Immunogenicity of mRNA-LNP vaccines encoding Mtb antigens including four unique signal peptides, and comparison of KC3OA / DPPS and ALC-0315 LNP formulations. Mtb-specific CD4 T cells were defined as any cells producing any of these 3 cytokines after peptide stimulation.

[0261] Figure 22B Immunogenicity of mRNA-LNP vaccines encoding Mtb antigens including four unique signal peptides, and comparison of KC3OA / DPPS and ALC-0315 LNP formulations. Mtb-specific CD8 T cells were identified as any cells producing IFN-γ; CD8 T cells producing TNF-a and IL-2 were present in the population producing IFN-γ.

[0262] Figure 23A Proportion of total vaccine-induced T cell responses for individual Mtb antigens or Mtb antigen subsets. Data correspond to cumulative T cell responses shown in Figure 22, and normalized to 100%. CD4 T cell response profiles using ALC-0315 comparator.

[0263] Figure 23BProportion of total vaccine-induced T cell responses against individual Mtb antigens or subsets of Mtb antigens. Data correspond to cumulative T cell responses shown in Figure 22 and normalized to 100%. CD4 T cell response profile using KC3-OA / DPPS LNP formulation.

[0264] Figure 23C Proportion of total vaccine-induced T cell responses against individual Mtb antigens or subsets of Mtb antigens. Data correspond to cumulative T cell responses shown in Figure 22 and normalized to 100%. CD8 T cell response profile using ALC-0315 comparator.

[0265] Figure 23D Proportion of total vaccine-induced T cell responses against individual Mtb antigens or subsets of Mtb antigens. Data correspond to cumulative T cell responses shown in Figure 22 and normalized to 100%. CD8 T cell response profile using KC3-OA / DPPS LNP formulation.

[0266] Figure 24A Cytokine polyfunctionality of vaccine-specific CD4 T cells, where CD4 T cell responses were induced by mRNA incorporating sec / MITD targeting nascent protein to endosomal compartments and signal peptide / transmembrane domain into LNP formulation. Boolean gated serial CD4 T cell responses across peptide pools for cells producing IFN-g, IL-2, and TNF-a. SP, single producer; DP, double producer; TP, triple producer.

[0267] Figure 24B Cytokine polyfunctionality of vaccine-specific CD4 T cells, where CD4 T cell responses were induced by mRNA incorporating LAMP-1 targeting nascent protein to late endosomal / lysosomal compartments into LNP formulation. Boolean gated serial CD4 T cell responses across peptide pools for cells producing IFN-g, IL-2, and TNF-a. SP, single producer; DP, double producer; TP, triple producer.

[0268] Figure 24CCytokine polyfunctionality of vaccine-specific CD4 T cells, where CD4 T cell responses were induced by mRNA that used a tPA signal peptide to direct protein secretion into LNP formulations. Boolean gating of IFN-g, IL-2, and TNF-a producing cells was performed on concatenated CD4 T cell responses across peptide pools. SP, single producer; DP, double producer; TP, triple producer.

[0269] Figure 25A Total CD4 T cell responses (cells IFN-g) induced by mRNA delivered with KC3-OA / DPPS or ALC-0315 LNP formulations. Data correspond to cumulative T cell responses shown in Figure 22. Mtb-specific T cell responses were concatenated across peptide pools.

[0270] Figure 25B Total CD8 T cell responses (cells IFN-g) induced by mRNA delivered with KC3-OA / DPPS or ALC-0315 LNP formulations. Data correspond to cumulative T cell responses shown in Figure 22. Mtb-specific T cell responses were concatenated across peptide pools.

[0271] Figure 26A Comparison of BCG (s.c.) versus KC3-OA / DPPS LNP (i.m.) CD4 T cell responses to individual Mtb antigens. Stim 1: EsxH / TB10.4 and Ag85B peptide pools, Stim 2: Mtb39a peptide pool, Stim 3: EsxW and EsxV peptide pools, Stim 4: EsxB / CFP10 and EsxA / ESAT-6 peptide pools, Stim 5: Peptide pool of the group of ten concatenated 15-mer minimal epitopes at the C-terminus. Mtb-specific CD4 T cells were defined as cells expressing IFN-g, TNF-a, IL-2, IL-17a, or a combination thereof.

[0272] Figure 26B Comparison of BCG (s.c.) versus KC3-OA / DPPS LNP (i.m.) CD4 T cell responses. Cumulative CD4 T cell responses to all peptide stimulation (Sim 1 + Stim 2 + Stim 3 + Stim 4 + Stim 5 - background). Mtb-specific CD4 T cells were defined as cells expressing IFN-g, TNF-a, IL-2, IL-17a, or a combination thereof.

[0273] Figure 27AComparison of CD8 T cell responses to single Mtb antigens by BCG (s.c.) versus KC3-OA / DPPS LNP (i.m.). Stim 1 : EsxH / TB10.4 and Ag85B peptide pools combined, Stim 2: Mtb39a peptide pool, Stim 3: EsxW and EsxV peptide pools combined, Stim 4: EsxB / CFP10 and EsxA / ESAT-6 peptide pools combined, Stim 5: Peptide pool of the group of ten C-terminal ten-in-one 15-mer miniepitopes. Mtb-specific CD8 T cells express IFN-γ, TNF-a, IL-2, IL-17a, or a combination thereof.

[0274] Figure 27B Comparison of CD8 T cell responses by BCG (s.c.) versus KC3-OA / DPPS LNP (i.m.). Cumulative CD8 T cell responses to all peptide stimulations (Sim 1 + Stim 2 + Stim 3 + Stim 4 + Stim 5 - background).

[0275] Figure 28 Vaccination with three different mRNA constructs encoding putative human MHC class I restricted Mtb epitopes in string-on-bead format generates antigen-specific CD8 T cell responses in CB6F1 mice.

[0276] Figure 29A Kinetics of vaccine-specific T cell responses. CB6F1 mice were immunized with mRNA encoding HLA-II directed fusion proteins consisting of 7 Mtb proteins plus 10 miniepitopes encapsulated in KC3-OA / DPPS LNP and boosted after 4 weeks; the antigens were flanked by sec / MITD sequences (SEQ ID NO. 37 and 38). Cumulative CD4 and CD8 T cell responses to all peptide pools are shown.

[0277] Figure 29B Kinetics of vaccine-specific CD8 T cell responses after immunization with mRNA encoding putative human MHC class I Mtb epitopes encapsulated in KC3-OA / DPPS LNP and boosting after 4 weeks. Cumulative CD8 T cell responses to all peptide pools are shown.

[0278] Figure 30AComparison of CD4 T cell responses between first and second generation HLA-II Mtb mRNA vaccine constructs encapsulated in KC3-OA / DPPS LNPs showing the total CD4 T cell responses (sum of all individual peptide pools minus background) accumulated after immunization of CB6F1 mice. One group was vaccinated with mRNA formulated with KC3-OA / DPPS LNPs containing an increased amount of 15 mol% DSPC compared to the typical 5 mol% DSPC at the expense of cholesterol.

[0279] Figure 30B Comparison of CD4 T cell responses between first and second generation HLA-II Mtb mRNA vaccine constructs encapsulated in KC3-OA / DPPS LNPs showing the proportion of total vaccine-induced CD4 T cell responses against a single Mtb antigen or Mtb antigen subset encoded by the mRNA after immunization of CB6F1 mice. Data correspond to the cumulative T cell responses shown in (Figures 13A-13D) and normalized to 100%. One group was vaccinated with mRNA formulated with KC3-OA / DPPS LNPs containing an increased amount of 15 mol% DSPC compared to the typical 5 mol% DSPC at the expense of cholesterol. Figure 30A

[0280] Figure 31A Comparison of CD8 T cell responses between first and second generation HLA-II Mtb mRNA vaccine constructs encapsulated in KC3-OA / DPPS LNPs showing the total CD8 T cell responses (sum of all individual peptide pools minus background) accumulated after immunization of CB6F1 mice. One group was vaccinated with mRNA formulated with KC3-OA / DPPS LNPs containing an increased amount of 15 mol% DSPC compared to the typical 5 mol% DSPC at the expense of cholesterol.

[0281] Figure 31B Comparison of CD8 T cell responses between first and second generation HLA-II Mtb mRNA vaccine constructs encapsulated in KC3-OA / DPPS LNPs showing the proportion of total vaccine-induced CD8 T cell responses against a single Mtb antigen or Mtb antigen subset encoded by the mRNA after immunization of CB6F1 mice. Data correspond to the cumulative T cell responses shown in (Figures 14A-14D) and normalized to 100%. One group was vaccinated with mRNA formulated with KC3-OA / DPPS LNPs containing an increased amount of 15 mol% DSPC compared to the typical 5 mol% DSPC at the expense of cholesterol. Figure 31A ​) and normalized to 100%. One group was vaccinated with mRNA formulated with KC3-OA / DPPS LNPs comprising an increased amount of 15 mol% DSPC (at the expense of cholesterol) compared to the typical 5 mol% DSPC.

[0282] Figure 32 is a scheme showing the synthesis of 2-((S)-2,2-di((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine (AKG-KC2-01) and 3-((S)-2,2-di((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine (AKG-KC3-01) according to some embodiments of the disclosure.

[0283] Figure 33 is a scheme showing the synthesis of 2-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine (AKG-KC2-OA), 2-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine (AKG-KC2-PA), 3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine (AKG-KC3-OA), and 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) according to some embodiments of the disclosure.

[0284] Figure 34 is a scheme showing the synthesis of 3-((S)-2,2-di((Z)-heptadec-8-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine, AKG-KC3-C17 (C8:1) and (S)-3-(2,2-bisheptadecyl-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine, AKG-KC3-C17 according to some embodiments of the disclosure.

[0285] Figure 35AComparison of splenic CD4 T cell responses in CB6F1 mice after vaccination with second generation HLA-II Mtb mRNA formulated in LNPs containing increasing amounts of phospholipid (PL). DPPS was kept constant at 5 mol% and the remaining mol% of PL consisted of DSPC. The increase in DSPC content was at the expense of cholesterol.

[0286] Figure 35B Comparison of splenic CD8 T cell responses in CB6F1 mice after vaccination with second generation HLA-II Mtb mRNA formulated in LNPs containing increasing amounts of PL. The CD8 T cell responses were quantified in the same mice as in Figure 35A

[0287] Figure 35C Comparison of splenic CD4 T cell responses in CB6F1 mice after vaccination with second generation HLA-II Mtb mRNA formulated in LNPs containing increasing amounts of PL. DPPS was kept constant at 5 mol% and the remaining mol% of PL consisted of DSPC. One group was immunized with mRNA produced without modified uridine formulated in 25 mol% PL; all other groups received mRNA in which all uridines were replaced by N1-methyl pseudouridines.

[0288] Figure 35D Comparison of splenic CD8 T cell responses in CB6F1 mice after vaccination with second generation HLA-II Mtb mRNA formulated in LNPs containing increasing amounts of PL. The CD8 T cell responses were quantified in the same mice as in Figure 35C

[0289] Figure 36A Comparison of splenic CD8 T cell responses in CB6F1 mice after vaccination with second generation HLA-I “mixed” mRNA formulated in LNPs containing increasing amounts of PL. DPPS was kept constant at 5 mol% and the remaining mol% of PL consisted of DSPC.

[0290] Figure 36B Comparison of splenic CD8 T cell responses in CB6F1 mice after vaccination with second generation HLA-I “Mtb only” mRNA formulated in LNPs containing increasing amounts of PL. DPPS was kept constant at 5 mol% and the remaining mol% of PL consisted of DSPC. ​​DETAILED DESCRIPTION

[0291] 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 present disclosure.

[0292] Liposome nanoparticle (LNP) compositions can comprise an ionizable lipid, a sterol, and one or more phospholipids. In some embodiments, the LNP compositions further comprise a nucleic acid, such as mRNA, for administration in a pharmaceutical composition, such as a vaccine. In some embodiments, the LNP compositions optionally further comprise a conjugated lipid.

[0293] Lipid nanoparticle (LNP) compositions comprising mRNA include Stabilized Nucleic Acid Lipid Particles (SNALP) that are used as carriers for the systemic delivery of mRNA or other nucleic acid therapeutics. SNALP compositions comprise a cationic lipid such as MC3 or KC2, which comprises a protonatable tertiary amine head group linked to a pair of straight-chain 18-carbon aliphatic chains comprising a pair of carbon-carbon double bonds separated by a single methylene group (e.g., linoleic acid). However, while the structure of these hydrocarbon chains, each comprising a pair of double bonds separated by a single methylene group, confers the desirable biological properties on SNALP compositions, this chemical substructure also results in the undesirable problem of increased sensitivity of the compounds to oxidative degradation. What is needed are new cationic lipids suitable for use in SNALP compositions but with enhanced resistance to oxidative degradation.

[0294] Some aspects of the present disclosure relate to lipid nanoparticle (LNP) targeting dendritic cells incorporating mRNA encoding a combination of specific CD8 and CD4 T cell epitopes present in Mycobacterium tuberculosis. In some embodiments, LNP comprising one or more cationic ionizable lipids can be used for delivery of mRNA, for targeting dendritic cells, or methods using these LNP compositions as vaccines for the prevention of tuberculosis or other mycobacterial infections. In some embodiments, the LNP can comprise phosphatidylserine or phosphatidylglycerol as targeting ligands to improve their recognition and activity in dendritic cells. In some embodiments, the mRNA is optimized for presentation of MHC-I epitopes and activation of CD8 T cells, while in other embodiments, the mRNA is optimized for presentation of MHC-II epitopes and activation of CD4 T cells. In some embodiments, the LNP vaccine incorporates mRNA sequences optimized for both MHC-I and MHC-II.

[0295] Disclosed herein are compounds, compositions, and methods related to treating mycobacterial infections. As used herein, the terms "compound," "drug," and "active agent" are used interchangeably. Some aspects of the disclosure relate to novel ionizable lipids or bioreducible ionizable lipids. These lipids are cationic (i.e., positively charged) at acidic pH, such as encountered intracellularly after endocytosis or phagocytosis by a cell. The same lipids and compositions comprising them are near neutral in charge when present at pH 7.4. These lipids can also have a single alkene group present in their alkyl or acyl groups.

[0296] Other aspects relate to compositions comprising a lipid nanoparticle comprising an ionizable cationic lipid, the lipid nanoparticle comprising a nucleic acid. In some embodiments, the nucleic acid is encapsulated into the lipid nanoparticle.

[0297] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid, a sterol, one or more phospholipids comprising at least one anionic phospholipid, a conjugated lipid, and one or more nucleic acid sequences encoding T cell epitopes from Mycobacterium tuberculosis (Mtb). In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid, a sterol, one or more phospholipids comprising at least one anionic phospholipid, a conjugated lipid, and one or more nucleic acid sequences encoding Mycobacterium tuberculosis antigens recognized by T cells.

[0298] Other aspects of the disclosure relate to lipid nanoparticles or targeted lipid nanoparticles incorporating mRNA encoding major histocompatibility complex class I (MHC-I) or class II (MHC-II) epitopes. In some embodiments, mRNA encoding MHC-I and MHC-II epitopes are incorporated into a single LNP vaccine. In some embodiments, epitopes present in Mycobacterium tuberculosis are enriched when compared to BCG or nontuberculosis mycobacterium (NTM). In some embodiments, the epitopes in the mRNA cassette are linked with non-immunogenic linkers. In other embodiments, the linkages between epitopes have been optimized to reduce the propensity to form neoepitopes.

[0299] Some aspects of the present disclosure provide improved compositions of 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 dendritic cells. Further incorporation of gemini ionizable lipids with a monounsaturated alkyl chain (mono-olefin) at the 2-position of the 1,3-dioxolane or ketal in LNP formulations show high levels of transfection in human dendritic cells and show good stability to oxidative damage compared to other ionizable lipids in the same family.

[0300] Definitions

[0301] For convenience, certain terms used in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0302] The following terms and phrases used herein are intended to have the following meanings:

[0303] As used herein, a noun clause without a quantifier is used to mean one or more than one (i.e., at least one) grammatical object. For example, “element” means one element or more than one element.

[0304] The term “comprising” or “including” and variations thereof as used herein is used to mean that the compositions, methods, and respective components thereof, exist in given embodiments, but also open-ended that additional elements not specified can also be present.

[0305] The term “consisting essentially of as used herein means those elements required for a given embodiment. This term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic of that embodiment of the disclosure.

[0306] The term “consisting of as used herein means the compositions, methods, and respective components thereof, as described herein, that do not include any element not recited in the description of the embodiments.

[0307] The term “comprising” as used in the specification includes “consisting of and “consisting essentially of.

[0308] If reference is made in this specification to “as noted above” or “as described above” or “the above”, reference is made to any page in the present specification where such textual material is found.

[0309] If reference is made in this specification to "as described herein," "thus described herein," "provided herein," or "as described in this text," or "as specified herein," reference is made to any page or pages in this specification front matter or back matter.

[0310] The term "about" as used herein means acceptable variations within 20%, within 10%, and within 5% of the stated value. In certain embodiments, "about" can mean + / - 1%, 2%, 3%, 4%, 5%, 10%, or 20% variation.

[0311] The term "effective amount" as used herein in reference to a compound or composition means an amount of active compound (also referred to herein as an active agent or a drug) sufficient to produce a bactericidal or bacteriostatic effect. In some embodiments, an effective amount is a "therapeutically effective amount," which means an amount of active compound sufficient to reduce the symptoms of a bacterial infection being treated.

[0312] The term "subject" (or alternatively "patient") as used herein refers to an animal, preferably a mammal, most preferably a human, who receives prophylactic or therapeutic treatment.

[0313] The term "administering" or variations thereof, as used herein, include all means of introducing a compound or pharmaceutical composition to a subject in need thereof, including, but not limited to, orally, intravenously, intramuscularly, intraperitoneally, subcutaneously, transdermally, by inhalation, buccally, ocularly, sublingually, vaginally, rectally, and the like. The administering of the compound or composition is suitably parenteral. For example, the compound or composition can be preferably administered intravenously, but can also be administered intraperitoneally or by inhalation, similar to the use of liposomal amikacin currently in clinical treatment for Mycobacterium avium (see Shirley et al., Amikacin Liposome Inhalation Suspension: A Review in Mycobacterium avium Complex Lung Disease. Drugs. 2019 Apr; 79(5):555-562).

[0314] The terms "treat," "treatment," and variations thereof, as used herein, refer to therapeutic or preventative measures, such as those described herein.

[0315] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present disclosure that retains the desired pharmacological activity of the parent compound.

[0316] The term "alkyl" means a saturated carbon chain having one to twenty carbon atoms, which can be straight-chained or branched or a combination thereof, unless the carbon chain is otherwise defined. Some examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, and t-butyl, pentyl, hexyl, heptyl, octyl, and the like. Unless specifically stated otherwise in the specification, alkyl groups are optionally substituted.

[0317] Unless specified otherwise in a particular example, the term "phosphatidylserine" and any of its acyl chain constituents refers to the L-isomer of serine in the head group.

[0318] The term "lipid conjugate" refers to a conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to: a polysarcosine (see, e.g., WO2021191265A1, which is incorporated by reference in its entirety for all purposes); a polyamide oligomer (e.g., an ATTA-lipid conjugate); a PEG-lipid conjugate, such as PEG coupled to dialkyloxypropyl, PEG coupled to diacylglycerol, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, PEG conjugated to ceramide (see, e.g., U.S. Patent No. 5,885,613, the disclosure of which is incorporated by reference in its entirety for all purposes); a cationic PEG lipid; and mixtures thereof. The PEG can be conjugated directly to the lipid, or can be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling PEG to a lipid can be used, including, e.g., non-ester-containing linker moieties and ester-containing linker moieties. In some preferred embodiments, a non-ester-containing linker moiety is used.

[0319] In embodiments, the abbreviation for ionizable cationic lipids can be taken from the abbreviations used in the table. For example, AKG-UO-1 can be referred to as UOl:

[0320]

[0321] For example, AKG-KC2-01 can be referred to as KC2-01.

[0322] The abbreviation UT used in various studies refers to an untreated sample.

[0323] The term "lipid nanoparticle" or "LNP" refers to a particle with a diameter of about 5 to 500 nm. In some embodiments, a lipid nanoparticle comprises one or more active agents. In some embodiments, a lipid nanoparticle comprises a nucleic acid. In some embodiments, the nucleic acid condenses inside the nanoparticle, which has a cationic lipid, a polymer or a multivalent small molecule, and an external lipid coating that interacts with the biological environment. Nucleic acids are naturally rigid polymers and prefer elongated conformation due to repulsive forces between phosphate groups. In cells, to cope with volume constraints, DNA can condense itself with the help of ions and other molecules under appropriate solution conditions. Generally, DNA condensation is defined as the collapse of elongated DNA strands into compact, ordered particles containing only one or a few molecules. By binding to phosphate groups, cationic lipids can condense DNA by neutralizing the phosphate charge and allowing it to pack tightly.

[0324] In some embodiments, the active agent is encapsulated in the 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 RNAs, and small interfering RNAs); nucleoproteins; peptides; nucleic acids; ribozymes; DNA-containing nucleoproteins, such as intact or partially deproteinized viral particles (virions); oligomeric and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins). In some embodiments, the active agent can be mixed with an excipient.

[0325] In LNP vaccine products, the active agent is typically contained inside the LNP. In some embodiments, the active agent comprises a nucleic acid. Generally, water-soluble nucleic acids condense with cationic lipids or polycationic polymers in the interior of the particle, and the particle surface is rich in neutral lipids or PEG-lipid derivatives. Additional ionizable cationic lipids can also be located at the surface and respond to acidification in the environment by becoming positively charged, facilitating endosomal escape.

[0326] With respect to LNPs, ionizable lipids can have different properties or functions. Due to the pKa of the amino group, the lipid molecule can become positively charged under acidic conditions. Under these conditions, the lipid molecule can electrostatically bind to the phosphate groups of the nucleic acid, which allows for the formation of LNPs and entrapment of the nucleic acid. In some embodiments, the pKa can be low enough that the surface charge of the LNP is essentially neutral in biological fluids such as blood at physiological pH. High LNP surface charge is associated with toxicity, rapid clearance from circulation by both fixed and free macrophages, hemolytic toxicity, including immune activation (Filion et al Biochim Biophys Acta. 1997 Oct 23; 1329(2):345-56).

[0327] In some embodiments, the pKa can be high enough such that the ionizable cationic lipid can adopt a positively charged form at the acidic endosomal pH. In this way, the cationic lipid can combine with endogenous endosomal anionic lipids to promote membrane lytic non-bilayer structures such as hexagonal HII phase, leading to more efficient intracellular delivery. In some embodiments, the pKa ranges from 6.2 to 7.5. For example, the pKa can be about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. The unsaturated tail also contributes to the ability of the lipid to adopt non-bilayer structures. (Jayaraman et al., Angew Chem Int Ed Engl. 2012 Aug 20; 51(34): 8529-33).

[0328] The release of nucleic acid from the LNP formulation, as well as characteristics such as liposome clearance and circulation half-life, can be improved by the presence of polyethylene glycol and / or a sterol (e.g., cholesterol) or other potential additives in the LNP, as well as the overall chemical structure, including the pKa of any ionizable cationic lipid included as part of the formulation.

[0329] The terms "encapsulated" and "entrapped" as used herein refer to the incorporation or association of an mRNA, DNA, siRNA, or other nucleic acid agent into a lipid nanoparticle. The term "encapsulated" as used herein refers to complete encapsulation or partial encapsulation. siRNAs can be capable of selectively knocking down or down-regulating the expression of a gene of interest. For example, siRNAs can be selected to silence a gene associated with a particular disease, disorder, or condition following administration of a nanoparticle composition comprising the siRNA to a subject in need thereof. The siRNA can comprise a sequence that is complementary to an mRNA sequence encoding a gene or protein of interest.

[0330] The term "mol%" with respect to cholesterol refers to the mole amount of cholesterol expressed as a percentage of the total of the mole amounts of cholesterol and non-PEGylated phospholipid. 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.

[0331] The term "mol%" with respect to PEG-lipid refers to the ratio of the mole amounts of PEG-lipid and non-PEGylated phospholipid expressed as a percentage. For example, "5 mol.% PEG-DSPE" in an LNP comprising HSPC and PEG-DSPE refers to a composition having 5 mol. parts PEG-DSPE / 100 mol. parts HSPC.

[0332] The term "pharmaceutically acceptable carrier, diluent or excipient" as used herein includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening, diluting, preserving, coloring, flavoring, surface-active, wetting, dispersing, suspending, stabilizing, isotonic, solvent, or emulsor agent approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0333] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to mean a sequence polymer of at least two amino acids covalently linked by amide bonds (also referred to herein as peptide bonds).

[0334] "Identity" as known in the art is a relationship between two or more polypeptide sequences or protein sequences or nucleic acid sequences, as determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence relatedness between polypeptides or proteins, as determined by the match between such sequence strings. "Identity" can be readily calculated by any bioinformatics method known in the art. "Percent (%) identity" is defined as the percentage of residues in the candidate amino acid sequence or nucleic acid sequence that are identical with the amino acid sequence or nucleic acid sequence in the second sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity.

[0335] The term "substantially identical" or "substantially similar" as used herein, when referring to nucleic acids or fragments thereof, means having nucleotide sequence identity in at least about 95% to 99% of the sequence when optimally aligned with appropriate nucleotide insertions or deletions with the other nucleic acid (or its complementary strand). The term "substantially identical" or "substantially similar" as used herein, when referring to proteins or fragments thereof, means having amino acid sequence identity in at least about 95% to 99% of the sequence when optimally aligned.

[0336] Aspects and embodiments are further described in the following subsections.

[0337] Ionizable cationic lipids

[0338] Provided herein are compounds useful for preparing lipid nanoparticle (LNP) compositions.

[0339] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable lipid having a chemical structure consisting of: a straight-chain polyunsaturated lipid tail pair covalently bound to a head group, the head group comprising a dialkylamino group; the head group comprising a heterocyclyl or alkyl moiety covalently bound to the dialkylamino group, and optionally further comprising a phosphate group; and each polyunsaturated lipid tail being unsaturated except for at least two olefins along the length of the lipid tail that are separated by at least two methylenes, and optionally comprising a single acyl group covalently bound to the head group at the end of the lipid tail. In some aspects, each lipid tail in the ionizable lipid is identical, and each lipid tail has a total of two olefins separated by only unsubstituted ethylene, n-propyl, or n-butyl groups. In some embodiments, each lipid tail of the ionizable lipid further comprises an acyl group connected to an oxygen of the head group to form an ester, and has a total of 16 or 18 carbon atoms including the acyl group.

[0340] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having a chemical structure of Formula (I):

[0341]

[0342] wherein:

[0343] R1is

[0344] 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;

[0345] R2and R3are each independently (C1-C4)alkyl optionally substituted with hydroxyl; and

[0346] n is an integer equal to 2, 3, or 4.

[0347] 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 R1hydrocarbon chain is C 15 -C 18 In some embodiments, the total length of the R1hydrocarbon chain is C 16 -C 18 In some embodiments, the total length of the R1hydrocarbon chain is C 16 or C 18 .

[0348] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein a is 0 and b is 1, 2, 3, or 4. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein a is 0 and b is 1 or 3. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein a is 0 and b is 1. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein a is 0 and b is 3.

[0349] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein a is 1 and b is 1, 2, 3, or 4. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein a is 1 and b is 1 or 3. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein a is 1 and b is 1. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein a is 1 and b is 3.

[0350] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein R 10 and R 12 are each independently (C1-C4)alkyl. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein R 10 and R 12 are each independently (C1-C4)alkyl. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein R 10 and R 12 are each independently (C1-C4)alkyl. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein R 10 and R 12 are each independently (C1-C4)alkyl. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I), wherein R 10 and R 12 are each independently (C1-C4)alkyl. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the 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 Each is independently selected from methyl, ethyl, -(CH2)(CH2)OH and -(CH2)2(CH2)OH.

[0351] 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.

[0352] In some embodiments, the ionizable cationic lipid comprises the chemical structure of Formula (II) or a pharmaceutically acceptable salt thereof:

[0353]

[0354] in:

[0355] Y is

[0356] n is an integer 2, 3, or 4;

[0357] R 22 is a single olefin with a total length of C 15 -C 18 hydrocarbon chains, and

[0358] R 10 and R 12 Each is independently a (C1-C4)alkyl group optionally substituted with hydroxy.

[0359] In some aspects, R in formula (II)22 is a polyene chain of Formula A.

[0360] In some aspects, R 10 and R 12 are each independently selected from the group consisting of methyl, ethyl, propyl, -(CH2)(CH2)OH, and -(CH2)2(CH2)OH. In some aspects, R 10 and R 12 are each independently methyl. In some aspects, R 10 and R 12 are each independently ethyl. In some aspects, R 10 and R 12 are each independently selected from the group consisting of methyl, ethyl, -(CH2)(CH2)OH, and -(CH2)2(CH2)OH. In some aspects, R 10 is methyl, and R 12 is selected from the group consisting of methyl, ethyl, -(CH2)(CH2)OH, and -(CH2)2(CH2)OH. In some aspects, R 10 is methyl, and R 12 is selected from the group consisting of -(CH2)(CH2)OH, and -(CH2)2(CH2)OH. In some aspects, in a compound comprising the chemical structure of Formula (II), R 10 is methyl, and R 12 is selected from the group consisting of -(CH2)(CH2)OH, and -(CH2)2(CH2)OH. In some aspects, R 10 and R 12 are independently selected from the group consisting of methyl or ethyl optionally substituted with one or more hydroxyl groups. In some aspects, in Formula (II), one or both of R 10 and R 12 is -(CH2)(CH2)OH or -(CH2)2(CH2)OH. In some aspects, R 10 is methyl, and R 12 is methyl or ethyl substituted with a hydroxyl group. In some aspects, one or both of R 10 is methyl, and R 12 is -(CH2)(CH2)OH. In some aspects, one or both of R 10 is methyl, and R 12 is -(CH2)2(CH2)OH.

[0361] In some embodiments, the compound has the structure of a compound listed in the following table. Table 1A shows some examples of cationic lipids.

[0362] Table 1A. Exemplary cationic lipids

[0363]

[0364] In some embodiments, the LNP composition comprises a KC3 ionizable cationic lipid. The term “KC3 ionizable cationic lipid” as used herein, unless otherwise indicated, refers to an ionizable cationic lipid having the following chemical formula:

[0365]

[0366] where each R1is the same or different, and is a linear C 15 to C 19 hydrocarbon chains, each comprising one or more unsaturated alkenyl double bonds within each polyolefin chain; R2and R3are each independently methyl; and n is 3. In some aspects, each R1in the KC3 ionizable cationic lipid is the same or different, and is a linear C 16 or C 18 hydrocarbon chains, each comprising one or more unsaturated alkenyl double bonds within each polyolefin chain.

[0367] In some embodiments, the LNP composition comprises a KC4 ionizable cationic lipid. The term “KC4 ionizable cationic lipid” as used herein, unless otherwise indicated, refers to an ionizable cationic lipid having the following chemical formula:

[0368]

[0369] where each R1is the same or different, and is a linear C 16 or C 18 hydrocarbon chains, each comprising one or more unsaturated alkenyl double bonds within each polyolefin chain; R2and R3are each independently methyl; and n is 4.

[0370] In some aspects, each R1in the KC3 or KC4 ionizable cationic lipid is the same or different, and is:

[0371]

[0372] 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. In some aspects, each R1in the KC3 or KC4 ionizable cationic lipid is the same or different, and is:

[0373]

[0374] wherein a is 1, and b is 1 or 3. In some aspects, each R1in the KC3or KC4ionizable cationic lipid is the same or different, and is

[0375]

[0376] wherein a is 1, and b is 1. In some aspects, each R1in the KC3or KC4ionizable cationic lipid is the same or different, and is:

[0377]

[0378] wherein a is 1, and b is 3.

[0379] In some aspects, each R1in the KC3or KC4ionizable cationic lipid is the same or different, and is:

[0380]

[0381] wherein a is 0, and b is 1 or 3. In some aspects, each R1in the KC3or KC4ionizable cationic lipid is the same or different, and is:

[0382]

[0383] wherein a is 0, and b is 1. In some aspects, each R1in the KC3or KC4ionizable cationic lipid is the same or different, and is

[0384]

[0385] wherein a is 0, and b is 3.

[0386] In some aspects, each R1in the KC3or KC4ionizable cationic lipid is the same or different, and is:

[0387]

[0388] wherein:

[0389] a is 1, 2, 3, or 4;

[0390] b is 2, 3, or 4; and

[0391] c is 3, 4, 5, 6, or 7,

[0392] with the proviso that the sum of a, b, and c is 10, 11, 12, or 13.

[0393] In some aspects, each R1in the KC3or KC4ionizable cationic lipid is the same or different, and is:

[0394]

[0395] wherein:

[0396] a is 1, 2, 3, or 4;

[0397] b is 4; and

[0398] c is 3, 4, 5, 6, or 7,

[0399] with the proviso that the sum of a, b, and c is 11 or 13.

[0400] In some aspects, each R1in the KC3 or KC4 ionizable cationic lipid is the same and is a straight chain C 16 hydrocarbon chain, each containing one unsaturated alkenyl double bond within each polyene chain. In some aspects, each R1in the KC3 or KC4 ionizable cationic lipid is the same and is a straight chain C 18 hydrocarbon chain, each containing one unsaturated alkenyl double bond within each polyene chain. In some aspects, each R1in the KC3 or KC4 ionizable cationic lipid is the same and is a straight chain C 16 hydrocarbon chain, each containing two unsaturated alkenyl double bonds within each polyene chain. In some aspects, each R1in the KC3 or KC4 ionizable cationic lipid is the same and is a straight chain C 16 hydrocarbon chain, each containing two unsaturated alkenyl double bonds within each polyene chain, wherein the alkenyl double bonds are separated by two or more saturated alkylene groups. In some aspects, each R1in the KC3 or KC4 ionizable cationic lipid is the same and is a straight chain C 18 hydrocarbon chain, each containing one unsaturated alkenyl double bond within each polyene chain. In some aspects, each R1in the KC3 or KC4 ionizable cationic lipid is the same or different and is a straight chain C 16 or C 18 hydrocarbon chain, each containing one or two unsaturated alkenyl double bonds within each polyene chain.

[0401] In some embodiments, the LNP composition comprises a mixture of a KC3 ionizable cationic lipid and a KC4 ionizable cationic lipid.

[0402] In some embodiments, the LNP composition comprises an ionizable lipid, wherein the ionizable lipid comprises:

[0403] (a) a dialkylamino moiety of the headgroup having the chemical structure of Formula (IV-A):

[0404]

[0405] wherein:

[0406] n in formula (IV-A) is 2, 3, or 4; and

[0407] R in formula (IV-A) 10 and R 12 are each independently selected from the group consisting of alkyl selected from the group consisting of methyl, ethyl, and n-propyl, wherein R 10 and R 12 alkyl are optionally substituted with one or more hydroxyl groups; and

[0408] (b) the ionizable lipid further comprises the chemical structure the chemical structure comprises an acyl group covalently bound to a moiety within each lipid tail distal to the head group relative to the dialkylamino moiety of formula (IV-A), wherein represents attachment to the head group of formula IV-A, and R 22 represents a moiety of each lipid tail covalently bound to the acyl group and having the chemical structure of formula A:

[0409]

[0410] wherein represents attachment of formula A to R 22 within each lipid tail; and

[0411] a is 4, 1, 2, or 3;

[0412] b is 4, 2, or 3; and

[0413] c is 4, 3, 5, 6, or 7,

[0414] with the proviso that the sum of a, b, and c in formula A is 12, 10, 11, or 13.

[0415] In some embodiments, the ionizable lipid is a compound of formula (IV-A), wherein R 10 and R 12 are each independently methyl, ethyl, -(CH2)(CH2)OH, or -(CH2)2(CH2)OH. In some embodiments, the ionizable lipid is a compound of formula (IV-A), wherein b is 4, and R 10 and R 12 are each methyl.

[0416] In some embodiments, the present disclosure provides compositions comprising ionizable cationic lipids. Some aspects of the present disclosure include compositions comprising 3-rac-2,2-bis((Z)-octadec-9-en-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylpropane-1-amine (AKG-KC3-OA racemate) or chiral purified forms of AKG-KC3-OA racemate, such as KC3-OA(S) and KC3-OA(R), and methods for their preparation. Some aspects of the present disclosure include compositions comprising 4-rac-2,2-bis((Z)-octadec-9-en-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylbutane-1-amine (AKG-KC4-OA). In some embodiments, the composition comprises an ionizable cationic lipid selected from one or more of the following: (a) a racemic mixture of 3-rac-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine (KC3-OA racemate), or a KC3-OA enantiomer; and (b) 4-rac-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylbutane-1-amine (AKG-KC4-OA racemate). In some embodiments, the composition comprises a mixture of (R) and (S) enantiomers of the KC3-OA ionizable cationic lipid, or a mixture of (R) and (S) enantiomers of the KC4-OA ionizable cationic lipid. In some embodiments, the composition comprises a mixture of the (R) and (S) enantiomers of a KC3-OA ionizable cationic lipid, or a mixture of the (R) and (S) enantiomers of a KC4-OA ionizable cationic lipid, and the mixture is racemic.

[0417] 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 mRNA molecules.

[0418] 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.

[0419] In some embodiments, the lipid nanoparticles are in an aqueous medium.

[0420] In some embodiments, the nucleic acid is encapsulated in a lipid nanoparticle with a compound disclosed herein, including a compound of Formula I, II, III, IV-B, V-A-l, or a combination thereof, wherein the nucleic acid is RNA. In some embodiments, the nucleic acid is encapsulated in a lipid nanoparticle with a compound disclosed herein, including a compound disclosed 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.

[0421] In some embodiments, the lipid nanoparticle comprises a membrane comprising phosphatidylcholine and a sterol. In some embodiments, the sterol is cholesterol. In some embodiments, the lipid nanoparticle comprises a membrane comprising phosphatidylcholine, an ionizable cationic lipid (ICL). In some embodiments, the ICL has a structure of Formula I, II, III, IV-B, V-A-l, and cholesterol, wherein the membrane separates the interior of the lipid nanoparticle from an aqueous medium. In some embodiments, the ICL has a structure as shown in Table 1. 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.

[0422] In some embodiments, the molar ratio of phosphatidylcholine to cholesterol is about 1:5 to about 1:2.

[0423] In some embodiments, the membrane further comprises a polymer-conjugated lipid.

[0424] In some embodiments, the lipid nanoparticle comprises an ICL, DSPC, cholesterol, and a polymer-conjugated lipid in a molar ratio of about 49.5:10.3:39.6:2.5.

[0425] In some embodiments, the polymer-conjugated lipid is PEG(2000)-dimyristyl glycerol (PEG-DMG) or PEG(molecular weight 2,000)-dimyristyl phosphatidylethanolamine (PEG-DMPE).

[0426] In some embodiments, the percentage of oxidative degradation products of the ionizable lipid is less than 50% of the percentage of oxidative degradation products of a DLin-KC2-DMA or DLin-MC3-DMA control formulation.

[0427] In some embodiments, the composition is a liquid pharmaceutical formulation for parenteral administration.

[0428] In some embodiments, the composition is a liquid pharmaceutical formulation for subcutaneous, intramuscular, or intradermal administration.

[0429] In some embodiments, the composition is in the form of a lyophilized powder, which is subsequently reconstituted with an aqueous medium prior to administration.

[0430] Further aspects of the disclosure relate to methods of preventing a bacterial or viral infection, the methods 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 of vaccinating a subject in need thereof, the methods comprising administering a composition comprising a nucleic acid encoding an antigenic protein.

[0431] In some embodiments, the composition is administered subcutaneously, intramuscularly, or intradermally.

[0432] In some embodiments, the bacterial infection is a Mycobacterium tuberculosis infection. In some embodiments, the bacterial infection is a form of non-tuberculous mycobacteria.

[0433] In some embodiments, the lipid nanoparticle is administered parenterally.

[0434] In some embodiments, the lipid nanoparticle composition is administered as part of a single injection.

[0435] In some embodiments, the lipid nanoparticle is administered in multiple injections at time intervals to optimize the T cell response to it.

[0436] In some embodiments, the lipid nanoparticle is administered intramuscularly (IM).

[0437] The disclosure features a lipid nanoparticle comprising an mRNA and a lipid. Some exemplary lipids include an ionizable cationic lipid (ICL), a phospholipid, a sterol lipid, an alkylene glycol lipid (e.g., a polyethylene glycol lipid), a sphingolipid, a glycerolipid, a glycerophospholipid, a prenol lipid, a sugar lipid, a fatty acid, and a polyketide. In some embodiments, the LNP comprises a single type of lipid. In some embodiments, the LNP comprises multiple (e.g., two or more) lipids. The LNP can comprise one or more of an ionizable cationic lipid, a phospholipid, a sterol, or an alkylene glycol lipid (e.g., a polyethylene glycol lipid).

[0438] In an embodiment, the LNP comprises an ionizable cationic lipid. As used herein, “ionizable cationic lipid,” “ionizable lipid,” and “ICL” are used interchangeably. An ICL is a lipid that comprises an ionizable moiety that is capable of carrying a charge (e.g., a positive charge, e.g., a cationic lipid) under certain conditions (e.g., within a certain pH range, e.g., under physiological conditions). The ionizable moiety can comprise an amine, and preferably a substituted amine. The ionizable lipid can be a cationic lipid or an anionic lipid. In addition to the ionizable moiety, the ionizable lipid can comprise an alkyl or alkenyl group, e.g., an alkyl or alkenyl group that is greater than 6 carbon atoms in length (e.g., greater than about 8 carbons, 10 carbons, 12 carbons, 14 carbons, 16 carbons, 18 carbons, 20 carbons, or more in length). Additional ionizable lipids that can be included in the LNP described herein are disclosed in Jayaraman et al. (Angew. Chem. Int. Ed. 51 :8529-8533 (2012)), Semple et al. Nature Biotechnol. 28: 172-176 (2010)), and U.S. Patent Nos. 8,710,200 and 8,754,062, each of which is incorporated herein by reference in its entirety.

[0439] In some embodiments, the LNP further comprises an ionizable lipid having the structure of Formula (IV-A), or a pharmaceutically acceptable salt thereof,

[0440]

[0441] wherein R 10 and R 12 each independently is (C1-C4)alkyl optionally substituted with hydroxyl; v is 0 or 1; q1 is 1 or 2; Y is R 22 is a is 1, 2, 3, 4, or 5; and c is 4, 5, 6, 7, or 8.

[0442] In some embodiments, for a compound of Formula (III), v is equal to 0. In some embodiments, for a compound of Formula (III), v is equal to 1. In some embodiments, for a compound of Formula (III), v is equal to 1 and q1 is equal to 1. In some embodiments, for a compound of Formula (III), v is equal to 1 and q1 is equal to 2.

[0443] In some embodiments, for a compound of Formula (III), R 22 the sum of a and c is 6, 7, 8, or 9. In some embodiments, for a compound of Formula (III), R 22 the sum of a and c is 6. In some embodiments, for a compound of Formula (III), R 22the 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.

[0444] In some embodiments, for the compound of Formula (III), v is equal to 0, and R 22 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 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 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.

[0445] In some embodiments, for the compound of Formula (III), R 10 and R 12 are each methyl, and R 10 and R 12 are each methyl, and R 22 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 are each methyl, v is 0, and R 22 the sum of a and c is 6, 7, 8, or 9.

[0446] In some embodiments, for the compound of Formula (IV-B), v is equal to 0, and R 22 In some embodiments, for the compound of Formula (III), v is equal to 0, and R 22 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 and a is 4 and c is 5. In some embodiments, for the compound of Formula (III), v is equal to 0, and R 22 and a is 1 and c is 8. In some embodiments, for the compound of Formula (III), v is equal to 0, and R 22 and a is 2 and c is 5.

[0447] ​​​​​The LNP can comprise an ionizable lipid at a concentration greater than, for example, about 0.1 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises an ionizable lipid at a concentration greater than, for example, about 1 mol%, about 2 mol%, about 4 mol%, about 8 mol%, about 20 mol%, about 40 mol%, about 50 mol%, about 60 mol%, about 80 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises an ionizable lipid at a concentration greater than about 20 mol%, about 40 mol%, or about 50 mol%. In one embodiment, the LNP comprises an ionizable lipid at a concentration of, for example, about 1 mol% to about 95 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises an ionizable lipid at a concentration of, for example, about 2 mol% to about 90 mol%, about 4 mol% to about 80 mol%, about 10 mol% to about 70 mol%, about 20 mol% to about 60 mol%, about 40 mol% to about 55 mol% of the total lipid content of the LNP. In one embodiment, the LNP 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%.

[0448] In one embodiment, the LNP comprises a phospholipid. Phospholipids are lipids that comprise a phosphate group and at least one alkyl, alkenyl, or heteroalkyl chain. The phospholipid can be naturally occurring or non-naturally occurring (e.g., a synthetic phospholipid). The phospholipid can comprise an amine, an amide, an ester, a carboxyl, a choline, a hydroxyl, an acetal, an ether, a carbohydrate, a sterol, or a glycerol. In some embodiments, the phospholipid can comprise a phosphocholine, a sphingomyelin, or an acetal phospholipid. 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), 1,2-diarachidonoyl-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(monoacylglycero) phosphate (BMP), L-a-phosphatidylcholine, 1,2-dimyristoleoyl-sn-glycero-3- phosphocholine (DHDPC), and 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC). Additional phospholipids that can 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 by reference herein in its entirety.

[0449] 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).

[0450] Incorporation of phosphatidylserine

[0451] LNP (e.g., as described herein) can comprise one or more of the following components: (i) an ionizable cationic lipid (ICL) comprising a C16 alkyl or C16 alkenyl or C18 alkyl or C18 alkenyl 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; (iii) cholesterol at a concentration of about 1 mol% to about 95 mol% (or any value therebetween, e.g., about 20 mol% to about 80 mol%); (iv) phosphatidylserine (PS) or phosphatidylglycerol (PG) added to the LNP lipid formulation at a concentration of about 0.5 mol% to about 20 mol%, about 2.5 mol% to about 10 mol%, about 4 mol% to about 8 mol%, or any value therebetween, of the total lipid content of the LNP, and (v) a polyethylene glycol (PEG)-2000 containing lipid (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 an embodiment, the LNP comprises two of (i) to (v). In an embodiment, the LNP comprises three of (i) to (v). In an embodiment, the LNP comprises four of (i) to (v). In an embodiment, the LNP comprises each of (i) to (v). In some embodiments, the LNP comprises (i) and (ii). In some embodiments, the LNP comprises (i) and (iii). In some embodiments, the LNP comprises (i) and (v). In some embodiments, the LNP comprises (ii) and (iii). In some embodiments, the LNP comprises (ii) and (v). In some embodiments, the LNP comprises (iii) and (iv). In some embodiments, the LNP comprises (iii) and (v). In some embodiments, the LNP comprises (i), (ii), and (iii). In some embodiments, the LNP comprises (i), (ii), and (v). In some embodiments, the LNP comprises (ii), (iii), and (v). In some embodiments, the LNP comprises (ii), (iii), (iv), and (v). In an embodiment, the LNP consists of or consists essentially of four of (i) to (v). In an embodiment, the LNP consists of or consists essentially of each of (i) to (v).In some embodiments, the LNP consists of or consists essentially of (i) and (ii). In some embodiments, the LNP consists of or consists essentially of (i) and (iii). In some embodiments, the LNP consists of or consists essentially of (i) and (v). In some embodiments, the LNP consists of or consists essentially of (ii) and (iii). In some embodiments, the LNP comprises (ii) and (v). In some embodiments, the LNP consists of or consists essentially of (iii) and (iv). In some embodiments, the LNP consists of or consists essentially of (iii) and (v). In some embodiments, the LNP consists of or consists essentially of (i), (ii), and (iii). In some embodiments, the LNP consists of or consists essentially of (i), (ii), and (v). In some embodiments, the LNP comprises (ii), (iii), and (v). In some embodiments, the LNP consists of or consists essentially of (ii), (iii), (iv), and (v).

[0452] The LNP can comprise a phospholipid at a concentration greater than, for example, about 0.1 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises a phospholipid at a concentration greater than, for example, about 0.5 mol%, about 1 mol%, about 1.5 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 8 mol%, about 10 mol%, about 12 mol%, about 15 mol%, about 20 mol%, about 50 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises a phospholipid at a concentration greater than about 1 mol%, about 5 mol%, or about 10 mol%. In one embodiment, the LNP comprises a phospholipid at a concentration of, for example, about 0.1 mol% to about 50 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises a phospholipid at a concentration of, for example, about 0.5 mol% to about 40 mol%, about 1 mol% to about 30 mol%, about 5 mol% to about 25 mol%, about 10 mol% to about 20 mol%, about 10 mol% to about 15 mol%, or about 15 mol% to about 20 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises a phospholipid at a concentration of about 5 mol% to about 25 mol%. In one embodiment, the LNP comprises a phospholipid at a concentration of about 10 mol% to 20 mol%.

[0453] 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).

[0454] Ionizable sterols

[0455] 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).

[0456] In some embodiments, the ionizable lipid can be a branched chain ionizable lipid selected from ALC-0315 and SM-102:

[0457]

[0458] 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%.

[0459] 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- poly(2-ethyl-2-oxazoline) (PEOZ), poly-N-vinylpyrrolidone (PVP), polyglycerol, poly(hydroxyethyl L-asparagine) (PHEA), and poly(hydroxyethyl L-glutamine) (PHEG). poly(2-ethyl-2-oxazoline) (PEOZ), poly-N-vinylpyrrolidone (PVP), polyglycerol, poly(hydroxyethyl L-asparagine) (PHEA), and poly(hydroxyethyl L-glutamine) (PHEG).

[0460] PEG-containing lipids can also comprise amines, amides, esters, carboxyls, phosphates, choline, hydroxyls, acetals, ethers, heterocycles, or carbohydrates. PEG-containing lipids can comprise at least one alkyl or alkenyl group, e.g., an alkyl or alkenyl group greater than 6 carbon atoms in length (e.g., greater than about 8 carbons, 10 carbons, 12 carbons, 14 carbons, 16 carbons, 18 carbons, 20 carbons, or more) in length, e.g., in addition to the PEG moiety. In one embodiment, the PEG-containing lipid comprises a PEG moiety comprising at least 20 PEG monomers, e.g., 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. Some exemplary PEG-containing lipids include PEG-DMG (e.g., DMG-PEG2k), PEG-c-DMG, PEG-DSG, PEG-DPG, PEG-DSPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, and PEG-DLPE. In some embodiments, the PEG-lipid comprises PEG-DMG (e.g., DMG-PEG2k), PEG-c-DMG, PEG-DSG, and PEG-DPG. Additional PEG-lipids that can be included in the LNPs described herein are disclosed in Fahy, E. et al. (J. Lipid. Res. 46:839-862 (2005), which is incorporated by reference herein in its entirety.

[0461] In some embodiments, the PEG-lipid is PEG-DMG (e.g., DMG-PEG2k). In some embodiments, the PEG-lipid is a-(3'-{[l,2-bis(myristyloxy)propoxy]carbonylamino}propyl)-w- methoxy, polyoxyethylene (PEG-c-DMG). In some embodiments, the PEG-lipid is PEG-DSG. In some embodiments, the PEG-lipid is PEG-DPG.

[0462] The LNP can comprise an alkylene glycol-containing lipid at a concentration greater than, for example, about 0.1 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration greater than, for example, about 0.5 mol%, about 1 mol%, about 1.5 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 8 mol%, about 10 mol%, about 12 mol%, about 15 mol%, about 20 mol%, about 50 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration greater than about 1 mol%, about 4 mol%, or about 6 mol%. In one embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration of, for example, about 0.1 mol% to about 50 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration of, for example, about 0.5 mol% to about 40 mol%, about 1 mol% to about 35 mol%, about 1.5 mol% to about 30 mol%, about 2 mol% to about 25 mol%, about 2.5 mol% to about 20%, about 3 mol% to about 15 mol%, about 3.5 mol% to about 10 mol%, or about 4 mol% to 9 mol% of the total lipid content of the LNP. In one embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration of about 3.5 mol% to about 10 mol%. In one embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration of about 4 mol% to 9 mol%.

[0463] In some embodiments, the LNP comprises at least two types of lipids. In one embodiment, the LNP comprises two of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP comprises at least three types of lipids. In one embodiment, the LNP comprises three of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP comprises at least four types of lipids. In one embodiment, the LNP comprises each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid.

[0464] An LNP (e.g., as described herein) can 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 an embodiment, the LNP comprises one of (i) to (iv). In an embodiment, the LNP comprises two of (i) to (iv). In an embodiment, the LNP comprises three of (i) to (iv). In an embodiment, 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).

[0465] An LNP (e.g., as described herein) can 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 an embodiment, the LNP comprises two of (i) to (iv). In an embodiment, the LNP comprises three of (i) to (iv). In an embodiment, 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 (ii), (iii), and (iv).

[0466] In one embodiment, the LNP comprises a ratio of ionizable lipid to phospholipid of about 50: 1 to about 1: 1 (e.g., 40: 1, 32:3, 6: 1, 7: 1, 5: 1, 24:5, 26:5, 10:3, 15:2, 16:7, 18: 1, 3: 1, 3:2, or 1: 1). In one embodiment, the LNP comprises a ratio of ionizable lipid to phospholipid of about 15:2. In one embodiment, the LNP comprises a ratio of ionizable lipid to phospholipid 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, 1:4, 4:5, 1:6, 5:6, 7:6, 7:8, or 8:9). In one embodiment, the LNP comprises a ratio of ionizable lipid to alkylene-containing lipid of about 1: 10 to about 10: 1 (e.g., 1:9, 1:8, 7:8, 7: 1, 7:5, 7:3, 6: 1, 6:5, 5: 1, 5:3, 4: 1, 4:3, 3: 1, 2: 1, 1: 1, 1:2, 1:3, 3:4, 1:4, 3:5, 1:4, 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, 1:4, 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, 3.:2, 22:5, 24:5, 26:5, 10:3, 15:2, 16:7, 18:, 3:, 3:2, or 1: 1).

[0467] In one embodiment, the LNP (e.g., described herein) comprises two of the ionizable lipid, the phospholipid, the sterol, and the alkylene glycol-containing lipid (e.g., PEG- containing lipid). In another embodiment, the LNP (e.g., described herein) comprises three of the ionizable lipid, the phospholipid, the sterol, and the alkylene glycol-containing lipid (e.g., PEG- containing lipid). In one embodiment, the LNP (e.g., described herein) comprises each of the ionizable lipid, the phospholipid, the sterol, and the alkylene glycol-containing lipid (e.g., PEG- containing lipid).

[0468] In some embodiments, the LNP described herein has a diameter of 5 to 500 nm, e.g., 10 to 400 nm, 20 to 350 nm, 25 to 325 nm, 30 to 300 nm, 50 to 250 nm, 60 to 200 nm, 75 to 190 nm, 80 to 180 nm, 100 to 200 nm, 200 to 300 nm, and 150 to 250 nm. The diameter of the LNP can be determined by any method known in the art, e.g., dynamic light scattering, transmission electron microscopy (TEM), or scanning electron microscopy (SEM). In some embodiments, the LNP has a diameter of 50 to 100 nm, 70 to 100 nm, and 80 to 100 nm. In one embodiment, the LNP has a diameter of about 90 nm. In some embodiments, the LNP described herein has a diameter of greater than about 30 nm. In some embodiments, the LNP has a diameter of greater than about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about 225 nm, about 250 nm, about 275 nm, or about 300 nm. In one embodiment, the LNP has a diameter of greater than about 70 nm. In one embodiment, the LNP has a diameter of greater than about 90 nm. In one embodiment, the LNP has a diameter of greater than about 180 nm.

[0469] In some embodiments, the plurality of LNP described herein has an average diameter of about 40 nm to about 180 nm. In some embodiments, the plurality of LNP described herein has an average diameter of about 50 nm to about 150 nm. In some embodiments, the plurality of LNP described herein has an average diameter of about 50 nm to about 120 nm. In some embodiments, the plurality of LNP described herein has an average diameter of about 60 nm to about 120 nm. In some embodiments, the plurality of LNP has an average diameter of about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm.

[0470] In some embodiments, the average neutral to negative surface charge of the nanoparticle or plurality of nanoparticles described herein is 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 neutral to negative surface charge of the nanoparticle or plurality of nanoparticles is -100 mv to 100 mv, -75 mv to 0, or -50 mv to -10 mv.

[0471] 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 plurality of 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 plurality of nanoparticles at pH 7.4 is -20 mv to +20, -10 mv to +10 mv, or -5 mv to +5 mv. Neutral charged LNPs have improved pharmacokinetic and biological performance compared to cationic LNPs.

[0472] Preparing lipid nanoparticles (LNPs)

[0473] Methods of preparing LNPs can include mixing a first solution with a second solution. Mixing can be achieved using standard liquid mixing techniques, e.g., propellor mixing, vortexing the solution, or preferably by microfluidic mixing or high efficiency T mixing. In some embodiments, the first solution comprises a lipid or plurality of lipids, and a nucleic acid, wherein all components are dissolved in a water / solvent system. The solvent can be any water miscible solvent (e.g., ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethylsulfoxide, dioxane, or tetrahydrofuran). In some embodiments, the first solution comprises a small percentage of water or pH buffered water. The first solution can comprise up to at least 60% by volume of water, e.g., up to at least about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% by volume of water. In one embodiment, the first solution comprises about 0.05% to 60% by volume of water, e.g., about 0.05% to 50% by volume, about 0.05% to 40% by volume, or about 5% to 20% by volume of water.

[0474] In some embodiments, the first solution comprises a single type of lipid, e.g., an ionizable lipid, a phospholipid, a sterol, or a PEG-containing lipid. In some embodiments, the first solution comprises a plurality of lipids. In some embodiments, the plurality comprises an ionizable lipid, a phospholipid, a sterol, or a PEG-containing lipid. In some embodiments, the plurality of lipids comprises cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DMG-PEG2k), or a-(3'-{[l,2-di(myristyloxy)propoxy]carbonylamino}propyl)-w-methoxy, polyoxyethylene (PEG2000-C-DMG), and an ionizable lipid. The plurality of lipids can be present in any ratio. In one embodiment, the plurality of lipids comprises an ionizable lipid or a sterol, a phospholipid, a sterol, a PEG-containing lipid, or a combination thereof in a particular ratio (e.g., a ratio described herein) of the aforementioned lipids.

[0475] In some embodiments, the second solution is water. In some embodiments, the second solution is an aqueous buffer having a pH of 3 to 6 (e.g., a pH of about 3, about 4, about 5, or about 6). The second solution can comprise a load component, e.g., a nucleic acid (e.g., mRNA). The second solution can comprise a small percentage of a water-miscible organic solvent. The second solution can comprise up to at least 60 vol% of at least one water-miscible organic solvent, e.g., up to at least about 0.05 vol%, 0.1 vol%, 0.5 vol%, 1 vol%, 2 vol%, 3 vol%, 4 vol%, 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol%, 10 vol%, 15 vol%, 20 vol%, 25 vol%, 30 vol%, 35 vol%, 40 vol%, 45 vol%, 50 vol%, 55 vol%, 60 vol%, or any vol% therebetween, of at least one organic solvent (e.g., a water-miscible organic solvent). In one embodiment, the second solution comprises about 0.05 vol% to 60 vol% of an organic solvent, e.g., about 0.05 vol% to 50 vol%, about 0.05 vol% to 40 vol%, or about 5 vol% to 20 vol% of an organic solvent (e.g., a water-miscible organic solvent). The aqueous buffer solution can be a citrate buffered aqueous solution. In some embodiments, the aqueous buffer solution is a citrate buffered 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 buffered solution having a pH of about 6.

[0476] In some embodiments, the solution comprising the mixture of the first and second solutions, the mixture comprising the LNP suspension, can be diluted. In some embodiments, the pH of the solution comprising the mixture of the first and second solutions, the mixture comprising the LNP suspension, can be adjusted. Dilution of the LNP suspension or adjustment of the pH of the LNP suspension can be achieved by the addition of water, acid, base, or an aqueous buffer. In some embodiments, neither dilution of the LNP suspension nor adjustment of the pH of the LNP suspension is performed. In some embodiments, both dilution of the LNP suspension and adjustment of the pH of the LNP suspension are performed.

[0477] In some embodiments, excess reagents, solvents, unencapsulated nucleic acids can be removed from the LNP suspension by tangential flow filtration (TFF), e.g., diafiltration. TFF can also be used to remove organic solvents (e.g., ethanol) and buffers from the LNP suspension. In some embodiments, the LNP suspension is subjected to dialysis instead of TFF. In some embodiments, the LNP suspension is subjected to TFF instead of dialysis. In some embodiments, the LNP suspension is subjected to both dialysis and TFF.

[0478] In one aspect, the disclosure features a method comprising treating a sample of LNP comprising a nucleic acid with a fluid comprising a detergent (e.g., Triton X-100, or an anionic detergent such as, but not limited to, sodium dodecyl sulfate (SDS), or a non-ionic detergent such as, but not limited to, beta-octyl glucoside, or a Zwittergent 3-14) for a period of time suitable to degrade the lipid layer and thereby release encapsulated and / or entrapped nucleic acid. In one embodiment, the method further comprises analyzing the sample for the presence, absence, and / or amount of released nucleic acid.

[0479] LNP comprising a ligand

[0480] Some aspects of the disclosure relate to LNP 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 LNP comprising a ligand as described herein.

[0481] LNP targeting can also be achieved by adding lipids to the formulation. For example, phosphatidylserine is known to redistribute to the outer surface of the plasma membrane during apoptosis and is a molecular 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 uptake and activation of dendritic cells. LNP targeting can also be achieved by adding certain anionic phospholipids to the formulation (Table 2A). For example, phosphatidylserine is known to redistribute to the outer surface of the plasma membrane during apoptosis and is a molecular 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 uptake and activation of dendritic cells (Caronni et al., Nat Comm. 2021 April 14; 12:2237-2253; Ischihashi et al., PLOS One 2013). While anionic phospholipids have been used previously in the context of liposomes, their inclusion in a lipid nanoparticle comprising a condensed nucleic acid is unexpected as the anionic head group can compete with the phosphate backbone of the mRNA for binding sites of the ionizable cationic lipids, can inhibit endosomal escape by altering surface charge, or can cause aggregation of the LNPs during formation or storage.

[0482] Table 2A. Anionic phospholipid targeting moieties

[0483]

[0484] Table 2B. Phosphatidylserine targeting moieties

[0485]

[0486] In some embodiments, the anionic targeting ligand is selected from the group of phosphatidylserine (PS), phosphatidylglycerol (PG), N-glutaryl-phosphatidylethanolamine (N-Glu-PE), or N-succinyl-phosphatidylethanolamine (N-Suc-PE). In some embodiments, the anionic phospholipid used is phosphatidylserine. In another embodiment, the phosphatidylserine comprises the L-isomer of serine. In another embodiment, the acyl chains of the phosphatidylserine are fully saturated, for example 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. The phosphatidylserine can also comprise an asymmetric acyl chain composition, for example where one acyl chain is stearic acid and the other is palmitic acid.

[0487] In some embodiments, the anionic phospholipid is selected from a group other than phosphatidylserine. In some embodiments, these non-PS anionic phospholipids include phosphatidylglycerol (PG), phosphatidic acid (PA), N-glutaryl-phosphatidylethanolamine (N-Glu-PE), N-succinyl-phosphatidylethanolamine (N-Suc-PE), and cardiolipin. In some embodiments, these anionic phospholipids include saturated acyl chains of 16 or 18 carbons, for example distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), N-succinyl-distearoylphosphatidylethanolamine (N-Suc-DSPE), N-glutaryl-distearoylphosphatidylethanolamine (N-Glu-DSPE), distearoylphosphatidic acid (DSPA), and cardiolipin.

[0488] Table 2C. Non-phosphatidylserine anionic phospholipids

[0489]

[0490] In some embodiments, the anionic phospholipid used is phosphatidylglycerol. In another embodiment, the acyl chains of the phosphatidylglycerol are fully saturated, for example the case of dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), or distearoylphosphatidylglycerol (DSPG). In a preferred embodiment, the PG used is DPPG or DSPG. The phosphatidylglycerol can also comprise an asymmetric acyl chain composition, for example where one acyl chain is stearic acid and the other is palmitic acid.

[0491] In some embodiments, the salt form of phosphatidylglycerol or phosphatidylserine is highly soluble in ethanol. In some embodiments, the salt form of phosphatidylserine is highly soluble in ethanol. In some embodiments, it is soluble at greater than 0.5 mg / ml, greater than 1 mg / mL, greater than 5 mg / mL, greater than 10 mg / mL, or greater than 20 mg / mL. In some embodiments, the salt form of phosphatidylglycerol or phosphatidylserine is soluble at least 0.3 mM, at least 0.4 mM, at least 0.5 mM, at least 0.6 mM, or at least 0.8 mM in 200 proof ethanol at 22 °C or lower, as determined by the flask-shaking method. In some embodiments, the salt is an ammonium salt. In some embodiments, the phosphatidylserine is added to the LNP lipid as an ammonium salt or a substituted ammonium salt. The substituted ammonium salt can be a monoalkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium having alkyl groups each having one to six, one to four, one to three, one, two, or three carbon atoms. One or more of the alkyl groups can be a normal alkyl group, or a branched alkyl group (e.g., such as isopropyl), or form a ring (e.g., such as cyclohexyl). The alkyl groups and the nitrogen ammonium atom can form a heterocyclic ring. The substituted ammonium salt can also be formed from an alkylene diamine. Tris(hydroxymethyl)aminomethane and triethanolamine can also be used as the amine base to form the PS salt. In some embodiments, the amine is selected from the group consisting of ammonia, dimethylamine, diethylamine, triethylamine, trimethylamine, 2-(dimethylamino)ethanol, diethanolamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methyl-glucamine, imidazole, histidine, lysine, arginine, 4-(2-hydroxyethyl)-morpholine, piperazine, 1-(2-hydroxyethyl)-pyrrolidine, triethanolamine, and tromethamine (tris(hydroxymethyl)aminomethane). In some embodiments, the targeting lipid is an ammonium salt of DPPS.

[0492] Table 2D. Ammonium and sodium salt forms of dipalmitoyl-phosphatidylserine or distearoyl-phosphatidylserine.

[0493]

[0494] 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 some embodiments, 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 evaporation of 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, PS as a sodium or potassium salt 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, such as HC1, to obtain the free acid form of PS, which is then neutralized with a substituted amine or ammonium hydroxide in the form of a free base. In yet another embodiment, an organic solution of PS as a sodium or potassium salt is treated with a cation exchange resin with ammonium in the form of a substituted ammonium. In yet another embodiment, PS is prepared in the form of a calcium or magnesium salt and treated with an ammonium salt or substituted ammonium salt of a chelating agent, such as EDTA, or with ammonium phosphate or substituted ammonium phosphate in the presence of an organic solvent, resulting in the displacement of calcium or magnesium ions in the form of a chelate or a less soluble phosphate salt, which is isolated, for example, by filtration, while the ammonium salt or substituted ammonium salt of PS remains in the organic (e.g., ethanol) solution.

[0495] In some embodiments, PS or PG is added to the LNP lipid formulation at a concentration of about 0.1 mol% to about 20 mol%, about 0.1 mol% to about 10 mol%, about 0.1 mol% to about 5 mol%, about 0.5 mol% to about 20 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 5 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 10 mol%, or about 1 mol% to about 5 mol% of the total lipid content of the LNP. In some embodiments, PS is added to the LNP lipid formulation at a concentration of about 1 mol% to about 20 mol%, about 2.5 mol% to about 10 mol%, about 3 mol% to about 9 mol%, or about 4 mol% to about 8 mol% of the total lipid content of the LNP.

[0496] In some embodiments, the PS or PG lipid is included in an LNP composition comprising an ionizable cationic lipid known in the art, including DODAP, AKG-OA-DM2, O-11769, DLin-MC3-DMA, DLin-KC2-NMA, DLin-KC3-DMA AL C-0315, and SM-102.

[0497] In another embodiment, the PS lipid is included in an LNP composition comprising an ICL of Formula I, II, III, IV-B, V-A-1, combinations 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.

[0498] In some aspects, methods of delivering a nucleic acid to a cell are provided, the methods comprising: contacting the cell with a composition comprising an LNP 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. 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, binding of the targeting ligand to a particular cell surface antigen induces internalization of the LNP when the targeting ligand is attached to the cell, when contacted and incubated under internalizing conditions with a cell expressing at least 100,000 or at least 1,000,000 molecules of the antigen.

[0499] Table 3A. Exemplary dialkyl and branched ionizable cationic lipids

[0500]

[0501] Table 3B. Exemplary dialkyl and branched ionizable cationic lipids (continued)

[0502]

[0503] Compositions

[0504] 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, V-A-1, combinations thereof, or a pharmaceutically acceptable salt thereof.

[0505] Further aspects of the present disclosure relate to the use of these ionizable lipids or lipid nanoparticle compositions comprising ionizable lipids in vaccines for the prevention of infectious diseases. In some embodiments, the compositions described herein can be used to prevent infections associated with tuberculosis. In some embodiments, the vaccines are used to prevent mycobacterium infections. Some embodiments relate to injectable pharmaceutical vaccine compositions comprising the compositions of the present disclosure. In some embodiments, the vaccines can be used to prevent tuberculosis, nontuberculous mycobacteria (NTM), nontuberculous pulmonary disease, leprosy, mycobacterium avium-intracellulare, mycobacterium kansasii, mycobacterium marinum, mycobacterium ulcerans, mycobacterium chelonae, mycobacterium fortuitum, or mycobacterium abscessus.

[0506] In some embodiments, the compounds and compositions described herein facilitate efficient uptake and transfection of target cells, including tissue macrophages and dendritic cells. The result is efficient delivery of nucleic acids encoding antigens specific to infectious viruses or bacteria, and subsequent presentation of the antigens to elicit the desired immune response, thereby protecting against the corresponding infection. In some embodiments, the nucleic acids are synthetic nucleic acids (e.g., engineered codon-optimized mRNAs) encoding Mycobacterium tuberculosis epitopes. In some embodiments, the epitopes are MHC class II epitopes contained in larger open reading frames (ORFs), such as Esx V (Rv3619), Esx W (Rv3620c), EsxB / CFP10 (Rv3874), EsxA / ESAT-6 (Rv3875), AMtb39A (Rv1196), Ag85B (Rv1886c), or AsxH / TB 10.4 (Rv0288). In some embodiments, the epitopes are shorter, non-overlapping MHC II minimal 15-mer epitopes identified in individuals with latent tuberculosis infection (LTBI) (U.S. Patent No. 10,703,784, incorporated by reference in its entirety herein, Arlehamn et al., (2013) PloS Pathog. 9:el003130 and Arlehamn et al., (2016) PloS Pathog. 12:el005760). Minimal MHC-II epitopes are defined as 12 to 20 residue long peptides containing a 9-residue core that is the main determinant of binding strength to the class II molecule binding groove. Flanking residues on each side of the core can vary due to the open class II binding groove. In some embodiments, these sequences are concatenated and encoded using a single mRNA. In some embodiments, the concatenated sequences are combinations of larger open reading frames and minimal 15-mer epitopes. In some embodiments, the combination of sequences contained in a single concatenated sequence is selected to remove redundant protein sequences. In some embodiments, the selection of minimal epitopes to be contained in a single concatenated sequence is selected to provide the most optimal HLA donor coverage. In some embodiments, the concatenated sequences are linked with non-immunogenic linkers, which reduce the likelihood of generating MHC class II neoepitopes. In some embodiments, the sequence of the peptide linker comprises GPGPG (SEQ ID NO: 228).

[0507] In some embodiments, the nucleic acid encodes MHC Class I epitopes. In some embodiments, the Class I epitopes are present in both M. tuberculosis and other non- M. tuberculosis, or in the Bacillus Calmette-Guerin (BCG) vaccine. In some embodiments, the epitopes are present in M. tuberculosis. In some embodiments, the MHC Class I sequences are concatenated and encoded using a single mRNA. In some embodiments, the concatenated MHC Class I sequences are a combination of larger open reading frames and minimal 9-10mer epitopes. In other embodiments, the mRNA cassette encodes only a concatenated sequence of minimal epitopes. In some embodiments, the combination of sequences included in a single concatenated sequence is selected to remove redundant protein sequences, and in some embodiments, the selection of minimal epitopes to be included in a single concatenated sequence is selected to provide optimal HLA donor coverage.

[0508] In some embodiments, the vaccine candidate includes both MHC-I and MHC-II mRNA cassettes. In some embodiments, both mRNAs are included in a single targeted LNP formulation. In some embodiments, the MHC-I and MHC-II mRNAs are combined in a 1:1 (wt:wt) ratio (MHC-I / MHC-II). In other embodiments, the mRNAs are included in a ratio of about 0.1 to 10 (wt:wt), about 0.2 to 5 (wt:wt), and about 0.5 to 2 (wt:wt).

[0509] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid, a sterol, one or more phospholipids comprising at least one anionic phospholipid, a conjugated lipid, and one or more nucleic acid sequences encoding T cell epitopes from Mycobacterium tuberculosis (Mtb). In some embodiments, the nucleic acid sequences encode peptides that bind to MHC molecules and are recognized by T cell receptors (typically 8-11 aa long for MHC Class I / CD8 and 12+ for MHC Class I / CD4).

[0510] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid, a sterol, one or more phospholipids comprising at least one anionic phospholipid, a conjugated lipid, and one or more nucleic acid sequences encoding T cell epitopes from Mycobacterium tuberculosis (Mtb). In some embodiments, the nucleic acid sequences encode peptides that bind to MHC molecules and are recognized by T cell receptors (typically 8-11 aa long for MHC Class I / CD8 and 12+ for MHC Class I / CD4).

[0511] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid, a sterol, one or more phospholipids comprising at least one anionic phospholipid, a conjugated lipid, and one or more nucleic acid sequences encoding Mtb proteins selected from the group consisting of: CFP10 / Rv3874, ESAT-6 / Rv3875, Mtb32A / Rv0125, Mtb39A / Rv1196, and Av85B / Rv1886c.

[0512] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid, a sterol, one or more phospholipids comprising at least one anionic phospholipid, a conjugated lipid, and one or more nucleic acid sequences selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 220. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid, a sterol, one or more phospholipids comprising at least one anionic phospholipid, a conjugated lipid, and a nucleic acid sequence comprising one or more nucleic acid sequences selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 220.

[0513] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid, a sterol, one or more phospholipids comprising at least one anionic phospholipid, a conjugated lipid, and one or more nucleic acid sequences encoding Mtb proteins selected from the group consisting of: Esx W / Rv3620c, Esx V / Rv3619c, PE13 / Rv1195, PPE30 / Rv1802, PPE40 / Rv2356c, and TB10.4 Rv0288. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid, a sterol, one or more phospholipids comprising at least one anionic phospholipid, a conjugated lipid, and one or more nucleic acid sequences selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO: 221, and SEQ ID NO: 222. In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid, a sterol, one or more phospholipids comprising at least one anionic phospholipid, a conjugated lipid, and a nucleic acid sequence comprising one or more nucleic acid sequences selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO: 221, and SEQ ID NO: 222.

[0514] UTR

[0515] In some tandem sequences, untranslated regions (3’ and 5’ UTRs) are selected to maximize mRNA stability and translation efficiency. UTRs can include those from viral proteins or human proteins such as hemoglobin alpha (HBA) or hemoglobin beta (HBB) chains.

[0516] In some embodiments, chemically modified nucleic acids are incorporated into tandem mRNA sequences. In some embodiments, mRNA comprises modified nucleosides. In some embodiments, the chemically modified residue incorporated is a modification of uridine. In some embodiments, the chemically modified nucleic acid incorporated is pseudouridine. In some embodiments, the chemically modified nucleic acid incorporated is N1-methylpseudouridine (also known as 1-methylpseudouridine). In some embodiments, the chemically modified nucleic acid incorporated is thiouridine. In some embodiments, the chemically modified nucleic acid incorporated is 5-methylcytidine. In some embodiments, the chemically modified nucleic acid incorporated is 5-methoxyuridine. In some embodiments, the chemically modified nucleic acid incorporated is 5-methylcytidine. In some embodiments, the chemically modified nucleic acid incorporated is N6-methyladenosine. In some embodiments, the chemically modified nucleic acid incorporated is 2'-O-methyluridine, 2-thiouridine.

[0517] In some embodiments, the mRNA sequence comprises a polyA tail of about 50 to 150 nucleotides in length, about 80 to 140 nucleotides in length, or about 100 to 140 nucleotides in length. In some embodiments, the polyA tail may be interrupted by a short sequence to improve stability.

[0518] Composition

[0519] In some embodiments, the composition further comprises a pharmaceutical excipient.

[0520] In some embodiments, the lipid nanoparticles are in an aqueous medium.

[0521] 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.

[0522] In some embodiments, the lipid nanoparticle comprises a membrane comprising phosphatidylcholine and a sterol. In some embodiments, the sterol is cholesterol. In some embodiments, the lipid nanoparticle comprises a membrane comprising phosphatidylcholine, an ionizable cationic lipid (ICL). In some embodiments, the ICL has the structure of Formula I and cholesterol, wherein the membrane separates the interior of the lipid nanoparticle from an aqueous medium. In some embodiments, the ICL has a structure shown in Table 1A. 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.

[0523] In some embodiments, the molar ratio of phosphatidylcholine to cholesterol is about 1:5 to about 1:2.

[0524] In some embodiments, the membrane further comprises a polymer-conjugated lipid.

[0525] In some embodiments, the lipid nanoparticle comprises an ICL, DSPC, cholesterol, and a polymer-conjugated lipid in a molar ratio of about 49.5:10.3:39.6:2.5.

[0526] In some embodiments, the polymer-conjugated lipid is PEG(2000)-dimyristyl glycerol (PEG-DMG) or PEG(molecular weight 2,000)-dimyristoyl phosphatidylethanolamine (PEG-DMPE).

[0527] The compositions of the present disclosure can be administered by a variety of routes, e.g., 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 a nucleic acid in vivo to a subject.

[0528] In some embodiments, the composition is a liquid pharmaceutical formulation for parenteral administration.

[0529] In some embodiments, the composition is a liquid pharmaceutical formulation for subcutaneous, intramuscular, or intradermal administration.

[0530] In some embodiments, the composition is in the form of a lyophilized powder, which is subsequently reconstituted with an aqueous medium prior to administration.

[0531] In some embodiments, the lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I):

[0532]

[0533] wherein:

[0534] R1is

[0535] 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;

[0536] R2and R3are each independently (Ci-C4)alkyl optionally substituted with hydroxyl; and

[0537] n is an integer equal to 2, 3, or 4.

[0538] wherein a and b in the two R1hydrocarbon chains are the same or different, or one of the two R1hydrocarbon chains is a saturated C 12 -C 18 alkyl.

[0539] In some embodiments, ionizable cationic lipid compositions are provided. In some embodiments, a lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I-A):

[0540]

[0541] wherein:

[0542] R1is

[0543] 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;

[0544] R2and R3are each independently (Ci-C4)alkyl optionally substituted with hydroxyl; and

[0545] n is an integer equal to 2, 3, or 4.

[0546] In some embodiments, a and b in the two R1hydrocarbon chains are the same.

[0547] In some embodiments, a and b in the two R1hydrocarbon chains are different.

[0548] In some embodiments, one of the two R1hydrocarbon chains is a saturated C 12 -C 18 alkyl.

[0549] In some embodiments, ionizable cationic lipid compositions are provided. In some embodiments, a lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I-A):

[0550]

[0551] wherein:

[0552] R1is

[0553] 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;

[0554] R2and R3are each methyl; and

[0555] n is an integer equal to 3.

[0556] In some embodiments, a and b in both R1hydrocarbon chains are the same.

[0557] In some embodiments, a and b in both R1hydrocarbon chains are different.

[0558] In some embodiments, one of the two R1hydrocarbon chains is a saturated C 12 -C 18 alkyl.

[0559] In some embodiments, ionizable cationic lipid compositions are provided. In some embodiments, a lipid nanoparticle (LNP) composition comprises an ionizable cationic lipid having the chemical structure of Formula (I-A):

[0560]

[0561] wherein:

[0562] R1is a saturated C15-C18 hydrocarbon chain,

[0563] R2and R3are each methyl; and

[0564] n is an integer equal to 3.

[0565] In some embodiments, a and b in both R1hydrocarbon chains are the same.

[0566] In some embodiments, a and b in both R1hydrocarbon chains are different.

[0567] In some embodiments, one of the two R1hydrocarbon chains is a saturated C 12 -C 18 alkyl.

[0568] In some embodiments, the present disclosure provides certain LNP compositions. In some aspects, the LNP composition comprises: a nucleic acid; 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; a sterol in a total amount of 0.5 to 50 mol% of the total lipid content in the LNP composition; one or more phospholipids in a total amount of 5 to 50 mol% of the total lipid content in the LNP composition; and a conjugated lipid in a total amount of 0.5 to 2.5 mol% of the total lipid content in the LNP composition. In some aspects, the LNP composition is further characterized in that: the nucleic acid is an mRNA; the ionizable cationic lipid is present in the LNP composition at an N / P ratio relative to the nucleic acid of 4 to 7; the sterol is cholesterol; and the conjugated lipid is a PEG-containing conjugated lipid. In some aspects, the one or more phospholipids in the LNP comprise at least two phospholipids having mismatched acyl chain lengths. In some aspects, the one or more phospholipids in the LNP 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 aspects, the phosphatidylserine (PS) lipids in the LNP consist of, consist essentially of, or comprise dipalmitoyl phosphatidyl-L-serine ((L-serine) DPPS). In some aspects, the one or more phospholipids in the LNP comprise a phospholipid selected from the group consisting of distearoylphosphatidylcholine (DSPC) and hydrogenated soy phosphatidylcholine (HSPC). In some aspects, the one or more phospholipids in the LNP consist of distearoylphosphatidylcholine (DSPC) and dipalmitoyl phosphatidyl-L-serine ((L-serine) DPPS). In some aspects, the PEG-containing conjugated lipid in the LNP is PEG(2000)-dimyristoylglycerol (PEG-DMG).

[0569] In some aspects, the LNP composition has 5 to 50 mol% total phospholipid, including compositions having 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mol% total phospholipid. In some embodiments, the LNP composition is further characterized by: a sterol in a total amount of 0.5 to 45.5 mol% of the total lipid content in the LNP composition; and one or more phospholipids in a total amount of 5 to 50 mol% of the total lipid content in the LNP composition. In some embodiments, the sterol in the LNP composition is cholesterol. In some embodiments, the ionizable cationic lipid is KC3-OA (racemic). In some embodiments, the ionizable cationic lipid is KC3-OA (S). In some embodiments, the ionizable cationic lipid is KC3-OA (R). In some embodiments, the ionizable cationic lipid is KC4-OA (racemic). In some embodiments, the ionizable cationic lipid is KC4-OA (S). In some embodiments, the ionizable cationic lipid is KC3-OA (R). In some embodiments, the ionizable cationic lipid is a mixture of KC3-OA and KC4-OA. In some embodiments, the LNP composition comprises a total of 48 to 54 mol% ionizable cationic lipid.

[0570] In some embodiments, the one or more phospholipids comprises a phosphatidylserine (PS) lipid. In some embodiments, the PS lipid in the LNP composition is DPPS. In some embodiments, the PS lipid in the LNP composition is present in a total of 5 mol%. In some embodiments, the LNP composition comprises a DSPC phospholipid. In some embodiments, the LNP composition comprises 7.5 to 20 mol% DSPC phospholipid.

[0571] In some embodiments, the sterol in the LNP composition is cholesterol. In some embodiments, the LNP composition comprises 25 to 40 mol% cholesterol. In some embodiments, the sterol in the LNP composition is beta sitosterol. In some embodiments, the LNP composition comprises 33 to 35.5 mol% beta sitosterol.

[0572] In some embodiments, the PEG-containing conjugated lipid in the LNP composition is PEG-DMG. In some embodiments, the LNP composition comprises 1.5 to 4.0 mol% PEG- containing conjugated lipid, which is PEG-DMG in the LNP composition. In some embodiments, the PEG-containing conjugated lipid in the LNP composition is PEG-DLG. In some embodiments, the LNP composition comprises 1.0 to 4.0 mol% PEG-containing conjugated lipid, which is PEG-DLG in the LNP composition.

[0573] In some embodiments, the lipid nanoparticle (LNP) vaccine composition comprises: a nucleic acid; a KC3 ionizable cationic lipid having an N / P ratio of 3 to 7 relative to the nucleic acid, the total amount of which is 46 to 54 mol% of the total lipid content in the LNP composition; one or more phospholipids, the total amount of which is 5 to 20 mol% of the total lipid content in the LNP composition; a conjugated lipid, the total amount of which is 1.0 to 3.5 mol% of the total lipid content in the LNP composition; and cholesterol. In some embodiments, the one or more phospholipids in the LNP comprise an anionic phospholipid having a total amount of 2 to 8 mol% of the total lipid content in the LNP composition. In some aspects, the anionic phospholipid is phosphatidylserine (PS). In some embodiments, the anionic phospholipid is an anionic phospholipid selected from distearoylphosphatidylglycerol (DSPG) and dipalmitoylphosphatidylglycerol (DPPG). In some embodiments, the anionic phospholipid is an anionic phospholipid selected from dipalmitoylphosphatidyl-L-serine (DPPS) or distearoylphosphatidyl-L-serine (DSPS).

[0574] How to use

[0575] Targeting dendritic cells

[0576] 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.

[0577] 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

[0578] CDla- / dim, CD11a+, CD11b++, CD11c+, CD14dim / -, CD16a- / dim, CD18+, CD32dim / -, CD33+, CD40+, CD45R0+, CD50+, CD54+, CD64- / dim, CD68+, CD71+, CD80dim, CD86+ / ++, MHC class I++ / +, HLA-DR++ / +, HLA-DP+, and HLA-DQ (Geiseler et al. Dev Immunol. 1998; 6(1-2): 25-39).

[0579] Alternatively, human primary blood dendritic cell lines have been developed and are commercially available from Creative Biolabs.

[0580] CD8+ T cells can produce IL2, IFN-gamma, and TNF, cytokines known to play a critical role during M. tuberculosis infection. Importantly, CD8+ T cells have cytolytic functions to kill M. tuberculosis infected cells either 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 M. tuberculosis. Thus, antigen-producing mRNA LNP delivered to DCs are expected to stimulate CD8+ T cell responses to combat M. tuberculosis infection.

[0581] CD8+ T cells are capable of recognizing M. tuberculosis specific antigens (as peptides) presented by classical and non-classical MHC molecules. Classical restricted CD8+ T cells have been identified that recognize antigens presented by antigen presenting cells in the context of classical MHC la (HLA-A, HLA-B, HLA-C) molecules. Non-classical restricted CD8+ T cells include those CD8+ T cells capable of recognizing Mg antigens in the context of HLA-E molecules (non-MHC la), glycolipids related to class 1 CD1 molecules, and MHC I related molecules (MR1) such as mucosal associated invariant T cells (MAIT). Finally, gd T cells represent a separate population of CD8 (and CD4) T cells that have both intrinsic and adaptive functions in response to M. tuberculosis infection. CD8+ T cells have been shown to play a direct function in response to M. tuberculosis infection, but they also play an important role in coordinating many different functional aspects in the overall host immune response (e.g., interactions to provide optimal CD4 T cell function).

[0582] In some embodiments, LNP are added to cultured human dendritic cells at an appropriate concentration (e.g., 1 to 5 pg / mL mRNA). After a period of time to allow cell uptake and antigen expression, human T cells (HemaCare) can be added, and cell culture media sampled at various times for INF-gamma by Elisa (R&D Systems, DIF50C). Alternatively, cells can be analyzed for CD8+ markers or intracellular IFy production by flow cytometry (PE anti-human IFN-gamma antibody, Biolegend).

[0583] In some embodiments, LNP are administered to a subject at a dose of about 0.01 to about 5 mg / kg mRNA by any of the routes of administration known in the art and / or outlined above. According to some embodiments, a portion of the LNP is taken up by DC cells, while the majority will accumulate in the liver and spleen. DC cells can express antigen peptides, which are processed for MHC I presentation and travel to lymph nodes for presentation to naive T cells, which induces education of memory T cells against the antigen.

[0584] In some embodiments, LNP have been modified with a targeting ligand, such as phosphatidylserine, are administered to a subject at a dose of about 1 pg to about 500 pg mRNA. In other embodiments, the targeting ligand is phosphatidylglycerol. In some embodiments, the targeting LNP are administered at a reduced dose of about 1 pg to about 100 ug mRNA. According to some embodiments, a higher proportion of the LNP can be taken up by DC cells, which allows for increased production of antigen peptides (compared to non-targeting LNP) and more effective vaccination against a pathogen. For example, assessing CD8+ reactivity to an antigen produced in vivo can be done by measuring INFy plasma levels from species-specific IFN-gamma Quantikine ELISA kits from R&D Systems.

[0585] Disclosed herein are methods for preventing infection by mycobacteria, such as Mycobacterium tuberculosis. Additional mycobacteria include, but are not limited to, Mycobacterium avium complex, Mycobacterium leprae, Mycobacterium gordonae, Mycobacterium abscessus, Mycobacterium abscessus, Mycobacterium mucogenicum, and Mycobacterium.

[0586] The administration of the vaccine for inducing a second immune response can provide MHC class II presented epitopes that are capable of eliciting a CD4+ helper T cell response against cells expressing the antigen from which the MHC presented epitopes are derived. Alternatively or additionally, the administration of the vaccine for inducing a second immune response can provide MHC class I presented epitopes that are capable of eliciting a CD8+ T cell response against cells expressing the antigen from which the MHC presented epitopes are derived. Furthermore, the administration of the vaccine for inducing a second immune response can provide one or more neoepitopes (including known neoepitopes) and one or more epitopes that are not cancer specific somatic mutations but are expressed by cancer cells and preferably induce an immune response, preferably a cancer specific immune response, against the cancer cells. In some embodiments, the administration of the vaccine for inducing a second immune response provides a neoepitope that is a MHC class II presented epitope and / or is capable of eliciting a CD4+ helper T cell response against cells expressing the antigen from which the MHC presented epitopes are derived; and provides an epitope that is not a cancer specific somatic mutation that is a MHC class I presented epitope and / or is capable of eliciting a CD8+ T cell response against cells expressing the antigen from which the MHC presented epitopes are derived. In some embodiments, the epitope is not a cancer specific somatic mutation.

[0587] As used herein, "cellular immune response", "cellular response", "cellular response against an antigen" or similar terms are meant to include a cellular response against cells characterized by the presentation of an antigen with class I or class II MHC. The cellular response is associated with cells known as 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 modulating 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 production of more diseased cells. In some embodiments, the present disclosure relates to stimulating an anti-Mycobacterium tuberculosis CTL response against Mycobacterium tuberculosis expressing one or more expressed antigens, and preferably with class I MHC presenting such expressed antigens.

[0588] An "antigen" according to some aspects of the present disclosure encompasses any substance that will elicit an immune response. In particular, an "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with an antibody or a T lymphocyte (T cell). 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 induces, optionally after processing, an immune response, preferably specific to the antigen, including cells expressing the antigen. According to some aspects of the present disclosure, any suitable antigen can be used as a candidate for an immune response, wherein the immune response is preferably a cellular immune response. In the context of embodiments of the present disclosure, an antigen is presented by a cell, e.g. by an antigen presenting cell, including a diseased cell, in particular a cancer cell, in the context of an MHC molecule, which leads to an immune response against the antigen. An antigen can be a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens can include tumor antigens.

[0589] An "antigen peptide" as used herein refers to a part or fragment of an antigen that is capable of stimulating an immune response, preferably a cellular response, against the antigen or against a cell characterized by expressing the antigen, preferably presenting the antigen, e.g. a diseased cell, in particular a cancer cell. Preferably, an antigen peptide is capable of stimulating a cellular response against a cell characterized by presenting the antigen with MHC class I, and preferably is capable of stimulating an antigen reactive cytotoxic T-lymphocyte (CTL). An antigen peptide according to some embodiments is a MHC class I and / or MHC class II presented peptide, or can be processed to yield a MHC class I and / or MHC class II presented peptide. In some embodiments, an antigen peptide comprises an amino acid sequence that substantially corresponds to an amino acid sequence of a fragment of an antigen. In some embodiments, the fragment of an antigen is a MHC class I and / or MHC class II presented peptide. In some embodiments, an antigen peptide comprises an amino acid sequence that substantially corresponds to an amino acid sequence of such a fragment, and is processed to yield such a fragment, i.e. a MHC class I and / or MHC class II presented peptide derived from an antigen. According to some embodiments, if a peptide is presented directly, i.e. without processing, in particular without cleavage, the length of the peptide is suitable for binding to a MHC molecule, in particular a MHC class I molecule. In some embodiments, the length of the peptide is 7 to 20 amino acids, 7 to 12 amino acids, 8 to 11 amino acids, e.g. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids.

[0590] The main types of professional antigen-presenting cells are dendritic cells, which have the broadest range of antigen presentation, and possibly the most important antigen-presenting cells, macrophages, B cells, and certain activated epithelial cells. Dendritic cells (DCs) are a population of white blood cells that capture antigens in peripheral tissues and present them to T cells via both MHC class II and MHC class I antigen presentation pathways. Dendritic cells are known to be potent inducers of immune responses, and activation of these cells is a critical step in inducing anti-tumor immunity. Dendritic cells are conveniently classified as "immature" and "mature" cells, which serves as a simple way to distinguish between two well-characterized phenotypes.

[0591] However, this nomenclature should not be interpreted as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as antigen-presenting cells with high antigen uptake and processing capacity, which correlates with high expression of Fc gamma receptors and mannose receptors. Typically, the mature phenotype is characterized by lower expression of these markers, but high expression of cell surface molecules responsible for T cell activation, such as class I and II MHC, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB). Dendritic cell maturation is considered a state of dendritic cell activation in which such antigen-presenting dendritic cells elicit T cell priming, whereas presentation by immature dendritic cells leads to tolerance. Dendritic cell maturation is primarily induced by biological molecules with microbial signatures detected by innate receptors (bacterial DNA, viral RNA, endotoxins, etc.), proinflammatory cytokines (TNF, IL-1, IFN), ligation of CD40 with CD40L on the surface of dendritic cells, and substances released from cells undergoing stress-induced 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 MHC class II proteins required for interaction with naive T cells; these are only expressed upon stimulation of non-professional antigen-presenting cells with certain cytokines such as IFN gamma. "Antigen-presenting cells" can be loaded with MHC class I presented peptides by transducing the cells with nucleic acids, preferably mRNA, encoding a peptide or a polypeptide comprising a peptide to be presented (e.g., a nucleic acid encoding an antigen).

[0592] In some embodiments, a pharmaceutical composition comprising a gene delivery vehicle that targets dendritic cells or other antigen presenting cells is administered to a patient, resulting in transfection occurring in vivo. As used herein, "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, the nucleic acid is RNA, such as in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to some aspects of the disclosure, nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. According to some aspects of the disclosure, nucleic acids can exist as single- or double-stranded, and as linear or covalently closed circular molecules. According to some aspects of the disclosure, nucleic acids can be isolated. In some embodiments, nucleic acids are (i) amplified in vitro, such as by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, such as by cleavage and separation by gel electrophoresis, or (iv) synthesized, such as by chemical synthesis. Nucleic acids can be used for introduction into cells, i.e., transfecting cells, particularly in the form of RNA that can be prepared by in vitro transcription from a DNA template. In addition, RNA can be modified prior to application by stabilization sequences, capping, and polyadenylation.

[0593] As used herein, the term "RNA" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or essentially of ribonucleotide residues. As used herein, the term "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of a B-D-ribofuranosyl group. As used herein, 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 differs from naturally occurring RNA by addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material to, for example, the termini or to internal positions, such as at one or more nucleotides of the RNA. Nucleotides in the RNA molecule can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally occurring RNA.

[0594] In some embodiments, the RNA is mRNA. The term "mRNA" as used herein means "messenger RNA" and refers to a "transcript" that can be generated 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 some aspects of the present disclosure, mRNA can be generated from a DNA template by in vitro transcription. In the context of RNA used in some aspects of the present disclosure, the term "modification" as used herein includes any modification of the RNA that is not naturally occurring. According to some embodiments, the RNA does not have uncapped 5'-triphosphates. Such uncapped 5'-triphosphates can be removed by treating the RNA with a phosphatase. The RNA according to some aspects of the present disclosure can have modified ribonucleotides to improve its stability and / or to reduce cellular toxicity. For example, in some embodiments, 5-methylcytidines in the RNA are partially or completely replaced by cytidines. In some embodiments, 5-methylcytidines in the RNA are completely replaced by cytidines. Alternatively or in addition, in some embodiments, pseudouridines in the RNA used are partially or completely replaced by uridines. In some embodiments, pseudouridines in the RNA used are completely replaced by uridines.

[0595] In some embodiments, RNA having a 5-cap or a 5'-cap analog can be provided. The term "5-cap" refers to a cap structure present at the 5' end of an mRNA molecule and typically consists of a guanosine nucleotide linked to the mRNA by an unusual 5' to 5' triphosphate linkage. In some embodiments, this guanosine is methylated at the 7-position. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, such as a 7-methylguanosine cap (m'G). In some embodiments, the 5'-cap comprises a 5'-cap analog which resembles the RNA cap structure and is modified to have the ability to stabilize and / or enhance translation of the RNA if linked thereto, preferably in vivo and / or in a cell.

[0596] According to some aspects of the present disclosure, the stability and translation efficiency of the RNA can be modified as desired. For example, by one or more modifications having a stabilizing effect and / or improving the translation efficiency of the RNA, the RNA can be stabilized and its translation improved. Such modifications are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference in its entirety. In order to improve the expression of the RNA used according to some aspects of the present disclosure, it can be modified within the coding region, i.e. the sequence encoding the expressed peptide or protein, preferably without changing the sequence of the expressed peptide or protein, in order to increase the GC content to improve mRNA stability and to perform codon optimization and thus to enhance translation in a cell.

[0597] Provided herein are lipid nanoparticle (LNP) compositions and methods of making and using the same. In some embodiments, the LNP compositions comprise a nucleic acid, such as a messenger ribonucleic acid (mRNA). In some embodiments, the LNP compositions are vaccines comprising an LNP formulation containing an mRNA encoding an epitope of the immune system or an antigen recognized by the immune system.

[0598] In some aspects, the LNP comprises a nucleic acid comprising a chemically modified mRNA, wherein the chemically modified mRNA comprises N1-methyl pseudouridine. In some aspects, the LNP comprises a nucleic acid comprising a 5' untranslated region (UTR) and a 3' UTR, a polyA tail of about 80 to about 140 nucleotides in length, and (i) a 5' enzymatic cap or (ii) a 5' clean cap. In some aspects, the LNP comprises a chemically modified mRNA, wherein the chemically modified mRNA comprises N1-methyl pseudouridine.

[0599] In some embodiments, the LNP composition comprises: (a) a nucleic acid; (b) an ionizable cationic lipid comprising KC3 ionizable cationic lipid at an N / P ratio of 3 to 7 relative to the nucleic acid, the ionizable cationic lipid being present in the LNP composition in a total amount of 46 to 54 mol% of the total lipid content in the LNP composition; (c) one or more phospholipids in a total amount of 5 to 20 mol% of the total lipid content in the LNP composition; (d) one or more anionic phospholipids in a total amount of 2 to 8 mol% of the total lipid content in the LNP composition; (e) a conjugated lipid in a total amount of 1 to 3.5 mol% of the total lipid content in the LNP composition; and (f) a sterol, e.g., cholesterol (e.g., in an amount to provide the remainder of the LNP composition). In some aspects, the one or more anionic phospholipids is phosphatidylserine (PS) or phosphatidylglycerol (PG). In some aspects, the one or more anionic phospholipids is selected from: dipalmitoylphosphatidyl-L-serine (DPPS), or distearoylphosphatidyl-L-serine (DSPS), distearoylphosphatidylglycerol (DSPG), and dipalmitoylphosphatidylglycerol (DPPG). In some aspects, the one or more phospholipids comprises distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC), or a combination thereof. In some aspects, the conjugated lipid is PEG(2000)-dimyristylglycerol (PEG-DMG). In some aspects, the sterol is cholesterol. In some aspects, the ionizable cationic lipid comprises 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 aspects, the ionizable cationic lipid further comprises a KC4 ionizable cationic lipid, e.g., 4- rac-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylbutane-1-amine (AKG-KC4-OA). In some aspects, the LNP composition consists of: 48 mol% KC3-OA, 5 mol% DPPS or DSPG, 5 to 10 mol% DSPC or HSPC, 1.5 mol% PEG-DMG, and cholesterol. In some aspects, the LNP composition consists of: 48 mol% KC3-OA, 5 mol% DPPS or DSPG, 5 mol% DSPC or HSPC, 1.5 mol% PEG-DMG, and 40.5 mol% cholesterol. In some aspects, the LNP composition consists of: 48 mol% KC3-OA, 5 mol% DPPS or DSPG, 10 mol% DSPC or HSPC, 1.5 mol% PEG-DMG, and 35.5 mol% cholesterol.

[0600] In some embodiments, methods of eliciting a T cell response in a host are provided, comprising administering to the host a nucleic acid sequence disclosed herein, or a nucleic acid having at least 90% sequence identity or complementarity to a sequence disclosed herein and / or a sequence encoding a T cell epitope from Mycobacterium tuberculosis (Mtb), or a polynucleotide sequence having at least 90% identity or complementarity to a sequence disclosed herein and / or a polynucleotide sequence of a Mtb antigen recognized by a T cell.

[0601] A lipid nanoparticle (LNP) composition consisting of: a messenger ribonucleic acid (mRNA) encoding one or more Mycobacterium tuberculosis (Mtb) proteins selected from the group consisting of:

[0602] CFP10 / Rv3874, ESAT-6 / Rv3875, Mtb32A / Rv0125, Mtb39A / Rv1196, Ag85B / Rv1886c, EsxW / Rv3620c, EsxV / Rv3619c, PE13 / Rv1195, PPE30 / Rv1802, PPE40 / Rv2356c, and TB10.4 / Rv0288; an ionizable cationic lipid comprising KC3 ionizable cationic lipid at an N / P ratio of 4 to 6 relative to the mRNA, the ionizable cationic lipid being present in the LNP composition in a total amount of 46 to 54 mol% of the total lipid content in the LNP composition; one or more phospholipids selected from the group consisting of: distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), and dipalmitoylphosphatidylcholine (DPPC), the total amount of the one or more phospholipids being 10 to 18 mol% of the total lipid content in the LNP composition; one or more anionic phospholipids selected from the group consisting of: dipalmitoylphosphatidyl-L-serine (DPPS), or distearoylphosphatidyl-L-serine (DSPS), distearoylphosphatidylglycerol (DSPG), and dipalmitoylphosphatidylglycerol (DPPG), the total amount of the one or more anionic phospholipids being 2 to 8 mol% of the total lipid content in the LNP composition; PEG(2000)-dimyristoylglycerol (PEG-DMG) in a total amount of 1 to 3.5 mol% of the total lipid content in the LNP composition; and cholesterol (e.g., 35.5 to 40.5 mol% cholesterol).

[0603] Some aspects of the present disclosure relate to a lipid nanoparticle (LNP) composition comprising a KC3 ionizable cationic lipid, cholesterol, one or more phospholipids comprising at least one anionic phospholipid, a conjugated lipid, and one or more nucleic acid sequences encoding a T cell epitope from Mycobacterium tuberculosis (Mtb).

[0604] In some embodiments, the LNP 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 phospholipid concentration of 10 mol%, wherein each mol% refers to mol% of the total lipid content in the LNP composition.

[0605] In some embodiments, the LNP composition comprises 48 mol% KC3 ionizable cationic lipid, 40.5 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP composition comprises 48 mol% KC3 ionizable cationic lipid, 38.5 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP composition comprises 45 mol% KC3 ionizable cationic lipid, 42.7 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP composition comprises 50 mol% KC3 ionizable cationic lipid, 38.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, and a total phospholipid concentration of 10 mol%, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP composition comprises 48 mol% KC3 ionizable cationic lipid, 40.5 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, and a total phospholipid concentration of 10 mol%, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP 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 phospholipid concentration of 10 mol%, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP composition comprises 46.5 mol% KC3 ionizable cationic lipid, 42 mol% cholesterol, 5 mol% (L-serine) DPPS lipid, where each mol% refers to mol% of total lipid content in the LNP composition. In some embodiments, the LNP composition comprises 15 mol% total phospholipid and 35.5 mol% cholesterol. In some embodiments, the LNP composition comprises 10 mol% total phospholipid and 40.5 mol% cholesterol. In some embodiments, the LNP composition comprises 40.5 mol% cholesterol, 5% anionic lipid (DPPS), and 5% PC (DSPC or DPPC) and a total phospholipid concentration of 10 mol%. In some embodiments, the LNP composition comprises 48 mol% cationic ionizable lipid, 5 mol% PC (DPPC), 5 mol% anionic lipid (DPPS), 40.5 mol% cholesterol, 1.5 mol% conjugated lipid (PEG-DMG).

[0606] In some aspects, the LNP comprises a nucleic acid sequence (e.g., mRNA) encoding a T cell epitope or Mtb antigen recognized by T cells from Mycobacterium tuberculosis (Mtb). In some aspects, the LNP comprises a nucleic acid sequence that is mRNA encoding a concatenated sequence of T cell epitopes or Mtb antigens recognized by T cells present in Mtb. In some aspects, the LNP comprises a nucleic acid sequence that is mRNA encoding one or more Mtb proteins selected from the group consisting of CFP10 / Rv3874, ESAT-6 / Rv3875, Mtb32A / Rv0125, Mtb39A / Rv1196, and Ag85B / Rv1886c. In some aspects, the LNP comprises a nucleic acid sequence that is mRNA comprising one or more nucleic acid sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 220. In some aspects, the LNP comprises a nucleic acid sequence that is mRNA encoding one or more Mtb proteins selected from the group consisting of EsxW / Rv3620c, EsxV Rv3619c, PE13 / Rv1195, PPE30 / Rv1802, PPE40 / Rv2356c, and TB10.4 / Rv0288. In some aspects, the LNP comprises a nucleic acid sequence that is mRNA comprising one or more nucleic acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO: 221, and SEQ ID NO: 222. In some aspects, the LNP comprises a nucleic acid sequence comprising a concatenated nucleic acid coding sequence comprising N-terminal and C-terminal signal peptides selected from the group consisting of Sec / MITD, Lamp1, HLA-Dra, or tPA. In some aspects, the LNP comprises a nucleic acid sequence that is mRNA having a sequence selected from the group consisting of SEQ ID NO: 34, 36, 38, 40, 42, 44, 224, and 226. In some aspects, the LNP comprises a nucleic acid that is mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 33, 35, 37, 39, 41, 43, 86-105, 207-210, 223, and 225.

[0607] In some embodiments, the one or more nucleic acids are mRNA. In some embodiments, the mRNA encodes a concatenated sequence of T cell epitopes present in Mtb. In some embodiments, the concatenated sequence of T cell epitopes comprises the amino acid sequences set forth in SEQ ID NOs: 1-17, 106-137, 138-203. In some embodiments, the concatenated sequence of T cell epitopes comprises an amino acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequences set forth in SEQ ID NOs: 1-17, 45-85, 106-137, 138-203. In some embodiments, the concatenated nucleotide sequence comprises two or more sequences encoding peptides or proteins that can elicit MHC class II restricted CD4 T cell responses.

[0608] In some embodiments, the two or more MHC class II epitopes are selected from the group of: EsxV (Rv3619), EsxW (Rv3620c), EsxB / CFP10 (Rv3874), EsxA / ESAT-6 (Rv3875), AMtb39A (Rv1196), Ag85B (Rv1886c), and EsxH / TB 10.4 (Rv0288). In some embodiments, the two or more MHC class II epitopes comprise a peptide or protein from:

[0609] EsxV (Rv3619), EsxW (Rv3620c), EsxB / CFP10 (Rv3874), EsxA / ESAT-6 (Rv3875), AMtb39A (Rv1196), Ag85B (Rv1886c), and EsxH / TB 10.4 (Rv0288) (SEQ ID NOs. 1-7).

[0610] In some embodiments, the concatenated nucleic acid coding sequence comprises, in order from N-terminus to C-terminus, seven proteins selected from the group of:

[0611] EsxB / CFP10 (Rv3874), EsxA / ESAT-6 (Rv3875), EsxH / TB10.4 (Rv0288), ΔAg85B (Rv1886c), ΔMtb39A (Rvl 196), EsxW (Rv3620c), and EsxV (Rv3619), or EsxB / CFP10 (Rv3874), EsxA / ESAT-6 (Rv3875), EsxW (Rv3620c), EsxV (Rv3619), EsxH / TB10.4 (Rv0288), ΔAg85B (Rv1886c), and ΔMtb39A (Rvl 196), or EsxB / CFP10 (Rv3874), ΔMtb39A (Rvl 196), EsxA / ESAT-6 (Rv3875), EsxW (Rv3620c), EsxH / TB10.4 (Rv0288), EsxV (Rv3619), and ΔAg85B (Rv1886c). (SEQ ID NO 18, 19, and 20).

[0612] In some embodiments, the composition comprises nucleic acids encoding 5 or more non-overlapping CD4 T cell epitopes in the form of peptides, wherein optionally the peptides are 12 to 50 amino acids long.

[0613] In some embodiments, the tandem nucleic acid coding sequence optionally comprises 10 selected MHC-II epitopes comprising:

[0614] AQIYQAVSAQAAAIH (SEQ ID NO. 9), PSPSMGRDIKVQFQS (SEQ ID NO. 10), GINTIPIAINEAEYV (SEQ ID NO. 11), AAFQGAHARFVAAAA (SEQ ID NO. 12), AGWLAFFRDLVARGL (SEQ ID NO. 13), ASIIRLVGAVLAEQH (SEQ ID NO. 14), MSFVTTQPEALAAAA (SEQ ID NO. 8), MHVSFVMAYPEMLAA (SEQ ID NO. 15), AYGSFVRTVSLPVGA (SEQ ID NO. 16), and LENDNQLLYNYPGAL (SEQ ID NO. 17).

[0615] In some embodiments, the tandem nucleic acid coding sequence comprises a GPGPG (SEQ ID NO. 228) linker sequence between each tandem epitope.

[0616] In some embodiments, the one or more nucleic acids comprise the nucleic acid sequence set forth in SEQ ID NO: 34, 36, 38, 40, 42, and 44.

[0617] In some embodiments, the one or more nucleic acids comprise a nucleic acid sequence having at least 90% identity, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 34, 36, 38, 40, 42, and 44.

[0618] In some embodiments, the tandem nucleic acid coding sequence comprises N-terminal and C-terminal signal peptides selected from Sec / MITD, Lamp1, HLA-Dr alpha, or tPA.

[0619] In some embodiments, the one or more nucleic acids comprise a chemically modified mRNA, wherein the chemically modified mRNA comprises N1-methylpseudouridine.

[0620] In some embodiments, the one or more nucleic acids comprise a 5’ untranslated region (UTR) and a 3’ UTR, a polyA tail of about 80 to about 140 nucleotides in length, and (i) a 5’ enzymatic cap or (ii) a 5’ clean cap.

[0621] In some embodiments, the one or more nucleic acids are mRNAs having a sequence selected from the group consisting of SEQ ID NO: 34, 36, 38, 40, 42, 44, 224, and 226.

[0622] In some embodiments, the one or more nucleic acids are mRNAs, and wherein the amino acid sequence encoded by the mRNA is selected from the group consisting of SEQ ID NO: 33, 35, 37, 39, 41, 43, 86 to 105, 207 to 210, 223, and 225.

[0623] In some embodiments, the nucleic acid encoded tandem sequence comprises two or more MHC class I epitopes selected from the group consisting of SEQ ID NO: 106 to 137 and 138 to 203.

[0624] In some embodiments, the nucleic acid encoded tandem sequence comprises two or more MHC class I epitopes present in Mycobacterium tuberculosis that remove epitopes present in BCG and are selected from the group consisting of SEQ ID NO: 86 to 95.

[0625] In some embodiments, the nucleic acid encoded tandem sequence comprises two or more MHC class I epitopes that are ordered to minimize the production of junction neoepitopes and are selected from the group consisting of SEQ ID NO: 86 to 105.

[0626] In some embodiments, the nucleic acid sequence is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to a nucleic acid sequence of the present disclosure. In some embodiments, the polypeptide sequence is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to a polypeptide sequence of the present disclosure.

[0627] 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.

[0628] 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.

[0629] In some embodiments, the bacterial infection is a Mycobacterium tuberculosis infection.

[0630] In some embodiments, lipid nanoparticles are administered parenterally.

[0631] Generally speaking, it is possible to administer the drug 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.

[0632] In some embodiments, the composition is administered by inhalation.In some embodiments, the composition is formulated as a nasal spray and / or aerosol.

[0633] 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.

[0634] As used herein in the context of administration, "parenterally" means modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinally, epidural, and intrasternal injections and infusions.

[0635] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral (i.e., through the digestive tract) and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, inhalation, subcapsular, subarachnoid, respiratory mucosal, intraspinally, epidural, and intrasternal injections and infusions. Intravenous injections and infusions are often, but not completely, used for liposomal drug administration.

[0636] The dosage regimen can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, one or more doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0637] 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.

[0638] The dosage of the compound and / or its pharmaceutically acceptable salt or the LNP comprising the compound and / or its pharmaceutically acceptable salt can vary within wide limits and in each particular case should be adjusted to the individual conditions and the pathogen to be controlled, naturally.

[0639] Additional embodiments

[0640] The following additional embodiments are provided for illustrative purposes.

[0641] Embodiment 1: A lipid nanoparticle (LNP) composition comprising a KC3 ionizable cationic lipid, cholesterol, one or more phospholipids comprising at least one anionic phospholipid, a conjugated lipid, and one or more nucleic acid sequences encoding a T cell epitope from Mycobacterium tuberculosis (Mtb).

[0642] Embodiment 2: The composition of embodiment 1, wherein the composition comprises:

[0643] 48 mol% of KC3 ionizable cationic lipids,

[0644] 40.5 mol% cholesterol,

[0645] 5 mol% (L-serine) DPPS lipid,

[0646] 5 mol% DSPC or DPPC; and

[0647] total 10 mol% phospholipid concentration;

[0648] Wherein each mol% refers to the mol% of the total lipid content in the LNP composition.

[0649] Embodiment 3: The composition of Embodiment 1 or Embodiment 2, wherein the one or more nucleic acids is mRNA.

[0650] Embodiment 4: The composition of Embodiment 3, wherein the mRNA encodes a tandem sequence of a T cell epitope present in Mtb.

[0651] Embodiment 5: The composition of Embodiment 4, wherein the tandem sequence of T cell epitopes comprises the amino acid sequence shown in SEQ ID NOs: 1 to 17, 106 to 137, 138 to 203.

[0652] Embodiment 6: The composition of Embodiment 4, wherein the tandem sequence of the T cell epitope comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 to 17, 45 to 85, 106 to 137, 138 to 203.

[0653] Embodiment 7: The composition of embodiment 5 or embodiment 6, wherein the tandem nucleotide sequence comprises two or more sequences encoding peptides or proteins that elicit an MHC class II restricted CD4 T cell response.

[0654] Embodiment 8: The composition of embodiment 7, wherein the two or more MHC class II epitopes are selected from the group consisting of: EsxV (Rv3619), EsxW (Rv3620c), EsxB / CFP10 (Rv3874), EsxA / ESAT-6 (Rv3875), AMtb39A (Rv1196), Ag85B (Rv1886c), and ExH / TB10.4 (Rv0288).

[0655] Embodiment 9: The composition of embodiment 7, wherein the two or more MHC class II epitopes comprise a peptide or protein from the group consisting of:

[0656] EsxV (Rv3619), EsxW (Rv3620c), EsxB / CFP10 (Rv3874), EsxA / ESAT-6 (Rv3875), AMtb39A (Rv1196), Ag85B (Rv1886c), and ExH / TB10.4 (Rv0288) (SEQ ID NO. 1-7).

[0657] Embodiment 10: The composition of embodiment 5 or embodiment 6, wherein the tandem nucleic acid coding sequence comprises, in order from N-terminus to C-terminus, seven proteins selected from the group consisting of:

[0658] EsxB / CFP10 (Rv3874), EsxA / ESAT-6 (Rv3875), EsxH / TB10.4 (Rv0288), AMtb39A (Rv1196), Ag85B (Rv1886c), EsxW (Rv3620c), and EsxV (Rv3619), or EsxB / CFP10 (Rv3874), EsxA / ESAT-6 (Rv3875), EsxW (Rv3620c), EsxV (Rv3619), EsxH / TB10.4 (Rv0288), AMtb39A (Rv1196), and Ag85B (Rv1886c), or EsxB / CFP10 (Rv3874), AMtb39A (Rv1196), EsxA / ESAT-6 (Rv3875), EsxW (Rv3620c), EsxH / TB10.4 (Rv0288), EsxV (Rv3619), and Ag85B (Rv1886c). (SEQ ID NO. 18, 19, and 20).

[0659] Embodiment 11: The composition of any one of embodiments 7-10, comprising nucleic acid encoding 5 or more non-overlapping CD4 T cell epitopes as peptides, wherein optionally the peptides are 12 to 50 amino acids long.

[0660] Embodiment 12: The composition of embodiment 5 or embodiment 6, wherein the tandem nucleic acid coding sequence optionally comprises 10 selected MHC-II epitopes comprising:

[0661] AQIYQAVSAQAAAIH (SEQ ID NO. 9), PSPSMGRDIKVQFQS (SEQ ID NO. 10), GINTIPIAINEAEYV (SEQ ID NO. 11), AAFQGAHARFVAAAA (SEQ ID NO. 12), AGWLAFFRDLVARGL (SEQ ID NO. 13), ASIIRLVGAVLAEQH (SEQ ID NO. 14), MSFVTTQPEALAAAA (SEQ ID NO. 8), MHVSFVMAYPEMLAA (SEQ ID NO. 15), AYGSFVRTVSLPVGA (SEQ ID NO. 16), and LENDNQLLYNYPGAL (SEQ ID NO. 17).

[0662] Embodiment 13: The composition of embodiment 7, wherein the tandem nucleic acid coding sequence comprises a GPGPG (SEQ ID NO: 228) linker sequence between each tandem epitope.

[0663] Embodiment 14: The composition of any one of embodiments 3-13, wherein the one or more nucleic acids comprise the nucleic acid sequences set forth in SEQ ID NOs: 34, 36, 38, 40, 42, and 44.

[0664] Embodiment 15: The composition of any one of embodiments 3-13, wherein the one or more nucleic acids comprise a nucleic acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to the nucleic acid sequences set forth in SEQ ID NOs: 34, 36, 38, 40, 42, and 44.

[0665] Embodiment 16: The composition of any one of embodiments 3-13, wherein the tandem nucleic acid coding sequence comprises N-terminal and C-terminal signal peptides selected from Sec / MITD, Lamp1, HLA-Dr alpha, or tPA.

[0666] Embodiment 17: The composition of any one of embodiments 3-16, wherein the one or more nucleic acids comprise a chemically modified mRNA, wherein the chemically modified mRNA comprises N1-methyl pseudouridine.

[0667] Embodiment 18: The composition of any one of embodiments 3-16, wherein the one or more nucleic acids comprise a 5’ untranslated region (UTR) and a 3’ UTR, a poly A tail of about 80 to about 140 nucleotides in length, and (i) a 5’ enzymatic cap or (ii) a 5’ clean cap.

[0668] Embodiment 19: The composition of any one of embodiments 3-16, wherein the one or more nucleic acids is an mRNA having a sequence selected from SEQ ID NO: 34, 36, 38, 40, 42, and 44.

[0669] Embodiment 20: The composition of any one of embodiments 3-16, wherein the one or more nucleic acids is an mRNA, and wherein the amino acid sequence encoded by the mRNA is selected from SEQ ID NO: 33, 35, 37, 39, 41, 43, 86-105, and 207-210.

[0670] Embodiment 21: The composition of embodiment 5 or embodiment 6, wherein the nucleic acid encoded tandem sequence comprises two or more Class I MHC epitopes selected from SEQ ID NO: 106-137 and 138-203.

[0671] Embodiment 22: The composition of embodiment 5 or embodiment 6, wherein the nucleic acid encoded tandem sequence comprises two or more Class I MHC epitopes present in Mycobacterium tuberculosis that removes epitopes present in BCG and is selected from SEQ ID NO: 86-95.

[0672] Embodiment 23: The composition of embodiment 5 or embodiment 6, wherein the nucleic acid encoded tandem sequence comprises two or more Class I MHC epitopes that are ordered to minimize the production of junction neoepitopes and is selected from SEQ ID NO: 86-105.

[0673] Embodiment 24: The composition of any one of embodiments 1-23, wherein the cationic lipid is KC3-OA, KC3-PA, KC3-Ol, KC3-C17(8:1), or KC3-C15(C8:1).

[0674] Embodiment 25: The composition of any one of embodiments 1-24, wherein the LNP comprises a total amount of conjugated lipid that is less than 2 mol% of the total lipid content in the LNP composition.

[0675] Embodiment 26: The composition of any one of embodiments 1-24, wherein the total amount of ionizable cationic lipid is 45-55 mol% of the total lipid content in the LNP composition.

[0676] wherein the total amount of cholesterol is 35 to 45 mol% of the total lipid content in the LNP composition;

[0677] wherein the total amount of the one or more phospholipids is 7 to 15 mol% of the total lipid content in the LNP composition;

[0678] wherein the one or more phospholipids consist of DSPC, the PS lipid is one or more lipids selected from DPPS and DSPS in L-serine configuration; and

[0679] the total amount of the PS lipid is about 5 mol% of the total lipid content in the LNP composition.

[0680] Embodiment 27: The composition of any one of embodiments 1 to 24, wherein the conjugated lipid is PEG-DMG; and

[0681] wherein the PS lipid is selected from DSPS (L-isomer) and DPPS.

[0682] Embodiment 28: The composition of any one of embodiments 1 to 27, wherein the ionizable cationic lipid is KC3-OA.

[0683] Embodiment 29: The composition of any one of embodiments 1 to 28, wherein the N / P ratio of the LNP composition is 4 to 7.

[0684] Embodiment 30: The composition of any one of embodiments 1 to 28, wherein the N / P ratio of the LNP composition is 5 to 6.

[0685] Embodiment 31: A nucleic acid lipid nanoparticle (LNP) composition comprising: an mRNA having at least 90% identity to the nucleic acid sequence set forth in SEQ ID NO: 34, 36, 38, 40, 42, and 44, an ionizable cationic lipid KC3-PA, and a (L-serine) PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition.

[0686] Embodiment 32: The composition of embodiment 31, wherein 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.

[0687] Embodiment 33: A nucleic acid lipid nanoparticle (LNP) composition comprising:

[0688] mRNA having at least 90% identity to the nucleic acid sequence set forth in SEQ ID NO: 34, 36, 38, 40, 42, and 44;

[0689] KC3 ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content in the LNP composition;

[0690] cholesterol in a total amount of 23.5 to 43.5 mol% of the total lipid content in the LNP composition;

[0691] (L-serine)PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content in the LNP composition;

[0692] DSPC or HSPC phospholipid in a total amount of 5 to 25 mol% of the total lipid content in the LNP composition; and

[0693] PEG-containing conjugated lipid in a total amount of 0.5 mol% to 2.5 mol% of the total lipid content in the LNP composition.

[0694] Embodiment 34: The composition of embodiment 1, comprising an ionizable lipid having the chemical structure:

[0695]

[0696] wherein

[0697] R1is 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; R2and R3are each independently methyl; and n is an integer equal to 2 or 3.

[0698] Embodiment 35: The composition of embodiment 34, wherein n is 3.

[0699] Embodiment 36: The composition of any one of the preceding embodiments, wherein the composition is a vaccine.

[0700] Embodiment 37: A pharmaceutical composition comprising the lipid nanoparticle of any one of the preceding embodiments and a pharmaceutically acceptable carrier.

[0701] Embodiment 38: A nucleic acid encoding a tandem amino acid sequence of T cell epitopes present in Mycobacterium tuberculosis, the nucleic acid having at least 90% identity to the nucleic acid sequence set forth in SEQ ID NO: 34, 36, 38, 40, 42, and 44.

[0702] Example

[0703] While the disclosure has been described in connection with certain embodiments thereof, and while it has been shown and described as being particularly applicable to the illustrated examples, it will be apparent to those of ordinary skill in the art that numerous modifications, substitutions and changes in the methods and apparatuses of the disclosure can be made without departing from the disclosure. Accordingly, it is intended that all such modifications, substitutions, and changes be included as further embodiments of the disclosure. In particular, any combination of the features, terms, or elements of the various illustrative components and examples described herein can be included in the disclosure.

[0704] Unless otherwise expressly stated, the isomeric form of the phosphatidylserine lipid used in the examples is phosphatidyl-L-serine.

[0705] Certain embodiments 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 embodiments.

[0706] Example 1: Synthesis of ionizable lipids

[0707] 1. 2-((S)-2,2-bis((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N- dimethylethan-1-amine (AKG-KC2-01, O-12095)

[0708] 2. 3-((S)-2,2-bis((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N- dimethylpropan-1-amine (AKG-KC3-01, O-12096)

[0709] 3. 2-((S)-2,2-bis((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1- amine (AKG-KC2-OA, O-11880)

[0710] 4. 2-((S)-2,2-bis((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1- amine (AKG-KC2-PA, O-11879)

[0711] 5. 3-((S)-2,2-bis((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1- amine (AKG-KC3-OA, O-11957)

[0712] 6. 3-((S)-2,2-bis((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1- amine (AKG-KC3-PA, O-12418)

[0713] 7. 3-((S)-2,2-bis((Z)-heptadec-8-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1- amine (AKG-KC3-C17(C8:1))

[0714] 8. (S)-3-(2,2-bisheptadecyl-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine (AKG-KC3-C17)

[0715] Following synthesis: 2-((S)-2,2-bis((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4- yl)-N,N-dimethylpropan-1-amine (AKG-KC2-01, O-12095)

[0716] 3-((S)-2,2-bis((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan- 1-amine (AKG-KC3-01, O-12096)

[0717] Figure 32 is a scheme showing the synthesis of AKG-KC2-01 and AKG-KC3-01.

[0718] Experimental procedure

[0719] Synthesis of (6Z,12Z)-1-bromooctadeca-6,12-diene, 2

[0720]

[0721] To a solution of (6Z, 12Z)-octadeca-6, 12-dien-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 x 100 mL). The combined organics were washed with brine, then dried over magnesium sulfate, 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 x 100 mL). The combined organics were washed with brine, then dried over magnesium sulfate, 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 10% ethyl acetate in n-hexane as eluent to give (6Z, 12Z)-1-bromooctadeca-6, 12-diene, 3 as a yellow oil (2.9 g, 8.89 mmol, 65%).

[0722] 1 H NMR (300 MHz, CDC13): 5.36-5.33 (m, 4H), 3.42-3.37 (t, J = 7.5 Hz, 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 = 6.6 Hz, 3H).

[0723] Synthesis of (6Z,12Z,25Z,31Z)-heptatriacontac-6,12,25,31-tetraen-19-ol, 3

[0724]

[0725] A solution of (6Z, 12Z)-1-bromooctadeca-6, 12-diene, 2 (2 g, 6.08 mmol) in ether (10 mL) was added to a mixture of magnesium turnings (162 mg, 6.69 mmol) and iodine in ether (2 mL) at room temperature under argon. The mixture was stirred at room temperature for 90 minutes (magnesium turnings consumed), 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 1 N HC1 solution. The mixture was extracted with ethyl acetate (2 x 100 mL) and the combined organics were washed with water, then brine. The organics were dried over magnesium sulfate, filtered, and the filtrate was concentrated under vacuum to give 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 2 N HC1. The aqueous solution was extracted with dichloromethane (2 x) and combined. 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 crude oil was purified on silica using 10% to 30% ethyl acetate in n-hexane as eluent to give (6Z, 12Z, 25Z, 31Z)-heptatriaconta-6, 12, 25, 31-tetraen-19-ol, 3 as a clear oil (0.29 g, 0.55 mmol, 18%).

[0726] 1 H NMR (300 MHz, CDC13): 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 = 6.6 Hz, 6H).

[0727] Synthesis of (6Z,12Z,25Z,31Z)-heptatriacontac-6,12,25,31-tetraen-19-one, 4

[0728]

[0729] 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 was added pyridinium chlorochromate (0.29 g, 1.32 mmol) at 0 °C. 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.

[0730] 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).

[0731] 2-((S)-2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)-1,3-dioxolane-4-yl)ethane- Synthesis of 1-ol, 7

[0732]

[0733] (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%).

[0734] 1H NMR (300 MHz, CDC13): 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), 2.23-2.19 (t, J = 6.6 Hz, 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 = 6.6 Hz, 6H).

[0735] 3-((S)-2,2-bis((9Z,12Z)-octadec-9,12-dien-1-yl)-1,3-dioxolane-4-yl)propane- Synthesis of 1-ol, 8

[0736]

[0737] A mixture of (6Z,12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-one, 4 (0.50 g, 0.95 mmol), (S)-(3)-(2,2-dimethyl-l,3-dioxolan-4-yl)propan-1-ol 6 (0.76 g, 4.75 mmol), and p-toluenesulfonic acid pyridinium (36 mg) in toluene (10 mL) was heated at reflux under a nitrogen overpressure. After 12 h, the mixture was concentrated under vacuum to give a crude oil. The resulting crude oil was purified by chromatography on silica using 20% to 40% ethyl acetate in n-hexane as eluent to give 3-((S)-2,2-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)-l,3-dioxolan-4-yl)propan-1-ol, 8 (0.48 g, 0.76 mmol, 80%) as a clear oil.

[0738] 1 H NMR (300 MHz, CDC13): 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), 2.23-2.19 (t, J = 6.6 Hz, 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 = 6.6 Hz, 6H).

[0739] 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)

[0740]

[0741] ​To a solution of 2-((S)-2,2-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxan-4- yl)ethan-1-ol, 7 (0.49 g, 0.79 mmol) in dichloromethane (10 mL) at 0 °C was added methanesulfonyl chloride (73 μL, 0.95 mmol) and triethylamine (0.26 mL, 1.2 mmol). The solution was allowed to warm to room temperature and stirred for an additional 1 h. The reaction was quenched with water and extracted with dichloromethane (2 x 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. A 2 M solution of dimethylamine (10 mL) was added to the resulting crude oil and allowed to stir for 24 h. The mixture was then quenched with water and extracted with dichloromethane (2 x 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 100% ethyl acetate in n-hexane as eluent to give 2-((S)-2,2-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxan-4-yl)-N,N- dimethylethan-1-amine, (AKG-KC2-01, O-12095), as a clear oil (206 mg, 0.32 mmol, 41%).

[0742] 1 H NMR (300 MHz, CDC13): 5.35-5.32 (m, 8H), 4.08-4.03 (m, 2H), 3.47 (t, J = 6.8 Hz, 1H), 2.36-2.27 (m, 2H), 2.21 (s, 6H), 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 = 3.7 Hz, 6H).

[0743] C 43 H 79 MS (APCI) for N02: 642.6

[0744] 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)

[0745]

[0746] The procedure has been described previously.

[0747] 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%).

[0748] 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).

[0749] C 44 H 81 MS (APCI) of NO2: 656.6

[0750] 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)

[0751] 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)

[0752] 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)

[0753] 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)

[0754] Figure 33 is a scheme showing the synthesis of AKG-KC2-OA, AKG-KC2-PA, AKG-KC3-OA, and AKG-KC3-PA.

[0755] Experimental procedures (refer to the previously described synthesis of AKG-KC2-01)

[0756] Synthesis of (Z)-1-bromooctadec-9-ene 3

[0757]

[0758] The procedure has been described previously.

[0759] (Z)-1-bromooctadec-9-ene (6.4 g, 19.33 mmol) as a clear oil.

[0760] 1 H NMR (300 MHz, CDC13): 5.36-5.32 (m, 2H), 3.41 (t, J = 7.5 Hz, 2H), 2.01-1.99 (m, 4H), 1.87-1.82 (m, 2H), 1.44-1.26 (m, 22H), 0.87 (t, J = 6.6 Hz, 3H).

[0761] (Z)-16-Bromohexadec-7-ene 4

[0762]

[0763] 1 H NMR (300 MHz, CDC13): 5.36-5.32 (m, 2H), 3.41 (t, J = 7.5 Hz, 2H), 2.01-1.99 (m, 4H), 1.87-1.82 (m, 2H), 1.44-1.26 (m, 22H), 0.87 (t, J = 6.6 Hz, 3H).

[0764] Synthesis of (9Z,28Z)-heptatriaconta-9,28-dien-19-ol 5

[0765]

[0766] The procedure has been described previously.

[0767] (9Z,28Z)-heptatriaconta-9,28-dien-19-ol (1.2 g, 2.25 mmol, 47%) as a solid.

[0768] 1 H NMR (300 MHz, CDC13): 5.36-5.29 (m, 4H), 3.57 (bs, 1H), 2.01-1.97 (m, 8H), 1.42-1.26 (m, 53H), 0.89 (t, J = 6.6 Hz, 6H).

[0769] (7Z,26Z)-Tritriaconta-7,26-dien-17-ol

[0770]

[0771] 1H NMR (300 MHz, CDC13): 5.36-5.29 (m, 4H), 2.03-1.98 (m, 8H), 1.42-1.26 (m, 44H), 0.90-0.89 (t, J = 6.6 Hz, 6H).

[0772] Synthesis of (9Z,28Z)-heptatriaconta-9,28-dien-19-one 7

[0773]

[0774] The procedure has been described previously.

[0775] (9Z,28Z)-heptatriaconta-9,28-dien-19-one as a clear oil (0.89 g, 1.67 mmol, 74%).

[0776] 1 H NMR (300 MHz, CDC13): 5.36-5.29 (m, 4H), 2.03-1.98 (m, 8H), 1.42-1.26 (m, 44H), 0.90-0.89 (t, J = 6.6 Hz, 6H).

[0777] (7Z,26Z)-Tritriaconta-7,26-dien-17-one 8

[0778]

[0779] 1 H NMR (300 MHz, CDC13): 5.36-5.29 (m, 4H), 2.03-1.98 (m, 8H), 1.42-1.26 (m, 44H), 0.90-0.89 (t, J = 6.6 Hz, 6H).

[0780] Synthesis of 2-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol become

[0781]

[0782] The procedure has been described previously.

[0783] 2-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol as a clear oil (0.39 g, 0.63 mmol, 74%).

[0784] 1H NMR (300 MHz, CDC13): 5.36-5.28 (m, 4H), 4.22-4.10 (m, 1H), 4.08-4.05 (m, 1H), 3.82-3.79 (m, 2H), 3.48 (t, J = 6.8 Hz, 1H), 2.24-2.21 (m, 1H), 2.01-1.99 (m, 8H), 1.81-1.80 (m, 2H), 1.59-1.54 (m, 6H), 1.34-1.26 (m, 45H), 0.87 (t, J = 6.3 Hz, 6H).

[0785] 2-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol, 10 synthesis

[0786]

[0787] The procedure has been described previously.

[0788] 2-((S)-2,2-bis((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol (1.02 g, 1.65 mmol, 51%) as a clear oil.

[0789] 1 H NMR (300 MHz, CDC13): 5.36-5.29 (m, 4H), 4.23-4.10 (m, 1H), 4.07-4.05 (m, 1H), 3.82-3.79 (m, 2H), 3.48 (t, J = 6.6 Hz, 1H), 2.24-2.12 (m, 1H), 2.01-1.97 (m, 8H), 1.84-1.78 (m, 2H), 1.57-1.55 (m, 8H), 1.34-1.29 (m, 35H), 0.87 (t, J = 6.3 Hz, 6H).

[0790] 3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol, 11 synthesis

[0791]

[0792] The procedure has been described previously.

[0793] 3-((S)-2,2-bis((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol (0.41 g, 0.65 mmol, 76%) as a clear oil.

[0794] 1H NMR (300 MHz, CDC13): 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 = 6.3 Hz, 6H).

[0795] Synthesis of 3-((S)-2,2-bis((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol

[0796]

[0797] The procedure has been described previously.

[0798] 3-((S)-2,2-bis((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol as a clear oil (0.9 g, 1.56 mmol, 80%).

[0799] 1 H NMR (300 MHz, CDC13): 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, 37H), 0.87 (t, J = 6.3 Hz, 6H).

[0800] Synthesis of 2-((S)-2,2-bis((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N- dimethylethan-1-amine (AKG-KC2-OA, O-11880) Synthesis of 3-((S)-2,2-bis((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol

[0801]

[0802] The procedure has been described previously.

[0803] 2-((S)-2,2-bis((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine as a clear oil (AKG-KC2-OA, O-11880) (200 mg, 0.31 mmol, 49%).

[0804] 1H NMR (300 MHz, CDC13): 5.38-5.28 (m, 4H), 4.08-4.01 (m, 2H), 3.48 (t, J = 6.8 Hz, 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, J = 6.3 Hz, 6H).

[0805] C 43 H 83 MS (APCI) for NO2: 646.7

[0806] Synthesis of 2-((S)-2,2-bis((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N- dimethylethan-1-amine (AKG-KC2-PA, O-11879)

[0807]

[0808] The procedure has been described previously.

[0809] 2-((S)-2,2-bis((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1- amine as a clear oil, (AKG-KC2-PA, O-11879), (195 mg, 0.33 mmol, 18%).

[0810] 1 H NMR (300 MHz, CDC13): 5.38-5.28 (m, 4H), 4.08-4.01 (m, 2H), 3.48 (t, J = 6.8 Hz, 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, J = 6.3 Hz, 6H).

[0811] C 39 H 75 MS (APCI) for NO2: 590.6

[0812] Synthesis of 3-((S)-2,2-bis((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N- dimethylpropan-1-amine (AKG-KC3-OA, O-11957)

[0813]

[0814] The procedure has been described previously.

[0815] 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.

[0816] 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)

[0817] C 44 H 85 MS (APCI) of NO2: 660.6

[0818] Synthesis of 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)

[0819]

[0820] The procedure has been described previously.

[0821] 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.

[0822] 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).

[0823] C 44 H 85 MS (APCI) for NO2: 604.6

[0824] 3-((S)-2,2-Di((Z)-heptadecan-8-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1- amine, synthesis of (S)-3-(2,2-Bisheptadecyl-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1- amine, synthesis of AKG-KC3-C17

[0825] Figure 34 is a scheme showing the synthesis of AKG-KC3-C17(C8:1) and AKG-KC3-C17.

[0826] Experimental procedure

[0827] Synthesis of (9Z,26Z)-tricosa-9,26-dien-18-one, 2

[0828]

[0829] To a stirred solution of oleoyl chloride (10 g, 33.3 mmol) in toluene (50 mL) at 0 °C was added triethylamine (5.8 mL, 33.3 mmol). A large amount of precipitate formed and the mixture was allowed to stir at room temperature for 8 hours. The mixture was quenched with 2% sulfuric acid solution and then extracted with ethyl acetate. 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 resulting oil was diluted with ethanol (20 mL) and [2N NaOH] (30 mL) was added. The mixture was heated at 100 °C for 12 hours and then cooled. The mixture was diluted with 2N HC1 solution until a pH of 4 was obtained. The mixture was extracted with ethyl acetate. 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. This oil was purified on silica using 10-20% ethyl acetate in n-hexane as eluent to give (9Z,26Z)-tricosa-9,26-dien-18-one, 2 as a yellow oil (3.8 g, 44%).

[0830] 1 H NMR (300 MHz, CDC13): δ ppm 5.35-5.31 (m, 4H), 2.39-2.34 (m, 4H), 2.0-1.85 (m, 8H), 1.57-1.52 (m, 4H), 1.27-1.25 (m, 40H), 0.88 (t, J = 6.6 Hz, 3H).

[0831] Synthesis of 3-((S)-2,2-bis((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol, 3 Synthesis of (S)-3-(2,2-bisheptadecyl-1,3-dioxolan-4-yl)propan-1-ol, 4

[0832]

[0833] Procedure for synthesis of AKG-KC2-01 described previously

[0834] 3-((S)-2,2-bis((Z)-heptadec-8-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol, 3 as a clear oil (0.7 g, 1.15 mmol, 65%).

[0835] 1 H NMR (300 MHz, CDC13): δ ppm 5.38-5.31 (m, 4H), 4.08-4.02 (m, 2H), 3.67-3.66 (m, 2H), 3.48-3.43 (m, 1H), 2.15-2.13 (m, 1H), 2.00-1.98 (m, 8H), 1.65-1.56 (m, 8H), 1.27-1.25 (m, 44H), 0.88 (t, J=6.6 Hz, 6H).

[0836] Final [mRNA] ng / mL

[0837]

[0838] A solution of 3-((S)-2,2-bis((Z)-heptadec-8-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol (1.3 g, 2.15 mmol) in methanol / ethyl acetate (20 mL, 1:1 / v:v) was hydrogenated under 1 atm (hydrogen balloon) over 10% palladium on carbon (100 mg) for 2 hours. The mixture was evacuated of hydrogen and flushed with nitrogen. The mixture was filtered over celite and the filtrate was concentrated under vacuum to give (S)-3-(2,2-bisheptadecyl-1,3-dioxolan-4-yl)propan-1-ol, 4 as a clear oil (1.3 g, quant.).

[0839] 1 H NMR (300 MHz, CDC13): δ ppm 5.38-5.31 (m, 4H), 4.08-4.02 (m, 2H), 3.67-3.66 (m, 2H), 3.48-3.43 (m, 1H), 2.15-2.13 (m, 1H), 2.00-1.98 (m, 8H), 1.65-1.56 (m, 8H), 1.27-1.25 (m, 44H), 0.88 (t, J=6.6 Hz, 6H).

[0840] 3-((S)-2,2-bis((Z)-heptadec-8-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1- amine, synthesis of AKG-KC3-C17(C8:1) (O-12620)

[0841]

[0842] Procedure previously described for the synthesis of AKG-KC2-01

[0843] 3-((S)-2,2-bis((Z)-heptadec-8-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1- amine as a clear oil, (AKG-KC3-C17(C8:1), O-12620), (290 mg, 0.46 mmol, 40%).

[0844] C 42 H 81 MS (APCI + ): 632.6

[0845] 1 H NMR (300 MHz, CDC13): δ ppm 5.38 - 5.28 (m, 4 H), 4.09 - 3.99 (m, 2 H), 3.48 - 3.41 (m, 1 H), 2.77 - 2.71 (m, 1 H), 2.55 (s, 6 H), 2.01 - 1.95 (m, 8 H), 1.88 - 1.78 (m, 2 H), 1.62 - 1.50 (m, 6 H), 1.27 - 1.24 (m, 44 H), 0.88 (t, J=6.8 Hz, 6 H).

[0846] (S)-3-(2,2-bisheptadecyl-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine, synthesis of AKG-KC3-C17 (O-12637)

[0847]

[0848] Procedure previously described for the synthesis of AKG-KC2-01

[0849] (S)-3-(2,2-bisheptadecyl-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine as a solid, (AKG-KC3-C17, O-12637), (275 mg, 0.43 mmol, 22%). C 42 H 85 MS (APCI + ): 636.6

[0850] 1 H NMR (300 MHz, CDC13): δ ppm 4.06-4.00 (m, 2 H), 3.47-3.43 (m, 1 H), 2.29-2.25 (m, 2 H), 2.21 (s, 6 H), 1.60-1.48 (m, 8 H), 1.29-1.24 (m, 60 H), 0.87 (t, J=6.6 Hz, 6 H).

[0851] Example 2. Preparation of Lipid Nanoparticles (LNPs).

[0852] mRNA modified with 5-methoxyuridine (5moU) and encoding mCherry (catalog number L-7203) was obtained from Trilink Biotechnologies (San Diego, CA). All uridine nucleosides were replaced with N1-methyl-pseudouridine. To produce the mRNA, a synthetic gene encoding the mRNA sequence was cloned into a DNA plasmid. The synthetic gene consisted of an RNA promoter, a 5’ untranslated region, a mCherry protein coding sequence, a 3’ untranslated region, and a poly(A) tail of approximately 120 A. The open reading frame sequence of the mCherry mRNA from TriLink (catalog number L-7203) corresponds to SEQ ID NO: 227:

[0853]

[0854] Stock solutions of each lipid were prepared. The ionizable lipids were weighed out in 4 mL glass vials (Thermo B7999-2) and dissolved in ethanol (Sigma-Aldrich 200 proof, RNase free) to a final concentration of 10 mM. The other lipids such as DSPC, DPPC-NH4, Cholesterol, and PEG-DMG were weighed out and dissolved in ethanol to a concentration of 1 mM. DSPS-Na was dissolved in methanol (Sulpelco, Omnisolve) at a concentration of 1 mM and briefly heated to 70 °C to complete its dissolution.

[0855] The lipid mix for each individual LNP was prepared by adding the desired volume of each lipid stock solution to a fresh vial, adding ethanol if necessary, to a final volume of 1.2 mL. For example, LNP formulation of AKG-UO-l / DSPC / DSPS / Chol / PEG-DMG (50 / 2.5 / 7.5 / 38.5 / 1.5 mol%) which contains 1500 nmol AKG-UO-l, 75 nmol DSPC, 225 nmol DSPS, 1155 nmol Chol, and 45 nmol PEG-DMG per 100 pg mRNA used at an N / P of 5.

[0856] mRNA solutions were prepared by thawing a vial of frozen mRNA (mCherry mRNA, Trilink) and diluting the mRNA to a final concentration of 0.033 mg / mL in 6.25 mM sodium acetate, pH 5.0. To prepare the LNPs, a NanoAssemblr benchtop microfluidics device (from Precision Nanosystems) was used. If the LNP contained the sodium or ammonium salt of DSPS, or the sodium salt of DPPS, a heating block accessory set to 70 °C was used, otherwise the LNP was mixed at room temperature. 3 mL of mRNA solution was loaded into a 3 mL disposable syringe (BD 309656) and 1 ml of lipid mixture was loaded into a 1 ml syringe (BD 309659) and placed in the NanoAssemblr heating block for 4 minutes, followed by mixing. LNP formation was achieved by pumping the liquid streams through a disposable microfluidic cassette at a mixing speed of 6 mL / min with a 3: 1 water:alcohol volume ratio. After mixing, 3.6 mL of LNP mixture was collected, while discarding 0.35 mL of the initial mixing volume and the final 0.05 mL of mixture. Ethanol was removed by buffer exchange in PBS (Cytivia, SH30256.01) using SpectraPor dialysis tubing (12 to 14 k MWCO), or by serial concentration and dilution using Amicon Ultra-4 centrifugal concentrators (10 k MWCO at 500g).

[0857] LNPs were typically exchanged into PBS, pH 7.4, and subsequently into 15 mM Tris, 20% sucrose, pH 7.4, concentrated to 20 to 50 pg / mL mRNA using Amicon-Ultra 4 (100,000 MWCO) spin columns, sterile filtered (Thermo Nalgene 0.2um #720-1320), then frozen by immersion in liquid nitrogen for 5 minutes and stored long-term at -80 °C.

[0858] Example 3. LNP characterization A. Determining mRNA concentration and relative encapsulation efficiency by fluorescent binding dye

[0859] Materials: Ribogreen reagent (Thermo #11491), 3 x 96-well plate with lid, PBS, dissociation buffer (PBS with 10% DMSO and 1% (wt / wt) zwittergent 3-14 (Sigma-Aldrich #693017), mRNA, general pipette tips & repetitive pipette tips.

[0860] 1. Prepare a 5 mL stock of 2 pg / mL mRNA in DPBS or PBS

[0861] 2. Prepare dilutions of standards in individual wells of a 96 well plate (Plate A) as follows;

[0862] Volume of 2 pg / mL stock (pL) Volume of PBS (pL) etc. 2000 400 0 1500 300 100 1000 200 200 500 100 300 0 0 400

[0863] 3. Using different wells in Plate A, estimate the sample mRNA concentration and dilute it to be within the standard curve. For example, if the approximate mRNA concentration in the sample should be about 30 pg / mL, then perform a 20x dilution (dilution factor). (Add 20 pL sample to 380 pL PBS in the well). No lid is used on Plate A. Mix the sample by pipetting up and down gently.

[0864] Example of Plate A

[0865] A 0 500 1000 1500 2000 B C S1 S2 S3 S4 S5 S6 S7 S8 etc. D E F G H

[0866] 4. Two additional plates are used: Plates B and C. Using a multichannel pipette, pipette 60 pL of each standard 2 into wells (in duplicate) and pipette the sample into 3 wells (in triplicate)

[0867] Example of Plates B and C

[0868] A 0 500 1000 1500 2000 B 0 500 1000 1500 2000 C S1 S2 S3 S4 S5 S6 S7 S8 etc. D S1 S2 S3 S4 S5 S6 S7 S8 etc. E S1 S2 S3 S4 S5 S6 S7 S8 Figure 1A F G H

[0869] 5. Calculate the number of wells used on each plate and add 4 to that number. For Plate B, prepare PBS with Ribogreen at 1 : 100 dilution. For example, for 40 wells, use 44 as the number. 44 x 60 pL = 2.64 mL of Ribogreen solution is needed, so it is 2.61 mL of PBS with 26.4 pL of Ribogreen.

[0870] 6. For Plate C, pipette 2.61 mL dissociation buffer and 26.4 pL Ribogreen.

[0871] 7. Using a repeat pipette set to 60 pL, add PBS + Ribogreen to each well on Plate B and 60 pL dissociation buffer + Ribogreen to Plate C. Both Plate B and Plate C are mixed on an orbital shaker (120 rpm) for 1 minute. Plate B is placed in the dark for 15 minutes. Plate C is incubated in the dark at 37°C for 10 minutes and then at room temperature for 5 minutes.

[0872] 8. Read both plates one after the other using Ex. 465, Em. 530 nm.

[0873] 9. Using the standard curve, calculate the slope and intercept, and calculate the mRNA concentration (mean and standard deviation) of the samples on plate B and C by extrapolation

[0874] 10. Calculate the percent encapsulation efficiency (% EE) by [mRNA] plate B / [mRNA] plate C x 100

[0875] B. LNP particle size

[0876] 1. Mix 30 pL of LNP with 1.5 mL PBS in a polystyrene cuvette (Sarstedt, #67.754) and analyze the size using ZetaSizer Pro (Malvern) using ZS Xplorer software version number 1.4.0.105. Record the Z-average size and polydispersity index values. Typically, LNP size measurements are performed after LNP mixing, after buffer exchange, and after sterile filtration.

[0877] C. LNP zeta potential

[0878] 1. Mix 30 pL LNP with 1.5 mL PBS and inject into disposable folded capillary cell (Malvern Nanoseries DTS1070) and measure zeta potential on ZetaSizer Pro at 25 °C.

[0879] Example 4. Determining LNP transfection efficiency in murine dendritic cells using mCherry mRNA. A. Cell propagation, transfection, harvesting, and staining protocol

[0880] 1. MutuDC1940 cells (Applied Biological Materials, T0528) were cultured in T75 flasks according to the supplier’s instructions. One day prior to transfection, they were plated at 180,000 cells / well into 24-well plates.

[0881] 2. LNP was added to each well in triplicate at the desired mRNA concentration (e.g., 1 pg / mL) in 1 mL of media, and after 24 hours the cells were washed once with DPBS (VWR 02-0119-1000).

[0882] 3. Then 0.2 mL DPBS (plus 5 mM EDTA, pH 7.4) was added to facilitate detachment.

[0883] 4. Cells were left at 37 °C for 3 minutes until detached.

[0884] 5. Add 0.5 ml DPBS to each well and transfer the liquid to a flow cytometry tube (Falcon 5 mL #352054).

[0885] 6. Centrifuge the tubes at 1100 rpm for 3 to 5 minutes and pour off the liquid.

[0886] 7. Add 100 pL Zombie Violet (Biolegend) diluted 1 :500 in PBS to each tube.

[0887] 8. Tap the tubes to resuspend the cells and let them sit at room temperature in the dark for 15 minutes.

[0888] 9. Add 0.5 mL (1 : 1 of 4% paraformaldehyde in PBS: DPBS) to the cells and flick the cells to resuspend and place them on ice for 30 minutes. Add another 2 ml PBS.

[0889] 10. Pellet the cells as above and resuspend them in 0.5 mL DPBS with 5% BSA and place in the freezer until needed.

[0890] B. Cell Analysis

[0891] Cell suspensions were analyzed for live / dead and mCherry fluorescence signals by an Attune NxT flow cytometer using VL1 and YL2, respectively. Gating analysis was performed on FloJo software.

[0892] Example 5. Effect of DSPS on efficiency of transfection of dendritic cells using LNP with KC2 as ionizable cationic lipid.

[0893] The objective of this study was to explore the effect of phosphatidylserine targeting using DSPS on transfection efficiency in murine dendritic cells. LNP were prepared as described in Example 9, particle size and zeta potential were characterized as described in Example 4, and transfection efficiency was evaluated in murine dendritic cells as described in Example 5. DLin-KC2-DMA was constant for all LNP at an N / P ratio of 5 and 50 mol% of total lipid, PS lipid was varied from 0 to 2.5 mol% initially, and DSPC phospholipid was varied from 0 to 7.5 mol% (total mol% of DSPC and DSPS constant at 10 mol%), and cholesterol was constant at 38.5 mol% (all as mol% of total lipid). Particle size, Polydispersity Index (PDI), and encapsulation efficiency for all formulations are shown in Tables 4 and 5 below.

[0894] Table 4. Example 6 and Mol% DSPSPhysical-chemical properties of KC2-containing LNPs used in Example 6, varying from 0 to 2.5 mol% of DSPS.

[0895] Particle size (nm) PDI % Entrapment ± SD Figure 1A 0 77.6 0.075 86.4±2.8 0.1 82.0 0.171 87.0±4.7 05 89.2 0.285 86.3±32 25 72.2 0263 871±68

[0896] Table 5. Physical-chemical properties of KC2-containing LNPs used in Example 6, varying from 0 to 2.5 mol% of DSPS. Figure 1B , Figure 1C and Mol% DSPS Table 5. Physical-chemical properties of KC2-containing LNPs used in Example 6, varying from 0 to 2.5 mol% of DSPS.

[0897] Particle size (nm) PDI % Entrapment ± SD Figure 1A 0 739 014 92.6±62 1.25 80.3 013 90.9±5.6 2.5 707 0.25 89.0±7.4 5 78.7 0.12 85.1±6.8 7.5 76.4 0.14 85.4±3.6

[0898] The initial set of LNPs containing DLin-KC2-DMA and phosphatidylserine in the form of DSPS varying from 0 to 2.5 mol% showed little transfection at 0 or 0.5 mol% DSPS, but increased 18-fold when DSPS was incorporated at 2.5 mol% ( Figure 1B ). A second series of LNPs prepared with 0 to 7.5 mol% of DSPS were evaluated at 0.1, 0.3 and 1 pg / mL mRNA concentration ( Figure 1C , Figure 1D and Figure 2 ). Transfection efficiency increased as the mol% of DSPS increased above 2.5 mol%, with a maximum at 7.5 mol% at 1 pg / mL mRNA, and at 5 mol% at both 0.1 and 0.3 pg / mL mRNA. These data suggest that the inclusion of phosphatidyl-L-serine can significantly increase the transfection efficiency of mRNA-containing LNPs, and that maximal uptake occurs between 5 and 7.5 mol% of DSPS as a % of total lipids.

[0899] Example 6. Effect of ICL and anionic phospholipid targeting ligands on mRNA transfection of dendritic cells.

[0900] The objective of this study was to see if other anionic phospholipids could also enhance the transfection efficiency of LNPs, and how LNPs prepared with different ICLs and PS targeting would transfect dendritic cells. LNPs were prepared as described in Example 2, particle size and (zeta potential were characterized as described in Example 3, and transfection efficiency was evaluated in murine dendritic cells as described in Example 4. The ICL of the LNP was varied (DLin-KC2-DMA, KC2-OA, KC3-OA or SM-102), with the N / P ratio constant at 5 and the total lipid constant at 50 mol%, the PS lipid was kept constant at 5 mol% and DSPC at 5 mol%, and cholesterol constant at 38.5 mol% (all as mol% of total lipids). Particle size, PDI and encapsulation efficiency of all formulations are shown in Table 6 below.

[0901] Table 6. Physicochemical properties of LNPs using different ICLs and having 5 mol% of anionic phospholipid.

[0902]

[0903] Transfection results are shown in Figure 2 , Figure 3 High transfection rates are shown for three different KC series ICLs (KC2, KC2-OA, and KC3-OA) and for LNPs made with a branched ICL (SM-102). The encapsulation efficiency was high for all formulations, including those made with alternative anionic phospholipids (Suc-DSPE or Glu-DSPE), and the particle size was below 100 nm for all. The data indicate that DSPS (L-serine) cannot be replaced by N-glutaryl-distearylphosphatidylethanolamine (Glu-DSPE) or N-succinyl-distearylphosphatidylethanolamine (Suc-DSPE) and provide the same high level of mRNA transfection, despite both phospholipids also containing two negative charges and both containing identical di-stearoyl (C18:0) fully saturated acyl chains. These studies also clearly indicate that the addition of DSPS can lead to high transfection efficiency of other ionizable cationic lipids, including those with a single unsaturation acyl gain (KC2-OA or KC3-OA) and those including branched ICLs such as SM-102. For example, the addition of DSPS to LNPs containing SM-102 led to a 22-fold increase in mCherry expression.

[0904] Example 7. PS targeting dependence on ICL and PS structure.

[0905] The objective of this study was to compare PS-targeting LNPs with the KC2 and KC3 series of ionizable cationic lipids having different acyl chain compositions. The KC2 series of lipids having a dimethylaminoethyl headgroup structure were compared to the KC3 series containing a dimethylaminopropyl-derived headgroup. LNPs contained a variety of ICLs (KC2, KC2-01, KC2-OA, KC2-PA, KC3-OA, and KC3-01) as ICLs at an N / P ratio of 5 and 50 mol% ICL, and a constant 1.5 mol% PEG-DMG. The cholesterol content was kept constant at 38.5 mol%, and the DSPC content was varied inversely with the mol% of DSPS, either 0 or 5 mol% (all lipid concentrations are used as mol% of total lipids). Transfection efficiency was evaluated in murine dendritic cells as described in Example 4.

[0906] Table 7. Physicochemical properties of LNPs using different ICLs and having 5 mol% of anionic phospholipid.

[0907]

[0908] Figure 4 Transfection results are shown in the middle and clearly show the positive impact of PS targeting on the transfection efficiency of various KC series ICLs. Here, the data show that ICLs containing both unsaturated C16 and C18 ICLs can be targeted by phosphatidyl-L-serine and lead to high transfection rates against dendritic cells. The highest transfection rates occur when PS and PC contain a mismatched acyl chain composition, and 5 mol% DPPC and 5 mol% DSPS, in combination with C16 ICL (KC2-PA).

[0909] Example 8. Effect of PEG on transfection efficiency of LNP containing AKG-UO-1.

[0910] The aim of this study was to explore the effect of PEG-lipid density on the transfection efficiency of non-targeted and phosphatidyl-L-serine targeted LNP. LNP were prepared as described in Example 2. LNP contained AKG-UO-1 as ICL with an N / P ratio of 5, and 0 or 5 mol% DSPS and 0.5 to 4.5 mol% PEG-DMG. The cholesterol content was kept constant at 38.5 mol% and the DSPC content was 10 mol% for formulations without DSPS and 5 mol% for formulations containing 5 mol% DSPS. At PEG-DMG contents higher than 1.5 mol%, the total cholesterol content was reduced due to the amount of PEG-DMG added, for example at a PEG-DMG content of 3.5 mol%, the cholesterol content was reduced from 38.5 mol% to 36.5 mol%. Particles with 0.5% PEG-DMG showed a negative zeta potential at pH 7.4 and a significant shift to a positive zeta potential at pH 5. LNP with 1.5 to 3.5 mol% PEG-DMG were essentially neutral at pH 7.

[0911] Table 8. Physicochemical properties of LNP containing AKG-UO-1, 0 or 5 mol% DSPS used in Example 8

[0912]

[0913] Figure 5AThe effect of PEG-DMG on LNP transfection of dendritic cells targeted by DSPS containing AKG-UO-1 ICL is shown in Figure 6. Formulations containing 0.5% PEG-DMG showed transfection efficiency in the presence of 5% DSPS that was 6 to 7 times higher than that observed at 1.5 to 2.5% PEG-DMG. Transfection was similar at 1.5 and 2.5% PEG-DMG but was significantly reduced at 3.5 and 4.5 mol% PEG-DMG. At each PEG density, the ratio of targeted to non-targeted transfection was different and was 12-fold at 0.5% PEG, 7-fold at 1.5% PEG, 37-fold at 2.5% PEG, and less than 5-fold at 3.5% and 4.5% PEG, likely due to high PEG shielding of the PS targeting moiety. The combination of these data shows that the optimal range of PEG density is 0.5 to 2.5% PEG-DMG, with the lower end of the range being optimal for total transfection efficiency and 2.5% being optimal for target specificity.

[0914] Example 9. Effect of N / P ratio on transfection efficiency of LNP containing mCherry mRNA.

[0915] The objective of this study was to explore the effect of different N / P ratios on transfection efficiency in dendritic cells. LNP were prepared as described in Example 2, particle size and zeta potential were characterized as described in Example 3, and transfection efficiency was evaluated in murine dendritic cells as described in Example 4. LNP all used KC2-01 as ICL, but the ratio of cationic lipid to mRNA phosphate (N / P) was varied from 4 to 7, the PS lipid was constant at 5 mol% and the DSPC phospholipid was constant at 5 mol%, and the cholesterol was constant at 38.5 mol%. The encapsulation efficiency of all formulations was 84 to 90%, indicating efficient mRNA encapsulation in LNP. Transfection efficiency was evaluated in murine dendritic cells as described in Example 4.

[0916] Table 9. Physicochemical properties of LNP used in Example 9

[0917]

[0918] Transfection activity of both DSPS-targeted and non-targeted KC-01 LNP formulations was evaluated at 1 pg / ml Figure 5B ) and 0.33 pg / ml Figure 6 ). These data show that LNP containing KC2-01 have high DSPS-mediated transfection efficiency over a wide range of N / P ratios, with maximal transfection efficiency observed at N / P of 7.

[0919] Example 10. Measure the effect of adding 10 and 25 mol% DOPS on LNP particle formation and activity in MutuDC1940 dendritic cell line.

[0920] The aim of this study was to explore the impact of including DOPS at compositions equal to or lower than previously shown in the literature (Gaitonde et al. (2011) Clin Immunol 138, 135-145; Rodriquez-Fernandez (2018) Front Immunol 9, 253) into mRNA LNP formulations on enhancing liposome (where present) uptake into dendritic cells. LNP were prepared at 25°C as described in Example 2 and analysed as described in Example 3. LNP contained KC2 at an N / P ratio of 5 as ICL, and 0, 10 to 25 mol% DOPS, and constant 1.5 mol% PEG-DMG. For 0% and 10% DOPS formulations, cholesterol content was kept constant at 38.5 mol% and DSPC content varied inversely with mol% of DOPS from 0 to 10 mol% (all lipid concentrations used as mol% of total lipid). In the 25 mol% DOPS formulation, there was no DSPC and cholesterol content was reduced by 15 mol% in total (from 38.5 mol% to 23.5 mol%). Transfection efficiency was evaluated in murine dendritic cells as described in Example 4.

[0921] Table 10. Physicochemical properties of LNP formulations containing DOPS

[0922]

[0923] Figure 7AThe effect of 10 to 25 mol% DOPS on LNP targeting of dendritic cells was evaluated. This study showed that inclusion of 10 mol% DOPS in KC2-based LNP formulations had a positive effect on mCherry expression levels in MutuDC1940 cells, while having no adverse effect on particle size or encapsulation of mRNA. However, when the DOPS content was increased to 25 mol%, mCherry expression was lower than in formulations without DOPS, and the size distribution was broadened (as indicated by an increase in PDI) and produced a distribution containing >400 nm particles. In summary, 25 mol% can have shown an enhancement in uptake of liposomes into dendritic cells in the literature, while in mRNA-containing LNP formulations it had a detrimental effect on both particle size and transfection activity. Importantly, the DOPS used here and in the literature contains an unsaturated acyl chain, in this case oleic acid. This is analogous to the typical case in many cells, where phosphatidylserine acyl chains are typically unsaturated at the sn-2 position, with multiple alkenes (2 to 4) in many cases. While there is a small enhancement at lower concentrations of DOPS, it was shown that this enhancement is significantly higher when the PS consists of saturated acyl chains, most preferably dipalmitoyl (C16) or distearoyl (C18).

[0924] Example 11. Effect of PEGylation and phosphatidylserine targeting on immunogenicity of SARS-CoV-2 spike protein mRNA vaccine constructs.

[0925] Mice and study design. In vivo studies were performed. Female BALB / c mice were purchased from Jackson Labs, allowed to acclimate in the animal facility for at least 7 days, and were 6 to 8 weeks old at the start of the study. On day 0 of the study, mice were injected intramuscularly in the right quadriceps with 1 pg of vaccine candidate (amount refers to mRNA) in a 50 pL volume. Study groups consisted of 5 mice and included vehicle control, comparator vaccine, and experimental vaccine candidate. Twenty-one days later, mice were given a second injection of the same vaccine candidate. Blood was collected from 5 randomly selected control mice at the start of the study and from all mice on study days 21 and 34, and serum was isolated. Serum was stored at -80 °C until antibody titers were analyzed. On day 34 of the study, mice were euthanized and spleens were harvested.

[0926] Design and preparation of mRNA. mRNA encoding SARS-CoV-2 full-length spike protein and flanked by the same UTRs used in the BNT162b2 (Comirnaty) vaccine was purchased from Vernal Biosciences. All uridine nucleosides were replaced with N1 -methyl-pseudo-uridines. To produce mRNA, a synthetic gene encoding the mRNA sequence (VRN029; SEQ ID NO: 221) was cloned into a DNA plasmid:

[0927]

[0928]

[0929] The synthetic gene consists of an RNA promoter, 5’ untranslated region, SARS-COV2 spike protein receptor binding domain, 3’ untranslated region, and a poly(A) tail of approximately 120A. The plasmid is propagated and amplified in E. coli cultures and subsequently isolated from clarified E. coli lysate via anion exchange chromatography. The purified plasmid is linearized using a type II restriction endonuclease that cuts at a site at the end of the poly(A) tail coding region. The plasmid is then incubated in a buffer with nucleotide triphosphates, RNA polymerase, and RNAse inhibitor. To stop the reaction, DNAse I is added to digest the linear plasmid template. The uncapped RNA is then purified using chromatography and subsequently incubated in another buffer with GTP, S-adenosyl methionine, guanalyltransferase, 2’-O-methyltransferase, and RNAse inhibitor. The capped mRNA is then purified using chromatography, the buffer is exchanged to water, and filled into vials.

[0930] Production of lipid nanoparticles (LNP) comprising mRNA. Stock solutions of each lipid were prepared. The ionizable lipid was weighed out in a 4 mL glass vial (Thermo B7999-2) and dissolved in ethanol (Sigma-Aldrich 200 proof, RNase free) to a final concentration of 10 mM. The other lipids, e.g. DSPC (Avanti Polar Lipids), cholesterol (Dishman), and PEG-DMG (NOF), were weighed out and dissolved in ethanol to a concentration of 1 mM. DSPS-Na (NOF) was dissolved in methanol (Sulpelco, Omnisolve) at a concentration of 1 mM and briefly heated to 70 °C to complete its dissolution.

[0931] The lipid mix for each individual LNP was prepared by adding the desired volume of each lipid stock solution to a new vial, adding ethanol if needed, to reach a final volume of 1.2 mL. For example, LNP formulation of AKG-UO-l / DSPC / DSPS / CHol / PEG-DMG (50 / 2.5 / 7.5 / 38.5 / 1.5 mol%) which contains 1500 nmol AKG-UO-l, 75 nmol DSPC, 225 nmol DSPS, 1155 nmol Chol, and 45 nmol PEG-DMG for every 100 pg mRNA used at N / P of 5. mRNA solution was prepared by thawing a frozen mRNA (SARS-CoV-2 Spike mRNA, Vernal) vial and diluting the mRNA in 6.25 mM sodium acetate, pH 5.0, to a final concentration of 0.033 mg / mL, where the concentration was determined by absorbance on a Nanodrop.

[0932] To prepare LNP, a NanoAssemblr benchtop microfluidic device (from Precision Nanosystems) was used. If the LNP contained DSPS, a heating block accessory set to 70 °C was used, otherwise the LNP was mixed at room temperature. 3 mL of mRNA solution was loaded into a 3 mL disposable syringe (BD 309656) and 1 ml of lipid mixture was loaded into a 1 ml syringe (BD 309659) and placed in the NanoAssemblr heating block for 4 minutes, followed by mixing. LNP formation was achieved by pumping the liquid streams through a disposable microfluidic cassette at a mixing speed of 6 mL / min at a 3: 1 water:alcohol volume ratio. After mixing, 3.6 mL of LNP mixture was collected, while discarding 0.35 mL of the initial mixing volume and the final 0.05 mL of mixture. Ethanol was removed by buffer exchange in PBS (Cytivia, SH30256.01) using SpectraPor dialysis tubing (12 to 14 k MWCO). LNP was typically exchanged into PBS at pH 7.4 and subsequently exchanged into 15 mM Tris, 20% sucrose at pH 7.4, concentrated to 20 to 50 pg / mL mRNA, sterile filtered (Thermo Nalgene 0.2um #720-1320), then frozen by immersion in liquid nitrogen for 5 minutes and stored long term at -20 °C. For this study, samples were concentrated to >40 pg / mL mRNA and diluted with different volumes of 15 mM Tris, 20% sucrose, pH 7.4 to a target concentration of 40 pg mRNA and subsequently frozen on LN2. Characterization of LNP was performed after thawing an aliquot of LNP and diluting 1 : 1 (volume:volume) with 15 mM Tris, pH 7.4 such that the final concentration was 20 pg / mL mRNA in 15 mM Tris, 10% sucrose, pH 7.4. This mimics the conditions of sample preparation prior to dosing animals by IM injection of 1 pg mRNA in a 50 pL volume into the hind limb.

[0933] LNP characterization. mRNA encapsulation and mRNA concentration within LNP was measured using a Ribogreen assay. Nanoparticle size and zeta potential were measured by a zetasizer (Malvern).

[0934] SARS-CoV-2 anti-spike antibody titers. A standard indirect ELISA was performed to analyze total IgG binding antibodies in serum samples against SARS-CoV-2 spike protein. For this assay, 100 pL of SARS-CoV-2 spike protein (Sino Biological, Cat# 40589-V08B1) was coated at 2 pg / mL in lx PBS at pH 7.4 to Nunc MaxiSorp 96-well plates. Plates were incubated statically at 37 °C for 12 hours. Unbound coating antigen was removed by washing the plates 3x with 100 pL PBS + 0.05% Tween 20. Plates were then blocked in PBS + 5% nonfat milk for 1 hour at 37 °C. Test and positive control samples were diluted to a starting dilution of 1 :20 in assay diluent (PBS, Tween-20, 1% nonfat milk), followed by a four-fold serial dilution using U-bottom dilution plates. Once blocking was complete, the blocking buffer was removed by inversion, and each sample was plated in duplicate. Plates were incubated statically at 37 °C for 2 hours, followed by 3x washing with 100 pL of PBS + 0.05% Tween 20 to remove unbound serum. 100 pL of secondary detection antibody (goat anti-mouse-HRP IgG, Abeam) was added to each well at a dilution of 1 :10,000. Plates were incubated statically at room temperature for 30 minutes, and then unbound antibody was removed, and plates were washed as described above. For development, 100 pL of One-Step Ultra TMB substrate was added to each well, and after approximately 10 minutes, the reaction was stopped with 50 pL TMB stop solution (SeraCare, Cat# 5150-0019). Plates were read with a Thermo LabSystems Multiskan spectrophotometer at 450 nm over 30 minutes. Titers were defined as the inverse of the dilution that produced an OD 450 at a specific cutoff value on the linear portion of the titer curve.

[0935] Ex vivo T cell responses. On day 34 of the study, spleens were mechanically dissociated into single cell suspensions. Cells were resuspended in cell stimulation media (RPMI containing L-glutamine and HEPES buffer, heat-inactivated fetal bovine serum, and Pen / Strep) and 2 x 10 6Individual cells were aliquoted into 96-well plates in a volume of 100 pL. Splenocytes from each mouse were stimulated individually with 100 pL of media at 37 °C for about 18 hours with a positive control cell stimulation cocktail (Cell Stimulation Cocktail) containing PMA and ionomycin (ThermoFisher, Cat# 00-4970-93), or 1 pg / mL of a peptide pool encompassing SARS-CoV-2 spike protein (JPT, Cat# PM-WCPV-S-2) treatment. GolgiStop (BD Biosciences, Cat# 554724) was added to each well after 1 hour of stimulation.

[0936] Flow cytometry. After stimulation, cells were washed with PBS and transferred to a 96-well deep well plate. Cells were stained with LIVE / DEAD near IR viability dye (ThermoFisher, Cat# L10119) diluted in PBS for 20 minutes at 4 °C. Cells were washed with FBS staining buffer (BD Biosciences, Cat# 554656) and incubated with Fc Block (BD Biosciences, Cat# 553142) for 10 minutes at 4 °C. Cells were then stained with a cell surface antibody cocktail consisting of CD3 BV605 (BD, Cat# 564009), CD4 BV421 (BD, Cat# 562891), and CD8 APC (BD, Cat# 553035) for 40 minutes at 4 °C. BD Brilliant stain buffer (Cat# 563794) was included in the staining buffer. Cells were washed with FBS staining buffer and subsequently fixed with Fix / Perm buffer (ThermoFisher, Cat# 00-5523-00) for 20 minutes at room temperature. Cells were washed with lx Perm buffer, incubated with Fc Block, and subsequently stained with an intracellular cytokine antibody cocktail consisting of IFN-g PE / Cy7 (BD, Cat# 557649), IL-2 PE (BioLegend, Cat# 503808), and TNF-a FITC (BD, Cat# 554418) for 40 minutes at 4 °C. Cells were then washed, resuspended in FBS staining buffer, and acquired on a MACSQuant 16 flow cytometer (Miltenyi Biotec). Flow data were analyzed using FlowJo v10.8.1 (BD Biosciences).

[0937] Table 11. Physical-chemical properties of LNPs used to evaluate immunogenicity of SARS-CoV-2 Spike protein mRNA vaccine constructs

[0938]

[0939] To evaluate the effect of PEG-lipid concentration on vaccine immunogenicity, BALB / c mice were immunized with mRNA-LNPs containing increasing amounts of PEG-lipid (PEG-DMG or PEG-DPPE). Serum was collected for antibody analysis on day 21 after the initial immunization and on day 13 after the boost (day 34 of the study). Splenocytes were stimulated with the spike peptide pool, and the percentage of IL-2-producing CD4 T cells ( Figure 7B and Figure 7A ). Two forms of PEG, PEG-DMG ( Figure 7B ) or PEG-DPPE( Figure 7C ) both negatively impacted mRNA-LNPA vaccine immunogenicity, with lower concentrations of PEG-lipid showing higher levels of both B cell and T cell responses.

[0940] To evaluate the effect of PEG-lipid acyl chain composition on the immunogenicity of mRNA-LNPs, BALB / c mice were immunized with mRNA-LNPs containing different PEG forms (PEG-DMG or PEG-DSG). Serum was collected on day 21 after the initial immunization and day 13 after the boost (day 34 of the study). Splenocytes were stimulated with the spike peptide pool and the percentage of CD4 T cells producing IL-2 was quantified using flow cytometry. The antibody titer data were log-transformed and subsequently statistically analyzed. The groups were compared using an unpaired t-test. For LNPs prepared with the ionizable lipid KC2OA, either PEG form performed similarly ( Figure 7D In contrast, LNPs using the ionizable lipid UO1 and PEG-DMG induced a better antibody response than LNPs containing PEG-DSG ( Figure 7E ).

[0941] To assess how incorporation of phosphatidylserine affects the immunogenicity of mRNA-LNPs, BALB / c mice were immunized with mRNA-LNPs containing a variety of ionizable lipids with or without DSPS. At day 34 (13 days after boosting), serum was collected for quantification of total IgG anti-spike antibodies by ELISA. Splenocytes were stimulated with peptides and the percentage of CD4 T cells producing IL-2 was quantified using flow cytometry.

[0942] The inclusion of DSPS and the comparator ionizable lipid ALC-0315 negatively affected total IgG anti-spike antibody levels ( Figure 7FDSPS had opposite effects on LNP containing ionizable lipids UOl and KC2OA and significantly increased the geometric mean antibody levels by 36-fold and 46-fold, respectively. DSPS also had an effect on CD4 T cell responses, where the responses were trending higher for LNP containing UOl and KC2OA and significantly higher for SM-102. In summary, inclusion of DSPS in the lipid nanoparticle can substantially impact the immunogenicity of the mRNA vaccine and the impact of DSPS is influenced by the type of ionizable cationic lipid.

[0943] The effect of different phosphatidylserine acyl chain compositions on serum antibody titers Figure 7F , Figure A) and magnitude of spike-specific CD4 T cell responses in the spleen Figure 7G , Figure B) was evaluated. Mice were immunized with LNP using the ionizable lipid UOl and phosphatidylserine in the form of DSPS or DPPS. Serum was collected on day 34 (13 days...

Claims

1. A lipid nanoparticle (LNP) composition, comprising: a. a messenger RNA (mRNA) encoding one or more Mycobacterium tuberculosis (Mtb) proteins selected from the group consisting of CFP10 / Rv3874, ESAT-6 / Rv3875, Mtb32A / Rv0125, Mtb39A / Rv1196, Ag85B / Rv1886c, EsxW / Rv3620c, EsxV / Rv3619c, PE13 / Rv1195, PPE30 / Rv1802, PPE40 / Rv2356c, and TB10.4 / Rv0288; b. an ionizable cationic lipid comprising a KC3 ionizable cationic lipid having an N / P ratio of 4 to 6 relative to the mRNA, wherein the ionizable cationic lipid is present in the LNP composition in a total amount of 46 to 54 mol% of the total lipid content in the LNP composition; c. one or more phospholipids selected from the group consisting of: distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC) and dipalmitoylphosphatidylcholine (DPPC), wherein the total amount of the one or more phospholipids is 10 to 18 mol% of the total lipid content in the LNP composition; d. one or more anionic phospholipids selected from the group consisting of dipalmitoylphosphatidyl-L-serine (DPPS), or distearoylphosphatidyl-L-serine (DSPS), distearoylphosphatidylglycerol (DSPG) and dipalmitoylphosphatidylglycerol (DPPG), wherein the total amount of the one or more anionic phospholipids is 2 to 8 mol% of the total lipid content in the LNP composition; e. PEG(2000)-dimyristoylglycerol (PEG-DMG), in a total amount of 1 to 3.5 mol% of the total lipid content in the LNP composition; and f. Cholesterol.

2. A lipid nanoparticle (LNP) composition comprising: a. a nucleic acid comprising a nucleic acid sequence encoding a T cell epitope from Mycobacterium tuberculosis (Mtb) or an Mtb antigen recognized by T cells; b. an ionizable cationic lipid comprising a KC3 ionizable cationic lipid having an N / P ratio of 3 to 7 relative to the nucleic acid, wherein the ionizable cationic lipid is present in the LNP composition in a total amount of 46 to 54 mol% of the total lipid content in the LNP composition; c. one or more phospholipids, the total amount of which is 5 to 20 mol% of the total lipid content in the LNP composition; d. one or more anionic phospholipids, the total amount of which is 2 to 8 mol% of the total lipid content in the LNP composition; e. conjugated lipids in an amount of 1 to 3.5 mol% of the total lipid content in the LNP composition; and f. Sterols.

3. The composition of claim 2, wherein the one or more anionic phospholipids is phosphatidylserine (PS) or phosphatidylglycerol (PG).

4. The composition of claim 3, wherein the one or more anionic phospholipids are selected from the group consisting of dipalmitoylphosphatidyl-L-serine (DPPS), or distearoylphosphatidyl-L-serine (DSPS), distearoylphosphatidylglycerol (DSPG), and dipalmitoylphosphatidylglycerol (DPPG).

5. The composition of claim 2, wherein the one or more phospholipids comprise distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC), or a combination thereof.

6. The composition of claim 5, wherein the conjugated lipid is PEG(2000)-dimyristoylglycerol (PEG-DMG).

7. The composition of claim 6, wherein the sterol is cholesterol.

8. The composition of claim 7, wherein the ionizable cationic lipid comprises 3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropane-1-amine (KC3-OA).

9. The composition of claim 8, wherein the ionizable cationic lipid further comprises a KC4 ionizable cationic lipid.

10. The composition of claim 9, wherein the ionizable cationic lipid is 4-rac-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylbutan-1-amine (AKG-KC4-OA).

11. The composition of claim 7, wherein the composition consists of: a.48mol%KC3-OA; b. 5 mol% DPPS or DSPG; c. 5 to 10 mol% DSPC or HSPC; d. 1.5 mol% PEG-DMG; and e. 35.5 to 40.5 mol% cholesterol.

12. The composition of any one of claims 2 to 11, wherein the nucleic acid sequence is an mRNA encoding a T cell epitope present in Mtb or a tandem sequence of an Mtb antigen recognized by T cells.

13. The composition of claim 12, wherein the mRNA encodes one or more Mtb proteins selected from the group consisting of: CFP10 / Rv3874, ESAT-6 / Rv3875, Mtb32A / Rv0125, Mtb39A / Rv1196, and Ag85B / Rv1886c.

14. The composition of claim 12, wherein the mRNA comprises one or more nucleic acid sequences selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:

220.

15. The composition of claim 12, wherein the mRNA encodes one or more Mtb proteins selected from the group consisting of: EsxW / Rv3620c, EsxV / Rv3619c, PE13 / Rv1195, PPE30 / Rv1802, PPE40 / Rv2356c, and TB10.4 / Rv0288.

16. The composition of claim 12, wherein the mRNA comprises one or more nucleic acid sequences selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO: 221, and SEQ ID NO:

222.

17. The composition of any one of claims 2 to 11, wherein the nucleic acid sequence encodes a tandem sequence, wherein the tandem nucleic acid encoding sequence comprises N-terminal and C-terminal signal peptides, and the N-terminal and C-terminal signal peptides are selected from Sec / MITD, Lamp1, HLA-Drα or tPA.

18. The composition of any one of claims 2 to 11, wherein the nucleic acid comprises a chemically modified mRNA, wherein the chemically modified mRNA comprises N1-methylpseudouridine.

19. The composition of any one of claims 2 to 11, wherein the nucleic acid comprises a 5' untranslated region (UTR) and a 3' UTR, a polyA tail of about 80 to about 140 nucleotides in length, and (i) a 5' enzymatic cap or (ii) a 5' clean cap.

20. The composition of any one of claims 2 to 11, wherein the nucleic acid is an mRNA having a sequence selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, 42, 44, 224, and 226.

21. The composition of claim 20, wherein the nucleic acid comprises a chemically modified mRNA, wherein the chemically modified mRNA comprises N1-methylpseudouridine.

22. The composition of any one of claims 2 to 11, wherein the nucleic acid is an mRNA encoding an amino acid sequence selected from SEQ ID NO: 33, 35, 37, 39, 41, 43, 86 to 105, 207 to 210, 223, and 225.

23. The composition of any one of claims 2 to 11, wherein the nucleic acid sequence encodes a tandem sequence, wherein the tandem sequence encoded by the nucleic acid comprises two or more MHC class I epitopes selected from SEQ ID NOs: 106 to 137 and 138 to 203.

24. The composition of any one of claims 2 to 11, wherein the nucleic acid sequence encodes a tandem sequence, wherein the tandem sequence encoded by the nucleic acid comprises two or more MHC class I epitopes present in Mycobacterium tuberculosis, the MHC class I epitopes excluding epitopes present in BCG and selected from SEQ ID NOs: 86 to 95.

25. The composition of any one of claims 2 to 11, wherein the nucleic acid sequence encodes a tandem sequence, wherein the tandem sequence encoded by the nucleic acid comprises two or more MHC class I epitopes, the MHC class I epitopes are ordered to minimize the generation of junctional neo-epitopes, and are selected from SEQ ID NOs: 86 to 105.

26. The composition of any one of claims 2 to 7, wherein the ionizable cationic lipid is KC3-OA, KC3-PA, KC3-01, KC3-C17(8:1), or KC3-C15(C8:1).

27. A lipid nanoparticle (LNP) composition comprising: a. a nucleic acid comprising a nucleic acid sequence encoding a polypeptide that is a T cell epitope from Mycobacterium tuberculosis (Mtb) or an Mtb antigen recognized by T cells, and wherein the nucleic acid sequence has at least 90% sequence identity with a nucleic acid sequence disclosed herein; b. an ionizable cationic lipid comprising a KC3 ionizable cationic lipid having an N / P ratio of 3 to 7 relative to the nucleic acid, wherein the ionizable cationic lipid is present in the LNP composition in a total amount of 46 to 54 mol% of the total lipid content in the LNP composition; c. one or more phospholipids, the total amount of which is 5 to 20 mol% of the total lipid content in the LNP composition; d. one or more anionic phospholipids, the total amount of which is 2 to 8 mol% of the total lipid content in the LNP composition; e. conjugated lipids in an amount of 1 to 3.5 mol% of the total lipid content in the LNP composition; and f. Sterols.

28. The composition of claim 27, wherein the nucleic acid is a messenger ribonucleic acid (mRNA) comprising a polynucleotide sequence encoding a polypeptide that is a T cell epitope from Mycobacterium tuberculosis (Mtb) or an Mtb antigen recognized by T cells, wherein the mRNA polynucleotide sequence is at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 220, or is at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO: 221, and SEQ ID NO:

222.

29. The composition of claim 1 or any one of claims 7 to 10, wherein the composition comprises cholesterol in a total amount of 35.5 to 42.7 mol% of the total lipids in the LNP composition.

30. The composition of claim 1 or any one of claims 7 to 10, wherein the composition comprises 48 mol% of the KC3 ionizable cationic lipid, 40.5 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, wherein each mol% refers to the mol% of the total lipid content in the LNP composition.

31. The composition of claim 1 or any one of claims 7 to 10, wherein the composition comprises 48 mol% of the KC3 ionizable cationic lipid, 38.5 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, wherein each mol% refers to the mol% of the total lipid content in the LNP composition.

32. The composition of claim 1 or any one of claims 7 to 10, wherein the composition comprises 45 mol% of the KC3 ionizable cationic lipid, 42.7 mol% cholesterol, and 5 mol% (L-serine) DPPS lipid, wherein each mol% refers to the mol% of the total lipid content in the LNP composition.

33. The composition of claim 1 or any one of claims 7 to 10, wherein the composition comprises 50 mol% of the 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.

34. The composition of claim 1 or any one of claims 7 to 10, wherein the composition comprises 48 mol% of the 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.

35. The composition of claim 1 or any one of claims 7 to 10, wherein the composition comprises 48 mol% of the 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.

36. The composition of claim 1 or any one of claims 7 to 10, wherein the composition comprises 46.5 mol% of the 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.

37. The composition of claim 1 or any one of claims 7 to 10, wherein the composition comprises 15 mol% total phospholipids and 35.5 mol% cholesterol.

38. The composition of claim 1 or any one of claims 7 to 10, wherein the composition comprises 10 mol% total phospholipids and 40.5 mol% cholesterol.

39. The composition of claim 1 or any one of claims 7 to 10, wherein the composition comprises 40.5 mol% cholesterol, 5% anionic lipid (DPPS) and 5% PC (DSPC or DPPC) and a total 10 mol% phospholipid concentration.

40. The composition of claim 1 or any one of claims 7 to 10, wherein the composition comprises 48 mol% cationic ionizable lipids, 5 mol% PC (DPPC), 5 mol% anionic lipids (DPPS), 40.5 mol% cholesterol, 1.5 mol% conjugated lipids (PEG-DMG).

41. The composition of any one of claims 1 to 40, wherein the composition is a vaccine.

42. A pharmaceutical composition comprising the LNP composition of any one of claims 1 to 40, and a pharmaceutically acceptable carrier.

43. A method comprising administering to a subject in need thereof the composition of claim 41 or claim 42 in an amount effective to induce an immune response against Mycobacterium tuberculosis infection in the subject.

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