Lipid nanoparticles for delivery of nucleic acids, and related methods of use

Abstract

To provide a lipid nanoparticle (LNP) for use in the delivery of therapeutic nucleic acids to cells.SOLUTION: Provided is use of (L-Serine) phosphatidylserine (PS) anionic targeting lipid in a total amount of 2.5-10 mol% of the total lipid content of a nucleic acid lipid nanoparticle (LNP) composition in the LNP composition.SELECTED DRAWING: Figure 15

Description

Technical Field

[0001] Related Applications This patent application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 118 ,534, filed on November 25, 2020, the entire content of which is incorporated herein by reference and made a part hereof.

[0002] Reference to Sequence Listing This specification includes a sequence listing submitted herewith together with this specification, which contains a file named 191016-010403_ST25.txt created on November 24, 2021 and having a size of 7,061 bytes, the content of which is incorporated herein by reference and made a part hereof. and made a part hereof.

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

Background Art

[0004] Lipid nanoparticles (LNPs) are used to deliver therapeutic nucleic acids to cells. For example, LNP pharmaceutical compositions are used in vaccines to deliver mRNA therapeutics. LNP formulations typically comprise an ionizable cationic lipid (ICL). However, certain ICL compounds are known in the art to be undesirably sensitive to oxidation during storage​​ Therefore, it has improved stability against oxidative degradation during storage and In some cases, when a therapeutic agent such as a nucleic acid is incorporated into an LNP, it induces desired transduction in cells. There is a need for improved ICL compounds that also provide inhibition activity or efficacy.

[0005] SNALP compositions are useful for delivery of nucleic acid therapeutics for a variety of infectious diseases. Infectious diseases such as HIV / AIDS, malaria, and COVID-19 are major threats to human health. For example, mycobacteria are the genus of bacteria responsible for tuberculosis (TB). According to the World Health Organization, TB is one of the top ten causes of death worldwide. It is the single most common cause of death among all pathogens. Despite current best efforts, The development of effective vaccines to prevent infectious diseases has presented a great challenge. New approaches in identifying peptides or antigen-peptide combinations may improve vaccine efficacy. Nevertheless, these antigen sequences were transferred to professional antigen-presenting cells such as dendritic cells. There is great potential in engineering adjuvants to help deliver and deliver to target cells efficiently. Antigen peptides or peptides combined with ionized cationic lipid nanoparticles remain mRNA coding for proteins represents a particularly promising strategy for vaccine development. SNALP PHARMACEUTICAL COMPOSITIONS FOR DELIVERY OF mRNA FOR THE TREATMENT AND PREVENTION OF VARIOUS DISEASES - Patent application There is a need for safe and effective treatments, including vaccine compositions. Summary of the Invention

[0006] In some embodiments, ionizable cationic lipids (ICLs) are provided. The cationic lipid is engineered to improve its stability against oxidative degradation during storage while retaining high transfection activity or efficacy intracellularly. Aspects of the disclosure are based, in part, on the discovery that having two or more methylene groups between a pair of alkynyl double bonds can improve unwanted oxidation and / or degradation of polyene-chain-containing ionizable lipids. The lipids disclosed herein include at least two carbon-carbon double bonds (olefins) spaced by at least two methylene groups or substituted methylene groups, where the substituted methylene is C(R1)(R2)-, where R1 and R2 are independently H, alkyl, or halogen. The lipids disclosed herein include two symmetric polyene hydrocarbon chains, each having two carbon-carbon double bonds (olefins) on either side of two, three, or four methylene groups. The olefins in the lipid tail separated by at least two methylene groups are much less susceptible to oxidation than compounds separated by one methylene group, such as DLin-MC3-DMA, and are considered an ultimate criterion in ionizable cationic lipid design and have been reported to have stability issues. In some embodiments, the compounds provided herein have a greater than 30%, greater than 50%, greater than 75%, greater than 90%, and greater than 95% reduction in oxidation byproducts when compared to a control LNP. In some embodiments, the compounds provided herein have a greater than 30%, greater than 50%, greater than 75%, greater than 90%, and greater than 95% reduction in oxidation byproducts when compared to a control LNP containing the DLin-KC2-DMA lipid. The olefins in the lipid tail separated by at least two methylene groups are much less susceptible to oxidation than compounds separated by one methylene group, such as DLin-MC3-DMA, and are considered an ultimate criterion in ionizable cationic lipid design and have been reported to have stability issues. In some embodiments, the compounds provided herein have a greater than 30%, greater than 50%, greater than 75%, greater than 90%, and greater than 95% reduction in oxidation byproducts when compared to a control LNP. In some embodiments, the compounds provided herein have a greater than 30%, greater than 50%, greater than 75%, greater than 90%, and greater than 95% reduction in oxidation byproducts when compared to a control LNP containing the DLin-KC2-DMA lipid. In some embodiments, the compounds provided herein have a greater than 30%, greater than 50%, greater than 75%, greater than 90%, and greater than 95% reduction in oxidation byproducts when compared to a control LNP. In some embodiments, the compounds provided herein have a greater than 30%, greater than 50%, greater than 75%, greater than 90%, and greater than 95% reduction in oxidation byproducts when compared to a control LNP containing the DLin-KC2-DMA lipid.

[0007] In some embodiments, an ionizable cationic lipid composition is provided. In some aspects, the ionizable cationic lipid can include two polyene hydrocarbon chains, each including one or two alkenyl double bond moieties. In some aspects, the ionizable cationic lipid can include two polyene hydrocarbon chains, each including two or more methylene groups between two alkenyl double bond moieties. In some aspects, the ionizable cationic lipid can contain two C 16 or C 18 polyene hydrocarbon chains.

[0008] In some embodiments, a liposomal composition is provided that includes an ionizable cationic lipid having a pair of linear polyene C or C hydrocarbon chains, each including an unsaturated linear ethylene, n-propylene, or n-butylene between two adjacent unsaturated alkynyl double bonds in each polyene hydrocarbon chain. 16 or C 18 In some embodiments, the liposomal composition can include an ionizable lipid having a chemical structure consisting of a pair of linear polyunsaturated lipid tails of 16 or 18 carbons covalently attached to a head group including a dialkylamino group having a pKa of 6-7, where the head group includes a heterocyclyl or alkyl moiety covalently attached to the dialkylamino group and optionally further includes a phosphate group, each polyunsaturated lipid tail is unsaturated except for at least two olefins separated by at least two methylene groups along the length of the lipid tail, and each lipid tail optionally includes a single acyl group at the terminus covalently attached to the head group. In some embodiments, ​​​​​​​​​In some embodiments, each lipid tail is the same and each lipid tail has a total of two olefins separated only by unsubstituted ethylene, n-propyl, or n-butyl. In some embodiments, each lipid tail further comprises an acyl group that forms an ester when combined with the oxygen of the head group.

[0009] In some embodiments, the dialkylamino moiety of the head group of the ionizable cationic lipid has a dialkylamino chemical structure of formula (IV-A).

[0010]

Chemical formula

[0011] In some embodiments, the ionizable cationic lipid comprises a chemical structure selected from the group consisting of

Chemical formula

Chemical formula

Chemical Structure

Chemical Structure

[0012] In some embodiments, the ionizable cationic lipid further comprises a pair of lipid tails linked to the head group, wherein each lipid tail comprises a hydrocarbon chain having a chemical structure of formula A or formula B

Chemical Structure

[0013]

Chemical Structure

[0014]

Chemical Structure

[0015] In some embodiments, the ionizable cationic lipid has the chemical structure of formula (IA): Yes.

[0016] [ka] [In the formula, a is 1, 2, 3, 4, 5 or 6; b is 2, 3 or 4; c is 3, 4, 5, 6, or 7, the sum of a, b, and c is 10 or 12, and q is 1 , 2, 3 or 4, and R 10 and R 12 Each of the following may be independently and optionally: is a (C1-C4) alkyl substituted with one or more hydroxyls; L is [ka] where v is 0 or 1, q is 1, 2 or 3, and q2 is 1 or 2. is] In some embodiments, in formula IA, when v is 0, q is 1, 2, or 3; When v is 1, q is 1, 2, 3, or 4.

[0017] In some embodiments, v in Formula I-A is 0. In some embodiments , v in Formula I-A is 0 and q is 1, 2, or 3. In some embodiments , v in Formula I-A is 0 and q is 1 or 2. In some embodiments, the ionizable lipid is a cationic lipid selected from the group consisting of Compounds 17-19 and 23-25.

[0018]

Chemical Structure

[0019] In some embodiments, the ionizable lipid is a cationic lipid selected from the group consisting of AKG-UO-1, AKG-UO-2 , AKG-UO-4, and AKG-UO-5. In some embodiments, the ionizable lipid is AKG-UO-1.

[0020]

Chemical Structure

[0021]

Chemical Structure

[0022]

Chemical Structure

[0023]

Chemical Structure

[0024]

Chem.

[0025]

Chem.

[0026]

Chem.

[0027]

Chem.

Chem.

[0028]

Chem.

[0029] In some embodiments, the ionizable lipid has a chemical structure of Formula II-A.

[0030] [Chemical formula] [wherein, a is 1, 2, 3, 4, 5 or 6, b is 2, 3 or 4, c is , 4, 5, 6, 7 or 8, R2 is [Chemical formula] , q is 1 or 2, R 10 and R 12 each is independently optionally (C1-C4) alkyl substituted with one or more hydroxyls

[0031] In some embodiments, the ionizable lipid is selected from the group consisting of Compounds 1-3 and Compounds 5-8. .

[0032] [Chemical formula]

[0033] In some embodiments, the ionizable lipid is selected from the group consisting of Compounds 1-8. .

[0034] [Chemical formula]

[0035] In some embodiments, the ionizable lipid has a chemical structure of Formula II-A.

[0036] ​[Chemical formula] [wherein, a is 1, 2, 3, 4, 5 or 6, b is 2, 3 or 4, and c is , 4, 5, 6, 7 or 8, R2 is [Chemical formula] and q' is 1 or 2, R 10 and R 12 each of which is independently, optionally, one or more hydroxyl substituted (C1-C4) alkyl]

[0037] In some embodiments, the ionizable lipid is a compound selected from the group consisting of Compounds 9-19 and is a compound.

[0038] [Chemical formula]

[0039] In some embodiments, the ionizable lipid has the chemical structure of Formula II-A.

[0040] [Chemical formula] [wherein, a is 1, 2, 3, 4, 5 or 6, b is 2, 3 or 4, and c is , 4, 5, 6, 7 or 8, R2 is [Chemical formula] and L is [Chemical formula] and where v is 0 or 1, q is 1, 2, 3 or 4, and q2 is 1 or 2, and R 10 and R 12 each independently is optionally substituted with one or more hydroxyl groups and is (C1-C4) alkyl] In some embodiments, in Formula II-A, when v is 0, q is 1, 2, or 3 or when v is 1, q is 3 or 4.

[0041] In some embodiments, the lipid is designed to be biodegradable, and thus the tolerance of the nanoparticles formed thereby in vivo is improved.

[0042] In some embodiments, the ionizable lipid has the chemical structure of Formula II-B.

[0043] [wherein a is 5, 6, or 7, c is 3, 4, or 5, R2 is R2 is [wherein q and q' are each independently 1 or 2, and R and R and R 10 and R 12 each independently is optionally substituted with hydroxyl and is (C 1-C4) alkyl]

[0044] In some embodiments, the ionizable cationic lipid is a compound selected from the group consisting of Compounds 29-34.

[0045] [wherein a is 5, 6, or 7, c is 3, 4, or 5,

[0046] ​In some embodiments, the ionizable lipid is a bio-reducible cationic lipid. In some embodiments, the ionizable lipid is a bio-reducible cationic lipid that includes a sterol chemical structure. In some embodiments, the ionizable lipid has the chemical structure of formula (VI-A) :

Chemical formula

Chemical formula

[0047]

Chemical formula

[0048] In some embodiments, the lipid nanoparticle composition includes a lipid and a nucleic acid, and the lipid nanoparticle contains an ionizable lipid of formula I, II, III, IV, or a combination thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid nanoparticle composition includes a lipid and a nucleic acid, and the lipid nanoparticle contains an ionizable lipid of formula I-A, II-A, II-B, V-A, or VI-A, or a combination thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid nanoparticle composition includes a lipid and a nucleic acid, and the lipid nanoparticle contains an ionizable lipid that includes a polyene hydrocarbon chain of formula A, formula A’, formula A”, or formula B, or a combination thereof, or a pharmaceutically acceptable salt thereof.

[0049] ​​​​​​​​​In some embodiments, the disclosure also provides compositions of lipid nanoparticles (LNPs) for delivering therapeutic nucleic acids to cells. Aspects of the disclosure are based in part on the surprising discovery that LNP compositions that combine certain amounts of phosphatidyl-L-serine with less than 20 mol% (e.g., 2.5 - 10 mol%) of the total lipids in the composition of various ionizable cationic lipids demonstrate a highly

[0050] enhanced targeting of encapsulated nucleic acids. In some embodiments, the LNP composition comprises (a) a nucleic acid, (b) an ionizable cationic lipid, (c) a sterol (e.g., cholesterol or a cholesterol derivative, or a plant sterol such as beta-sitosterol), (d) a phospholipid comprising phosphatidylserine (e.g., a mixture of phosphatidylserine and DSPC), and (e) a co-lipid (e.g., PEG-DMG). In one aspect, the LNP composition comprises (a) a nucleic acid, (b) an ionizable cationic lipid, (c) a sterol (e.g., cholesterol or a cholesterol derivative, or a plant sterol such as beta-sitosterol), (d) phosphatidylserine lipid in an amount of 1 - 10 mol% (e.g., 2.5 - 10 mol%, 3 - 9 mol%, 5.0 - 7.5 mol%) of the total lipids in the composition and additional phospholipids (e.g., DSPC), and (e) a co-lipid (e.g., PEG-DMG). , phosphatidylserine lipid in a total amount of 2.5 to 10 mol%, 3 to 9 mol%, 5.0 to 7.5 mol%), and additional phospholipids (e.g., DSPC), and (e ) a total amount of 0.5 to 4.5 mol% (e.g., 0.5 to 2.5 mol%, 1.5 mol ) of the total lipid in the composition of complex lipids (e.g., PEG-DMG). In one aspect, the LNP composition comprises (a) a nucleic acid, (b) an ionizable cationic lipid in a total amount of 40 to 65 mol% (e.g., 50 mol% ) of the total lipid in the composition, (c) a sterol in a total amount of 25 to 40 mol% (e.g., ) of the total lipid in the composition (e.g., cholesterol or a cholesterol derivative, or a plant sterol such as beta-sitosterol), (d) phosphatidylserine lipid in a total amount of 1 to 10 mol% (e.g., 2.5 to 10 mol%, 3 to 9 mol%, 5.0 to 7.5 mol ) of the total lipid in the composition, and additional phospholipids (e.g., DSPC) including phospholipids, and (e) a total amount of 0.5 to 4.5 mol% (e.g., 0 .5 to 2.5 mol%, 1.5 mol%) of the total lipid in the composition of complex lipids (e.g., PEG-DMG). In one aspect, the LNP composition comprises (a) a nucleic acid, (b) an ionizable cationic lipid in a total amount of 40 to 6 5 mol% (e.g., 50 mol%) of the total lipid in the composition, (c) a sterol in a total amount of 25 to 40 mol% (e.g., 38.5 mol%) of the total lipid in the composition (e.g., cole sterol or a cholesterol derivative, or a plant sterol such as beta-sitosterol), (d) a total amount of 5 to 25 mol% of the total lipid in the composition of phospholipids, wherein the composition contains phosphatidylserine lipid in a total amount of 1 to 10 mol% (e.g., 2.5 to 10 mol%, 3 to 9 mol%, 5.0 to 7 .5 mol%) of the total lipid in the composition, and additional phospholipids (e.g., DS ) of the total lipid in the composition, (c) a sterol in a total amount of 25 to 40 mol% (e.g., 38.5 mol%) of the total lipid in the composition (e.g., cole sterol or a cholesterol derivative, or a plant sterol such as beta-sitosterol), (d) a total amount of 5 to 25 mol% of the total lipid in the composition of phospholipids, and (d) phosphatidylserine lipid in a total amount of 1 to 10 mol% (e.g., 2.5 to 10 mol%, 3 to 9 mol%, 5.0 to 7 sterol or a cholesterol derivative, or a plant sterol such as beta-sitosterol), (d) a total amount of 5 to 25 mol% of the total lipid in the composition of phospholipids, and (d) phosphatidylserine lipid in a total amount of 1 to 10 mol% (e.g., 2.5 to 10 mol%, 3 to 9 mol%, 5.0 to 7 sterol), and additional phospholipids (e.g., DS ) of the total lipid in the composition, and (e) a total amount of 1 to 10 mol% (e.g., 2.5 to 10 mol%, 3 to 9 mol%, 5.0 to 7 .5 mol%) of the total lipid in the composition of phosphatidylserine lipid, and additional phospholipids (e.g., DS phospholipids containing PC (e.g., 10 mol% of the total lipids in the composition), and (e) a composition containing 0.5 to 4.5 mol% (e.g., 0.5 to 2.5 mol%, 1.5 mol%) of the total amount of complex lipids (e.g., PEG-DMG) of the total lipids in the composition. In one aspect, the LNP composition comprises (a) nucleic acid, (b) each of which has, between two adjacent unsaturated alkynyl double bonds in each polyene hydrocarbon chain, a pair of linear polyenes containing unsaturated linear ethylene, n-propylene or n-butylene C

[0051] or C hydrocarbon chain and is an ionizable cationic lipid present in the composition in a total amount of 40 to 65 mol% of the total lipids in the composition, (c) 25 to 40 mol% of the total amount of cholesterol in the composition, (d) 5 to 2 16 or C 18 phospholipids which are phospholipids containing phosphatidylserine lipids (e.g., phosphatidyl-L-serine lipids) and additional phospholipids (e.g., DSPC in a total amount of 10 mol% of the total lipids in the composition) in a total amount of 5 to 25 mol% of the total lipids in the composition, and (e) 0.5 to 2.5 mol% of the total amount of complex lipids (e.g., PEG-D MG) of the total lipids in the composition. In one aspect, the LNP composition comprises (a) mRNA nucleic acid, (b) in the composition 40 to 65 mol% of the total amount of the ionizable cationic lipid of formula (I-A), formula (II-A), or formula (II-B) when v is 0, (c) 25 to 40 mol% of the total amount of cholesterol in the composition, (d) 1 to 10 mol% (e.g., 2 .5 to 10 mol%, 3 to 9 mol%, 5.0 to 7.5 mol%) of the total amount of L-serine phospha tidyl lipids in the composition, and (e) 0.5 to 2.5 mol% of the total amount of complex lipids (e.g., PEG-D MG) of the total lipids in the composition. (e) containing 0.5 to 2.5 mol% of the total amount of complex lipids (e.g., PEG-D MG) of the total lipids in the composition. In one aspect, the LNP composition comprises (a) mRNA nucleic acid, (b) in the composition 40 to 65 mol% of the total amount of the ionizable cationic lipid of formula (I-A), formula (II-A), or formula (II-B) when v is 0, (c) 25 to 40 mol% of the total amount of cholesterol in the composition, (d) 1 to 10 mol% (e.g., 2 .5 to 10 mol%, 3 to 9 mol%, 5.0 to 7.5 mol%) of the total amount of L-serine phospha Chidylserine lipids (e.g., DPPS or DSPS), and 5 to 25 mol% of the total amount of DSPC in the composition, and (e) 0.5 to 2.5 mol% of the total amount of the complex lipid (e.g., PEG-DMG) in the composition is included. In one embodiment, the LNP composition , (a) mRNA nucleic acid, (b) the total amount of 40 to 65 mol% of the total lipid in the composition, when v is 0 of the ionizable cationic lipid of formula (I-A), (c) 25 to 40 mol of the total lipid in the composition % of the total amount of cholesterol, (d) 3 to 9 mol% of the total amount of L-ser ine phosphatidylserine lipid (e.g., DPPS or DSPS), and the total in the composition lipid of 5 to 25 mol% of the total amount of DSPC, and (e) 0.5 to 2 .5 mol% of the total amount of the complex lipid (e.g., PEG-DMG) is included.

[0052] In some embodiments, the composition comprises (a) a polyunsaturated ionizable cationic lipid, and (b) a charged phospholipid phosphatidylserine lipid.

[0053] In some embodiments, the composition comprises (a) an ionizable cationic lipid of formula IV-A , and (b) DSPS (L isomer), DPPS (L isomer), DMPS (L isomer) , DOPS (L isomer), DSPS (D isomer), DSPG, DPPG, N-Glu-D SPE, and an anionic phospholipid targeting moiety selected from the group consisting of N-Suc-DSPE portion. In some embodiments, the composition comprises (a) an ionizable cationic lipid of formula IV-A thionic lipid, and (b) an anionic phospholipid targeting moiety of formula V-A. In some embodiments, the composition comprises (a) an ionizable cationic lipid of formula IV-A, and (b) An anionic phospholipid targeting moiety selected from the group consisting of DSPS (L - isomer) and DPPS (L - isomer). It contains an anionic phospholipid targeting moiety.

[0054] In some embodiments, the composition comprises (a) an ionizable cationic lipid of formula IV, and (b) an anionic phospholipid targeting moiety of formula V - A. In some embodiments, the composition comprises (a) an ionizable cationic lipid of formula IV, as well as (b) DSPS (L - isomer), DPPS (L - isomer), DMPS (L - isomer), DOPS (L - isomer), DSP S (D - isomer), DSPG, DPPG, N - Glu - DSPE, and D - Suc - DS PE, and an anionic phospholipid targeting moiety selected from the group consisting thereof. In some embodiments, the composition comprises (a) an ionizable cationic lipid of formula IV, and (b) an anionic phospholipid targeting moiety of formula V - A. In some embodiments, the composition comprises (a ) an ionizable cationic lipid of formula IV - A, as well as (b) an anionic phospholipid targeting moiety selected from the group consisting of DSPS (L - isomer) and D PPS (L - isomer). It contains an anionic phospholipid targeting moiety selected from the group consisting of DSPS (L - isomer) and D PPS (L - isomer).

[0055] In one aspect, the LNP composition comprises (a) mRNA nucleic acid, (b) a total amount of 4 0 - 65 mol% of the total lipids in the composition, an ionizable cationic lipid selected from the group consisting of AKG - KC2 - OA, AKG - KC3 - OA, Dlin - K C2 - DMA and Dlin - KC3 - DMA, (c) a total amount of 25 - 40 mol% of cholesterol (or its derivative) of the total lipids in the composition, (d) a mixture of two or more phospholipids in a total amount of 5 - 25 mol% of the total lipids in the composition, wherein the phospholipids are 3 - 9 mol% (e.g., 5.0 - 7, of the total lipids in the composition), A mixture of phospholipids containing a total amount of L-serine phosphatidylserine lipids (e.g., DPPS or DS PS) of 5 mol%, and (e) a total amount of 0.5 to 2.5 mol% of the total lipids in the composition of complex lipids (e.g., PEG-DMG).

[0056]

Chemical formula

[0057] In one aspect, the nucleic acid lipid nanoparticle (LNP) composition comprises a nucleic acid, an ionizable cationic lipid AKG-UO-1, and a total amount of (L-serine) PS lipids of 2.5 to 10 mol% of the total lipid content of the LNP composition. In some embodiments, the nucleic acid is mRNA, and 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, and DOPC. The LNP composition further comprises 0.5 to 1.5 mol% of PEG-DMG or PEG-DSG with respect to the total lipid content in the LNP composition.

[0058] In one aspect, the nucleic acid lipid nanoparticle (LNP) composition comprises a nucleic acid, KC2OA, KC2, KC2-01, ALC-0315, and an ionizable cationic lipid selected from SM102, and a total amount of (L-serine ) PS lipids of 2.5 to 10 mol% of the total lipid content of the LNP composition. In some embodiments, the LNP composition has an N / P ratio of 3 to 8 (e.g., a ratio of 5 to 7 or 5).

[0059] In one aspect, the nucleic acid lipid nanoparticle (LNP) composition comprises a nucleic acid, AKG-UO-6 and ​​​​An ionizable cationic lipid selected from AKG-UO-7, and a total amount of (L-serine)PS lipid of 2.5 to 10 mol% of the total lipid content of the LNP composition. In some embodiments, the N / P ratio is 3 to 8 (e.g., a ratio of 5 to 7 or 5 or 7). In some embodiments, the N / P ratio is 3 to 8 (e.g., a ratio of 5 to 7 or 5 or 7).

[0060] In one aspect, the nucleic acid lipid nanoparticle (LNP) vaccine composition has an N / P ratio of 3 to 8 and contains an mRNA nucleic acid, a total amount of ALC-0315 ionizable cationic lipid of 46 to 65 mol% of the total lipid content of the LNP composition, a total amount of cholesterol of 25 to 40 mol% of the total lipid content of the LNP composition, a total amount of (L-serine)PS lipid of 2.5 to 10 mol% of the total lipid content of the LNP composition, a total amount of DSPC phospholipid of 5 to 25 mol% of the total lipid content of the LNP composition, and a total amount of PEG-DMG of 0 to 2.5 mol% of the total lipid content of the LNP composition. In one aspect, the nucleic acid lipid nanoparticle (LNP) vaccine composition has an N / P ratio of 3 to 8 and contains an mRNA nucleic acid, a total amount of Dlin-KC2-DMA ionizable cationic lipid of 40 to 65 mol% of the total lipid content of the LNP composition, a total amount of cholesterol of 25 to 40 mol% of the total lipid content of the LNP composition, a total amount of (L-serine)PS lipid of 2.5 to 10 mol% of the total lipid content of the LNP composition, a total amount of DSPC phospholipid of 5 to 25 mol% of the total lipid content of the LNP composition, and a total amount of PEG-DMG of 0 to 2.5 mol% of the total lipid content of the LNP composition. In one aspect, the lipid nanoparticle (LNP) vaccine composition has an N / P ratio of 3 to 8 and contains an mRNA nucleic acid, a total amount of KC3-OA of 40 to 65 mol% of the total lipid content of the LNP composition and other components as described above.

[0061] In one aspect, the nucleic acid lipid nanoparticle (LNP) vaccine composition has an N / P ratio of 3 to 8 and contains an mRNA nucleic acid, a total amount of Dlin-KC2-DMA ionizable cationic lipid of 40 to 65 mol% of the total lipid content of the LNP composition, a total amount of cholesterol of 25 to 40 mol% of the total lipid content of the LNP composition, a total amount of (L-serine)PS lipid of 2.5 to 10 mol% of the total lipid content of the LNP composition, a total amount of DSPC phospholipid of 5 to 25 mol% of the total lipid content of the LNP composition, and a total amount of PEG-DMG of 0 to 2.5 mol% of the total lipid content of the LNP composition. In one aspect, the nucleic acid lipid nanoparticle (LNP) vaccine composition has an N / P ratio of 3 to 8 and contains an mRNA nucleic acid, a total amount of cholesterol of 25 to 40 mol% of the total lipid content of the LNP composition, a total amount of (L-serine)PS lipid of 2.5 to 10 mol% of the total lipid content of the LNP composition, a total amount of DSPC phospholipid of 5 to 25 mol% of the total lipid content of the LNP composition, and a total amount of PEG-DMG of 0 to 2.5 mol% of the total lipid content of the LNP composition. In one aspect, the lipid nanoparticle (LNP) vaccine composition has an N / P ratio of 3 to 8 and contains an mRNA nucleic acid, a total amount of KC3-OA of 40 to 65 mol% of the total lipid content of the LNP composition and other components as described above.

[0062] In one aspect, the lipid nanoparticle (LNP) vaccine composition has an N / P ratio of 3 to 8 and contains an mRNA nucleic acid, a total amount of KC3-OA of 40 to 65 mol% of the total lipid content of the LNP composition Ionized cationic lipid, cholesterol in an amount of 25 to 40 mol% of the total lipid content of the LNP composition Sterol, (L-serine) in an amount of 2.5 to 10 mol% of the total lipid content of the LNP composition PS lipid, DSPC phospholipid in an amount of 5 to 25 mol% of the total lipid content of the LNP composition, and PEG-DMG in an amount of 0 to 2.5 mol% of the total lipid content of the LNP composition.

[0063] In one aspect, a nucleic acid lipid nanoparticle (LNP) vaccine composition has an N / P ratio of 3 to 8 and contains an mRNA nucleic acid, ionized cationic lipid in an amount of 40 to 65 mol% of the total lipid content of the LNP composition, cholesterol in an amount of 25 to 40 mol% of the total lipid content of the LNP composition, (L-serine) PS lipid in an amount of 2.5 to 10 mol% of the total lipid content of the LNP composition, and DSPC phospholipid in an amount of 5 to 25 mol% of the total lipid content of the LNP composition, and PEG-DMG in an amount of 0 to 2.5 mol% of the total lipid content of the LNP composition.

[0064] One aspect of the present disclosure relates to the use of (L-serine) PS lipid in an amount of 2.5 to 10 mol% of the total lipid content of the LNP composition for targeting LNP to dendritic cells. In some embodiments, the LNP contains mRNA. In some embodiments, the LNP further contains cholesterol. In some embodiments, the LNP further contains ICL. In some embodiments, the LNP further contains one or more additional phospholipids including DSPC. In some embodiments, the LNP further contains complex lipids. In some embodiments, the LNP contains an mRNA nucleic acid having an N / P ratio of 3 to 8 and ionized cationic lipid (ICL) in an amount of 40 to 65 mol% of the total lipid content of the LNP composition ​​​​​​​ ) Cholesterol in an amount of 25 to 40 mol% of the total lipid content of the LNP composition, the LNP composition (L-serine)PS lipid in an amount of 2.5 to 10 mol% of the total lipid content of the LNP composition, the LNP composition DSPC phospholipid in an amount of 5 to 25 mol% of the total lipid content of the LNP composition, and the total lipid of the LNP composition It contains a complex lipid in an amount of 0 to 2.5 mol% of the content.

Brief Description of the Drawings

[0065]

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Figure 17

Mode for Carrying Out the Invention

[0066] Both the foregoing summary and the following detailed description are for illustrative and explanatory purposes only, and it is to be understood that the compositions and methods of the present disclosure are not limited thereby.

[0067] Stabilized nucleic acid lipid particles (SNALP) are used as a vehicle for the systemic delivery of mRNA or other nucleic acid therapeutics. The SNALP composition is separated by a single methylene group A pair of straight-chain 18-carbon aliphatic chains containing a pair of carbon-carbon double bonds ( e.g., linoleic acid) and a protonatable tertiary amine head group bound thereto, including a cationic lipid such as MC3 or KC2. However, the structure of these hydrocarbon chains, each containing a pair of double bonds separated by a single methylene group confers the desired biological properties of the SNALP composition, but this chemical substructure also presents an undesirable problem of increased susceptibility of the compound to oxidative degradation. For example, FIG. 1 is a diagram of the oxidative degradation mechanism of a lipid ester of linoleic acid containing a plurality of conjugated unsaturates particularly sensitive to oxidation. Suitable for use in SNALP compositions, there is a need for novel cationic lipids with enhanced resistance to oxidative degradation.

[0068] Disclosed herein are compounds, compositions, and methods related to the treatment of bacterial infections. As used herein, the terms "compound," "drug," and "active agent" are used interchangeably. Some aspects of the present disclosure relate to novel ionizable lipids or bioreducible ionizable lipids. These lipids are cationic at acidic pH (i.e., carry a positive charge) and are encountered intracellularly, for example, after endocytosis or phagocytosis by cells. The same lipids and compositions containing them have a nearly neutral charge when present at pH 7.4. These lipids may also have a plurality of olefins separated by at least two methylene groups present in their alkyl or acyl groups.

[0069]

[0070] ​​​​​​​​Other aspects relate to compositions comprising lipid nanoparticles comprising an ionizable cationic lipid, wherein the lipid nanoparticles contain nucleic acid. In some embodiments, the nucleic acid is encapsulated within the lipid nanoparticles .

[0071] Other aspects of the disclosure relate to the use of these ionizable lipids or lipid nanoparticle compositions comprising ionizable lipids in vaccines for the prevention of infectious diseases or cancer. In some embodiments, the infectious disease may be a bacterial or viral infection. In some embodiments, the compositions described herein are used to prevent infectious diseases associated with tuberculosis, HIV / AIDS, malaria, or coronavirus-related infections such as COVID-19. In other embodiments, the infectious disease is influenza, hepatitis B, hepatitis C, dengue fever, human papillomavirus (HPV), norovirus, mumps, measles, meningococcal disease, pneumococcal disease, polio, rotavirus, respiratory syncytial virus (RS V), rubella, herpes zoster / herpesvirus, tetanus, or whooping cough.

[0072] In some embodiments, the compounds and compositions described herein can promote efficient uptake and transfection of target cells, including tissue macrophages and dendritic cells. As a result, efficient delivery of nucleic acids encoding antigens specific for infectious viruses or bacteria and subsequent presentation of the antigens that elicit the desired immune response that protects against the corresponding infectious disease occur. In some embodiments, the nucleic acid is an epitope of a coronavirus such as SARS -CoV, MERS-CoV or SARS-CoV-2 . . ​It may be a synthetic nucleic acid encoding a -peptide (e.g., codon-optimized mRNA). In several embodiments, the nucleic acid is an S-protein (spike protein) of a coronavirus such as SARS-CoV, MERS-CoV or SARS-CoV-2 or a fragment thereof, and may be a

[0073] Definition For convenience, the specific terms used in this specification, the examples, and the appended claims are summarized herein. Unless otherwise specified, all technical and scientific terms used

[0074] in this specification shall have the same meaning as commonly understood by one of ordinary skill

[0075] in the technical field to which this disclosure pertains. As used in this specification, the following terms and phrases are intended to have

[0076] the following meanings. The articles "a" and "an" are used to refer to one or more (i.e., at least one) grammatical objects of the article. By way of

[0077] example, "an element" means one element or more refers to the element. This term allows for the presence of additional elements that do not substantially affect the basis and novel or functional characteristics of that embodiment of the present disclosure.

[0078] The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding all elements not recited during the description of the embodiment.

[0079] As used herein, the term "comprising" includes "consisting of" and "consisting essentially of".

[0080] When reference is made in the description to "as described above" or "above-mentioned", "the foregoing", it refers to any of the disclosures made in the specification on any of the previous pages.

[0081] When reference is made in the description to "as described herein", "described herein", "provided herein" or "as described in this text", or "as defined herein", it refers to any of the disclosures made in the specification on any of the previous or subsequent pages.

[0082] As used herein, the term "about" means an acceptable variation within 20%, within 10% and within 5% of the specified value. In certain embodiments, "about" can mean a variation of + / - 1%, 2%, 3%, 4%, 5%, 10% or 20%.

[0083] As used herein in connection with a compound or composition, the term "effective amount" means an amount of the active compound (also referred to herein as the active agent or active drug) sufficient to produce a bactericidal or bacteriostatic effect. In one embodiment, the effective amount is the symptoms of the bacterial infection being treated ​​​​​​​ It refers to a "therapeutically effective amount" of the active compound sufficient to alleviate.

[0084] As used herein, the term "subject (alternatively, "patient")" refers to an animal, preferably a mammal, most preferably a human, that is to receive either a prophylactic or a therapeutic treatment.

[0085] As used herein, the term "administration" or "administering" means all means of introducing a compound or pharmaceutical composition into a subject in need thereof, including, but not limited to, oral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, inhalation, buccal, ophthalmic, sublingual, vaginal, and rectal. Administration of the compound or composition is preferably parenteral. For example, the compound or composition can preferably be administered intravenously, but currently, as used clinically for liposomal amikacin in the treatment of Mycobacterium avium, it can also be administered intraperitoneally or via inhalation (see Shirley et al., Amikacin Liposome Inhalation Suspension: A Review in Mycobacterium avium Complex Lung Disease. Drugs. 2019 Apr;79(5):555 - 562).

[0086] As used herein, the terms "treat", "treating", and "treatment" refer to therapeutic or prophylactic measures such as those described herein.

[0087] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic inorganic or organic acid addition salt of the compounds of the present disclosure that has the desired pharmacological activity.

[0088] The term "alkyl" means a saturated carbon chain having 1 to 20 carbon atoms, which may be linear, branched, or a combination thereof, unless otherwise specified in the carbon chain. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, and octyl. Unless otherwise specified herein, alkyl groups are optionally substituted.

[0089] The term "phosphatidylserine" refers to the L-isomer of serine in the head group, together with any of its acyl chain compositions, unless otherwise specified in a particular embodiment.

[0090] The term "lipid complex" refers to a complex lipid that inhibits aggregation of lipid particles. Such lipid complexes include, but are not limited to, polysarcosine (see, e.g., WO2021191265A, which is hereby incorporated by reference in its entirety for all purposes), polyamide oligomers (e.g., ATTA-lipid complexes), PEG-lipid complexes (PEG coupled to dialkyloxypropyl, PEG coupled to diacylglycerol, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, PEG conjugated to ceramide, etc.) (see, e.g., U.S. (e.g., which is hereby incorporated by reference in its entirety for all purposes). (see Japanese Patent No. 5,885,613), cationic PEG lipids, and mixtures thereof include. PEG can be conjugated directly to the lipid or to the lipid via a linker moiety bonded. Any linker moiety suitable for coupling PEG to the lipid (e.g., including non-ester-containing linker moieties and ester-containing linker moieties) can be used . In preferred embodiments, non-ester-containing linker moieties are used.

[0091] The abbreviations of ionizable cationic lipids can be truncated from those used in the tables in the examples. For example, AKG-UO-1 or AKG-KC2-01 may be referred to as UO1 or KC 2-01.

[0092] The abbreviation UT used in various studies refers to untreated samples.

[0093] The term "lipid nanoparticles" or "LNP" refers to particles having a diameter of about 5 to 500 nm. In some embodiments, the lipid nanoparticles contain one or more active agents. In some embodiments, the lipid nanoparticles contain nucleic acids. In some embodiments, the nucleic acid is condensed inside nanoparticles having an external lipid coat that interacts with cationic lipids, polymers, or multivalent small molecules and the biological environment. Due to the repulsion between phosphate groups , nucleic acids are necessarily rigid polymers and preferably have an elongated shape. Inside cells, to cope with volume constraints, DNA can pack itself with the help of ions and other molecules under appropriate solution conditions. Usually, DNA condensation is defined as the collapse of extended DNA strands into compact, systematic particles containing only one or a few molecules. Phosph ​​By binding to the ester group, the cationic lipid can neutralize the phosphate charge and densely pack the DNA by condensing it.

[0094] In some embodiments, the active agent is encapsulated in the LNP. In some embodiments the active agent may be an anionic compound, for example, DNA, RNA, natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNAs and short interfering RNAs), nuclear proteins, peptides, nucleic acids, ribozymes, DNA-containing nuclear proteins, for example, intact or partially deproteinized virus particles (virions), oligomers and polymeric anionic compounds other than DNA (for example, acidic polysaccharides and glycoproteins)), but are not limited thereto. In some embodiments, the active agent may be mixed with an adjuvant.

[0095] In LNP vaccine products, the active agent is generally contained inside the LNP. In some embodiments, the active agent includes nucleic acids. Typically, water-soluble nucleic acids are condensed with the cationic lipid or polycationic polymer inside the particle, and the surface of the particle is rich in neutral lipid or PEG-lipid derivatives. Additional ionizable cationic lipids may also be on the surface and, by having a positive charge, respond to acidification in the environment and promote endosomal escape.

[0096] The ionizable lipid may have properties or functions different from those of the LNP. Due to the pKa of the amino group, the lipid molecule may become positively charged under acidic conditions. Under these conditions, the lipid molecule can electrostatically bind to the phosphate group of the nucleic acid, facilitating the formation of the LNP and the In some embodiments, the pKa is at a physiological pH value, such as blood. The surface charge of the LNP may be low enough to render it substantially neutral in any biological fluid. The P surface charge is responsible for toxicity, rapid removal from the circulation by sessile and free macrophages, and hemolysis. Associated with toxicity (including immune activation) (Filion et al. Biochim B iophys Acta.1997 Oct 23;1329(2):345-56).

[0097] In some embodiments, the pKa is determined so that the ionized cationic lipid is capable of reacting with an acidic endothelium. The pH of the solution may be high enough to allow the cation to assume a positive charge at the room pH value. The anionic lipids, in combination with endogenous endosomal anionic lipids, form membrane-soluble structures such as the hexahedral HII phase. Dissociation of non-bilayer structures can be promoted, resulting in more efficient intracellular transport. In some embodiments, the pKa is in the range of 6.2 to 6.5. a may be about 6.2, about 6.3, about 6.4, about 6.5. The unsaturated tail may be a non-dicyclic It also contributes to the ability of lipids to form molecular layer structures (Jayaraman et al., Ang ew Chem Int Ed Engl.2012 Aug 20;51(34):8 529-33).

[0098] Among other properties, such as liposome clearance and circulation half-life, the release of nucleic acids from LNP formulations The extract may be polyethylene glycol and / or a sterol (e.g., cholesterol) or The presence of other potential excipients in the LNP or LNP, as well as the overall chemical structure (as part of the formulation) The lipids can be modified by any ionizable cationic lipid (including the pKa of any ionizable cationic lipids included in the lipids). do.

[0099] The term "bioreductive" refers to a compound that undergoes accelerated degradation due to cleavage of disulfide bonds in a reducing environment. Unlike other nucleic acid therapeutics such as siRNA, the success of mRNA-based therapeutics is determined by the utility of a safe and efficient delivery vehicle that encapsulates the mRNA. mRNA is fragile and requires a protective coating to maintain its active state until it reaches the target site. mRNA-containing LNPs are a promising option for COVID-19 immunity (Jackson et al., Preliminary Repor t. N Engl J Med. 2020 Nov 12;383(20):1920- 1931). The efficiency and tolerability of LNPs are attributed to amino lipids, and unlike many biomaterial applications that may require weeks or months of dosing, functional LNP-mediated delivery of mRNA occurs within hours, eliminating the need for persistent lipids. Indeed, this is particularly important for applications that require long-term dosing. LNPs have been shown to enter cells via endocytosis and accumulate in the endosomal compartment. Ionizable cationic lipids (ICLs) are susceptible to enzymatic hydrolysis by lipases or hydrolysis induced by the reducing environment of late endosomes / lysosomes and are completely biodegradable, but can efficiently deliver mRNA to the cytosol after endocytosis. The extracellular space is a relatively acidic environment, while the intracellular space is a reducing environment, allowing disulfide bond molecules to remain intact in the extracellular space but be rapidly reduced upon internalization (Huang et al. , Mol Ther. 2005 Mar;11(3):409-17, 2005). Several One embodiment is a biodegradable disulfide bond ICL molecule (see Compounds 29-36 (Table 2)) that is stable in the LNP formulation during circulation but cleaves in the reducing environment of lysosomes. Such compounds and compositions can promote rapid biological destruction of lipids and prevent potential toxic accumulation of ICL lipids (as observed in the case of formulations with DLin-MC3-DMA (Sabins et al., Mol Ther. 2018 Jun 6;26(6):1509-1519). When used herein, the terms "encapsulation" and "encapsulated" refer to the incorporation of mRNA, DNA, siRNA or other nucleic acid pharmaceuticals into lipid nanoparticles or their association with lipid nanoparticles. As used herein, the term "encapsulated" refers to complete encapsulation or partial encapsulation. siRNA can selectively knockdown or downregulate the expression of a target gene. For example, siRNA can be selected to silence a gene associated with a particular disease, disorder,

[0100] or condition when a nanoparticle composition containing the siRNA is administered to a subject in need thereof. siRNA may include a sequence complementary to the mRNA sequence encoding the target gene or protein. When used herein, the term "mol %" related to cholesterol refers to the molar amount of cholesterol relative to the total molar amount of cholesterol and non-PEGylated phospholipids expressed in percentage points. For example, "55 mol % cholesterol" in a liposome containing cholesterol and HSPC refers to a composition of 55 parts by mole of cholesterol per 45 parts by mole of HSPC.

[0101] ​​​​​​​​​​refers to.

[0102] The related term "mol%" of PEG-lipid refers to the ratio of the molar amounts of PEG -lipid and non-PEGylated phospholipid. For example, "5 mol% of PEG-DSPE" in an LNP containing HSPC and PEG- DSPE refers to a composition having 5 mol parts of PEG-DSPE per 100 mol parts of HS PC.

[0103] As used herein, the term "pharmaceutically acceptable carrier, diluent or excipient" refers to any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent that has been approved by the US Food and Drug Administration as acceptable for use in humans or livestock, and includes, but is not limited to, these.

[0104] Various aspects and embodiments are described in further detail in the following subsections.

[0105] Compound Provided herein are compounds, compositions and methods for the treatment or prevention of infectious diseases including tuberculosis. According to aspects of the present disclosure, the cationic lipid includes a compound having formula I, II, III or IV or a pharmaceutically acceptable salt thereof. According to aspects of the present disclosure the ionizable cationic lipid includes one or more chemical substructures selected from the group consisting of formula IV, formula IV-A, formula A, formula A', formula A'', formula A''', and / or formula B. In some aspects of the present disclosure, the ionizable cationic lipid has the formula I, formula I-A, formula I-A', formula I-A'', formula II, formula II-A, formula II-A', formula II ​ -B, a compound having formula II-B', formula III, or formula III-A, or a pharmaceutically acceptable salt thereof. In some embodiments of the present disclosure, the LNP can comprise a compound having formula V or formula V-A, or a pharmaceutically acceptable salt thereof. In some embodiments of the present disclosure, the LNP can comprise a compound having formula VI or formula VI-A, or a pharmaceutically acceptable salt thereof. In some embodiments of the present disclosure, the LNP can comprise a compound having formula VII, or a pharmaceutically acceptable salt thereof. In some embodiments of the present disclosure, the LNP can comprise a compound having formula VIII, or a pharmaceutically acceptable salt thereof. According to an embodiment of the present disclosure, the cationic lipid is (a) an ionizable cationic lipid selected from a compound of formula I, formula I-A, formula I-A', formula I-A'', formula II, formula II-A, formula II-A', formula II-B, formula II-B', formula III, or formula III-A, or a sterol lipid of formula VI-A, or a branched lipid of formula V-III, and (b) has a sterol of formula VI, and optionally (c) further comprises a compound comprising an alkylene glycol lipid of formula VII. In some embodiments, the LNP further comprises a phospholipid of formula V or formula V-A. In some embodiments, the LNP further comprises an anionic phospholipid targeting moiety of Table 3. can include. In some embodiments of the present disclosure, the LNP can comprise a compound having formula V or formula V-A, or a pharmaceutically acceptable salt thereof. can include. In some embodiments of the present disclosure, the LNP can comprise a compound having formula V or formula V-A, or a pharmaceutically acceptable salt thereof. can include. In some embodiments of the present disclosure, the LNP can comprise a compound having formula VI or formula VI-A, or a pharmaceutically acceptable salt thereof. can include. In some embodiments of the present disclosure, the LNP can comprise a compound having formula VI or formula VI-A, or a pharmaceutically acceptable salt thereof. can include. In some embodiments of the present disclosure, the LNP can comprise a compound having formula VII, or a pharmaceutically acceptable salt thereof. can include. In some embodiments of the present disclosure, the LNP can comprise a compound having formula VIII, or a pharmaceutically acceptable salt thereof. can include. According to an embodiment of the present disclosure, the cationic lipid is (a) an ionizable cationic lipid selected from a compound of formula I, formula I-A, formula I-A', formula I-A'', formula II, formula II-A, formula II-A', formula II-B, formula II-B', formula III, or formula III-A, or a sterol lipid of formula VI-A, or a branched lipid of formula V-III, and (b) has a sterol of formula VI, and optionally (c) further comprises a compound comprising an alkylene glycol lipid of formula VII. can include. In some embodiments, the LNP further comprises a phospholipid of formula V or formula V-A. In some embodiments, the LNP further comprises an anionic phospholipid targeting moiety of Table 3. from the selected ionizable cationic lipid, or a sterol lipid of formula VI-A, or a branched lipid of formula V-III, and (b) has a sterol of formula VI, and optionally (c) further comprises a compound comprising an alkylene glycol lipid of formula VII. from the selected ionizable cationic lipid, or a sterol lipid of formula VI-A, or a branched lipid of formula V-III, and (b) has a sterol of formula VI, and optionally (c) further comprises a compound comprising an alkylene glycol lipid of formula VII. III, and (b) has a sterol of formula VI, and optionally (c) further comprises a compound comprising an alkylene glycol lipid of formula VII. can further include a compound comprising an alkylene glycol lipid of formula VII. In some embodiments, the LNP further comprises a phospholipid of formula V or formula V-A. In some embodiments, the LNP further comprises an anionic phospholipid targeting moiety of Table 3. In some embodiments, the LNP further comprises a phospholipid of formula V or formula V-A. In some embodiments, the LNP further comprises an anionic phospholipid targeting moiety of Table 3. In some embodiments, the LNP further comprises an anionic phospholipid targeting moiety of Table 3.

[0106] Also provided herein are compounds, compositions, and methods for the treatment or prevention of infectious diseases, including tuberculosis. According to an embodiment of the present disclosure, the cationic lipid comprises a compound having formula A, or a pharmaceutically acceptable salt thereof. In some embodiments, the cationic lipid Also provided herein are compounds, compositions, and methods for the treatment or prevention of infectious diseases, including tuberculosis. According to an embodiment of the present disclosure, the cationic lipid comprises a compound having formula A, or a pharmaceutically acceptable salt thereof. comprises a compound having formula A, or a pharmaceutically acceptable salt thereof. In some embodiments, the cationic lipid The sex lipid contains two fatty acyl groups as in Formula II, II, III or IV.

[0107] Disclosed herein are compounds of Formula I, Formula II, Formula III, Formula IV or pharmaceutically acceptable salts thereof useful in the preparation of vaccines. Also disclosed herein are compositions comprising a cationic lipid of Formula I, Formula II, Formula III, Formula IV or a pharmaceutically acceptable salt thereof. In some embodiments, the vaccine is used for the prevention of mycobacterium infection. In some embodiments, the vaccine is tuberculosis, non-tuberculous mycobacteria (NTM), non-tuberculous lung disease, leprosy, mycobacterium avium-intracellulare, mycobacterium kansasii, mycobacterium marinum, mycobacterium ulcerans, mycobacterium chelonae, mycobacterium fortuitum, mycobacterium abscessus, and other infectious diseases such as coronaviruses (COVID-19, SARS CoV2, SARS-CoV, MERS-CoV), diphtheria, Ebola, flu (influenza), hepatitis, Hib disease, HIV / AIDS, HPV (human papillomavirus), malaria, measles, meningococcal disease, otitis ​​​​​​​​​​​​​​​​Influenza, norovirus, plague, diseases caused by Streptococcus pneumoniae, polio, respiratory syncytial virus ( RSV), rotavirus, rubella (German measles), herpes zoster (herpes), tetanus (lockjaw ), whooping cough (pertussis), and Zika can be used for prevention.

[0108] Disclosed herein are compounds, compositions and methods for treating or preventing infectious diseases, including tuberculosis. According to aspects of the present disclosure, the cationic lipid includes a compound having formula I, II, III or IV or a pharmaceutically acceptable salt thereof. In some embodiments, the cationic lipid includes two fatty acyl groups of formula I, II, III or IV.

[0109] One aspect of the present disclosure provides a lipid including one or more polyunsaturated polyene hydrocarbon chains of formula A

[0110] [Chemical formula] [wherein, a is 1, 2, 3 or 4, b is 2, 3 or 4, and c is 3, 4 , 5, 6 or 7] In some aspects, the ionizable lipid can include two polyunsaturated polyene hydrocarbon chains where b is 4. In some aspects, the ionizable lipid can include two polyunsaturated polyene hydrocarbon chains of formula A, wherein the sum of a, b and c is 10, 11, 12 or 13. In some aspects, the ionizable lipid can include two polyunsaturated polyene hydrocarbon chains of formula A, wherein a is 4, b is 4, and c is 4 or 5. In some aspects, the ionizable lipid can include two polyunsaturated poly ene hydrocarbon chains of formula A, wherein It can contain a saturated polyene hydrocarbon chain, where a is 1, 2, or 3, and b is 4 and c is 3, 4, 5, 6, or 7. In some embodiments, the ionizable lipid can contain two polyunsaturated polyene hydrocarbon chains of Formula A, where a is 5 or 6 and b is 2, 3, or 4, and c is 3, 4, 5, 6, or 7. In some embodiments the ionizable lipid can contain two polyunsaturated polyene hydrocarbon chains of Formula A where the sum of a, b, and c is 10, 11, 12, or 13. In some embodiments the ionizable lipid can contain two polyunsaturated polyene hydrocarbon chains of Formula A where the sum of a, b, and c is 12. In some embodiments, the ionizable lipid can contain two polyunsaturated polyene hydrocarbon chains of Formula A, where b is 2 and the sum of a, b, and c is 12. In some embodiments, the ionizable lipid is can contain two polyunsaturated polyene hydrocarbon chains of Formula A, where b is 3, and a, b and c sum to 12. In some embodiments, the ionizable lipid is two of Formula A can contain polyunsaturated polyene hydrocarbon chains, where b is 4, and a, b and c sum to 12. In some embodiments, the ionizable lipid can contain two polyunsaturated polyene hydrocarbon chains of Formula A.

[0111] One aspect of the present disclosure provides a lipid containing one or more polyunsaturated polyene hydrocarbon chains of Formula A for.

[0112]

Chemical formula

[0113] One aspect of the present disclosure provides a lipid comprising one or more polyunsaturated polyene hydrocarbon chains of formula A’’ In some embodiments, the ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of formula A’ where the sum of a, b, and

[0114] [Chemical formula] [wherein a is 4, b is 4, and c is 4 or 5] In some embodiments, the ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of formula A’ where the sum of a, b, and c is 12

[0115] One aspect of the present disclosure provides a lipid comprising one or more polyunsaturated polyene hydrocarbon chains of formula A’’’ In some embodiments, the ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of formula A’ where the sum of a, b, and

[0116] [Chemical formula]

[0117] [wherein a is 5 or 6, b is 2, 3, or 4, and c is 3, 4, 5, 6, or 7] In some embodiments, the ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of formula A’’’ where the sum of a, b, and c is 10, 11, 12, or 13. In some embodiments, the ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of formula A where the sum of a, b, and c is 12 It can contain two polyunsaturated polyene hydrocarbon chains of “’’’. In some embodiments the ionizable lipid can contain two polyunsaturated polyene hydrocarbon chains of formula A’’’ where b is 2 and the sum of a, b and c is 12 It can contain two polyunsaturated polyene hydrocarbon chains of “’’’. In some embodiments the ionizable lipid can contain two polyunsaturated polyene hydrocarbon chains of formula A’’’ where b is 3 and the sum of a, b and c is 12 It can contain two polyunsaturated polyene hydrocarbon chains of “’’’. In some embodiments, the ion izable lipid can contain two polyunsaturated polyene hydrocarbon chains of formula A’’’ where b is 4 and the sum of a, b and c is 12 It can contain two polyunsaturated polyene hydrocarbon chains of “’’’. In some embodiments, the ionizable lipid can contain two polyunsaturated polyene hydrocarbon chains of formula A’’’.

[0118] One aspect of the present disclosure provides a lipid containing one or more polyunsaturated polyene hydrocarbon chains of formula B

[0119] [Chemical formula] [wherein, a is 5, 6 or 7, and c is 3, 4 or 5] In some embodiments, the ionizable lipid can contain two polyunsaturated polyene hydrocarbon chains where b is 4. In some embodiments, the ionizable lipid can contain two polyunsaturated polyene hydrocarbon chains of formula B where the sum of a and c is 9, 10 or 11

[0120] In some embodiments, the ionizable lipid has the chemical structure of formula (IV-A) [Chemical formula] or a pharmaceutically acceptable salt thereof, wherein Y is [Chemical formula]​​ and n is an integer of 2, 3 or 4, and R 22 is a polyene hydrocarbon chain of formula A, formula A', formula A'', or formula A''' or formula B, and R 10 and R 12 each is independently, optionally substituted with hydroxyl, (C1-C4) alkyl .

[0121] In some embodiments, R in formula (IV-A) 22 is a polyene hydrocarbon chain of formula A . In some embodiments, R in formula (IV-A) 22 is a polyene hydrocarbon chain of formula A' . In some embodiments, R in formula (IV-A) 22 is a polyene hydrocarbon chain of formula A'' . In some embodiments, R in formula (IV-A) 22 is a polyene hydrocarbon chain of formula A''' . In some embodiments, R in formula (IV-A) 22 is , a polyene hydrocarbon chain of formula B

[0122] In some embodiments, in formula (IV-A), R 10 and R 12 are each independently selected from methyl, ethyl, propyl, -(CH2)(CH2)OH, and -(CH2)2( CH2)OH. In some embodiments, in formula (IV-A), R 10 and R 12 are each independently methyl. In some embodiments, in formula (IV-A ), R 10 and R 12 are each independently ethyl. In some embodiments, in formula (IV-A), R 10and R 12 At least one of which is optionally hydro substituted n-propyl with xylyl. In some embodiments, formula (IV-A) in, R 10 is methyl, and R 12 is selected from methyl, ethyl, -(CH2)(CH2)OH, and -(CH2)2(CH2)OH. In some embodiments, formula (I V-A), R 10 is methyl, and R 12 is selected from -(CH2)(CH2)OH, and - (CH2)2(CH2)OH. In some embodiments, in a compound containing the chemical structure of formula (IV-A) , R 10 is methyl, and R 12 is selected from -(CH2)(C H2)OH, and -(CH2)2(CH2)OH. In some embodiments in, in formula (IV-A), R 10 and R 12 are independently selected from methyl or ethyl, optionally substituted with one or more hydroxyls. In some embodiments in, in formula (IV-A), R 10 and R 12 one or both of which are -(CH2)(CH2)OH or -(CH2)2(CH2)OH in formula (IV-A) . In some embodiments, in formula (IV-A), R 10 is methyl, and R 12 is methyl or ethyl substituted with hydroxyl . In some embodiments, one or both of R in formula (IV-A) are methyl, and R 10 one or both of which are methyl, and R 12 is -(CH2) (CH2)OH in formula (IV-A). In some embodiments, one or the other of R 10 in formula (IV-A) Both are methyl, and R 12 is -(CH2)2(CH2)OH in formula (IV-A) exists.

[0123] In some embodiments, the ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalently attached to the Y moiety of formula (IV-A), wherein Y is and n is an integer of 2, 3 or 4, and R

Chemical formula

Chemical formula

Chemical formula

[0124] In some embodiments, the ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalently attached to the Y moiety of formula (IV-A), wherein Y is [Chemical formula] 22 and n is an integer of 2, 3 or 4, R is a polyene hydrocarbon chain of formula A, formula A', formula A'', formula A''' 10 or formula B, and each of R 12 and R is independently optionally substituted with hydroxyl and is (C1-C4) alkyl. In some embodiments, the ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalently attached to the Y moiety of formula (IV-A), wherein Y is [Chemical formula] 22 and n is an integer of 2, 3 or 4, R is a polyene hydrocarbon chain of formula A, formula A', formula A'', or 10 formula A''', and each of R 12 and R is independently optionally substituted with hydroxyl and is (C1-C4) alkyl. In some embodiments, the ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalently attached to the Y moiety of formula (IV-A), wherein Y is [Chemical formula] and n is an integer of 2, 3 or 4, R​​​​​​22 is a polyene hydrocarbon chain of formula B , R 10 and R 12 each is independently optionally substituted with hydroxyl and is (C1-C4) alkyl.

[0125] In some embodiments, the ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalently attached to the Y moiety of formula (IV-A), wherein Y is

Chemical formula

Chemical formula

[0126] In some embodiments, the ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalently attached to the Y moiety of formula (IV-A), wherein Y is

Chemical formula

[0127] In some embodiments, the ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalently attached to the Y moiety of formula (IV-A), wherein Y is

Chemical formula

[0128] In some embodiments, the ionizable lipid is of formula (IV):

Chemical formula

Chemical formula

[0129] One aspect of the present disclosure provides a compound of formula I or a pharmaceutically acceptable salt thereof.

[0130] [Chemical formula] [wherein Y is independently a methyl or ethyl group, here, the two fatty acyl groups have 16 to 18 carbons and contain two non-conjugated olefins

[0131] Another aspect of the present disclosure provides a composition comprising an ionizable lipid, the lipid nanoparticles comprising the ionizable lipid of formula I or a pharmaceutically acceptable salt thereof.

[0132] [Chemical formula] ​[wherein Y is independently a methyl or ethyl group, wherein the two fatty acyl groups together have 16 to 18 carbons and contain two olefins separated by 2 to 4 methylene groups]

[0133] In some embodiments, the two fatty acyl groups have 16 carbons. In some embodiments, the two fatty acyl groups have 17 carbons. In some embodiments the two fatty acyl groups have 18 carbons.

[0134] In some embodiments, there is provided an ionizable lipid of Formula I-A or a pharmaceutically acceptable salt thereof.

[0135] [Chemical formula] [wherein a is 1, 2, 3, 4, 5 or 6, b is 2, 3 or 4, c is , 3, 4, 5, 6 or 7, the sum of a, b and c is 10 or 12, and L is [Chemical formula] and each of R 10 and R 12 is independently optionally hydroxylated (C1-C4) alkyl, v is 0 or 1, q is 1, 2, 3 or 4, and q2 is 1 or 2] In some aspects, in the ionizable lipid of Formula I-A, v is 0, q is 1, 2 or 3, and the sum of a, b and c is 12. In some aspects, in the ionizable lipid of Formula I- A, v is 1, q is 3 or 4, and the sum of a, b and c is 12. In some aspects, in the ionizable lipid of Formula I-A', R In some aspects, in the ionizable lipid of Formula I-A, v is 1, q is 3 or 4, and the sum of a, b and c is 12. In some aspects, in the ionizable lipid of Formula I-A', R In some aspects, in the ionizable lipid of Formula I-A', R10 and R 12 is independently selected from methyl, ethyl, and propyl, each of which is , optionally substituted with a single hydroxyl. In some embodiments, a, the sum of b and c is 12, and R 10 and R 12 are independently selected from methyl, ethyl, - (CH2)(CH2)OH, and -(CH2)2(CH2)OH.

[0136] In some embodiments, there is provided an ionizable lipid of formula I-A’, or a pharmaceutically acceptable salt thereof.

[0137] [Chemical formula] [wherein a is 1, 2, or 3, c is 3, 4, 5, 6, or 7, L is [Chemical formula] , Y’ is methyl or ethyl, v is 0 or 1, q is 2, 3, or 4 and q2 is 1 or 2] In some embodiments, in the ionizable lipid of formula I-A’, v is 0, q is 1, 2, or 3, and the sum of a and c is 6 or 8. In some embodiments, in the ionizable lipid of formula I-A ’, v is 1, q is 3 or 4, and the sum of a and c is 6 or 8.

[0138] In some embodiments, there is provided an ionizable lipid of formula I-A’’, or a pharmaceutically acceptable salt thereof.

[0139] [Chemical formula] [wherein, a is 4, 5 or 6, b is 2, 3 or 4, c is 3, 4, 5, 6 or 7, L is and, v is

Chemical formula

[0140] Another aspect of the present disclosure provides a compound of formula II or a pharmaceutically acceptable salt thereof.

[0141]

Chemical formula

[0142] In some embodiments, the two fatty acyl groups have 16 carbons. In some embodiments, the two fatty acyl groups have 17 carbons. In some embodiments the two fatty acyl groups have 18 carbons.

[0143] Another aspect of the disclosure provides a compound of formula II-A or a pharmaceutically acceptable salt thereof.

[0144] [Chemical formula] [wherein a is 1, 2, 3, 4, 5 or 6, b is 2, 3 or 4, c is 4, 5, 6, 7 or 8, R2 is [Chemical formula] and q and q' are each independently 1 or 2, R 10 and R 12 are each methyl or ethyl] In some embodiments, in the ionizable lipid of formula I-A, the sum of a, b and c is 11 or 13. In some embodiments, the ionizable lipid of formula I-A is characterized by one or more of the following: a is 1, 2 or 3, q is 2, q' is 1, and at least one of R 10 or R 12 is ethyl. In some embodiments in the ionizable lipid of formula II-A, b is 4. In some embodiments, in the ionizable lipid of formula II-A a is 4, b is 4 and c is 4. In some embodiments​​​ In the ionizable lipid of formula II-A, a is 1, b is 4, and c is 8. How many In some embodiments, in the ionizable lipid of formula II-A, a is 2, b is 4, and c is 5 is.

[0145] Another aspect of the present disclosure provides a compound of formula II-A' or a pharmaceutically acceptable salt thereof. is.

[0146] [Chemical formula] [wherein, a is 1, 2 or 3, b is 2, 3 or 4, c is 4, 5, 6, 7 or 8, and R2 is [Chemical formula] is, q and q' are each independently 1 or 2, and R 10 and R 12 are each methyl or ethyl] In some embodiments, in the ionizable lipid of formula I-A', the sum of a, b and c is 11 or 13. In some embodiments, the ionizable lipid of formula I-A is characterized by one or more of the following: q is 2, q' is 1, and R or R 10 or R 12 At least one of is ethyl. In some embodiments, in the ionizable lipid of formula II-A', b is 4. In some embodiments, in the ionizable lipid of formula II-A', a is 4, b is 4, and c is 4. In some embodiments, in the ionizable lipid of formula II-A', a is 1, b is 4, and c is 8. In some embodiments, In the ionizable lipid of formula II-A', a is 2, b is 4, and c is 5.

[0147] ​ Another aspect of the present disclosure provides a compound of formula II-B or a pharmaceutically acceptable salt thereof. .

[0148] [Chemical formula] [wherein a is 5, 6 or 7, c is 3, 4 or 5, and R2 is [Chemical formula] and q and q' are each independently 1 or 2, and R 10 and R 12 are each methyl or ethyl] In some embodiments, in the ionizable lipid of formula I-B, the sum of a and c is 9 or 11. In some embodiments, the ionizable lipid of formula I-B is characterized by one or more of the following: q is 2, q' is 1, and at least one of R 10 or R 12 is ethyl. In some embodiments, the ionizable lipid of formula I-B is characterized by one or more of the following: q is 1, q' is 2, and R and R are each methyl. In some embodiments, in the ionizable lipid of formula II-B, c is 4. In some embodiments, in the ionizable lipid of formula II-B, 10 and R 12 a is 5 or 7 and c is 4. In some embodiments, in the ionizable lipid of formula II-B, a is 5 and c is 4. In some embodiments, in the ionizable lipid of formula II-B, a is 7 and c is 4.

[0149]

[0149] Another aspect of the present disclosure provides a compound of formula II-B' or a pharmaceutically acceptable salt thereof. .

[0150] [Chemical formula] [wherein, a is 5 or 7, c is 3 or 4, and R2 is [Chemical formula] and q and q' are each independently 1 or 2, and R 10 and R 12 are each methyl] In some embodiments, in the ionizable lipid of formula I-B', the sum of a and c is 9 or 1 11. In some embodiments, in the ionizable lipid of formula I-B', c is 4. In some embodiments, in the ionizable lipid of formula II-B', a is 5 or 7 and c is 4 In some embodiments, in the ionizable lipid of formula II-B, a is 5 and c is 4 In some embodiments, in the ionizable lipid of formula II-B', a is 7 and c is 4 In some embodiments, in the ionizable lipid of formula II-B, a is 5 and c is 3 In some embodiments, in the ionizable lipid of formula II-B', a is 7 and c is 3.

[0151] Another aspect of the present disclosure provides a compound of formula III or a pharmaceutically acceptable salt thereof.

[0152] [Chemical formula] [wherein, Y is a methyl or ethyl group, the two fatty acyl groups are disulfide fatty acyl groups having 16 to 18 carbons and containing a single olefin]

[0153] In some embodiments, the two fatty acyl groups have 16 carbons. In some embodiments, the two fatty acyl groups have 17 carbons. In some embodiments the two fatty acyl groups have 18 carbons.

[0154] Another aspect of the present disclosure provides a compound of formula III-A or a pharmaceutically acceptable salt thereof.

[0155] [Chemical formula] [wherein, a is 5, 6 or 7, c is 3, 4 or 5, q is 2 or 3, R and R 10 and R 12 are methyl or ethyl] In some aspects, the ionizable lipid can include a compound of formula III-A where a is 5 or 7. In some aspects, the ionizable lipid can include two polyunsaturated polyene hydrocarbon chains of formula A where the sum of a and c is 8, 9 or 10.

[0156] Another aspect of the present disclosure provides a compound of formula III-A' or a pharmaceutically acceptable salt thereof.

[0157] [Chemical formula] [wherein, a is 5 or 7, c is 3, 4 or 5, q is 2 or 3, R 10 and R 12 are methyl or ethyl] In some aspects, the ionizable lipid can include two polyunsaturated polyene hydrocarbon chains where c is 3. In some aspects, the ionizable lipid has a sum of a and c of ​​​​​comprising two polyunsaturated polyene hydrocarbon chains of formula III-A' where the sum is 8 or 10 In some embodiments, the ionizable lipid has q = 2, and the sum of a and c is 8 or 10, and can comprise two polyunsaturated polyene hydrocarbon chains of formula III-A'. In some embodiments, the ionizable lipid has q = 2, R 10 and R 12 are each methyl, and the sum of a and c is 8 or 10, and can comprise two polyunsaturated polyene hydrocarbon chains of formula III -A'. In some embodiments, the ionizable lipid has q = 2, R and 10 R 12 are each methyl, c is 3, and can comprise two polyunsaturated polyene hydrocarbon chains of formula III-A'. .

[0158] In some embodiments, the compounds in Formulas I-III have a pKa of 6-7. In some embodiments, the lipid nanoparticle composition comprises a lipid and a nucleic acid, and the lipid nanoparticle comprises a compound of Formula I, II, III, a combination thereof, or a pharmaceutically acceptable salt thereof.

[0159] In some embodiments, the LNP has the formula (IV):

Chemical formula

Chemical formula

Chem.

[0160] In some embodiments, Y is

Chem.

[0161] In some embodiments, the compound of Formula IV has a pKa of 6 to 7.

[0162] In some embodiments, the ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains of Formula (IV-A):

Chem.

Chem.

[0163] ​ In some embodiments, the ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains of formula (IV-A);

Chemical formula

Chemical formula

[0164] In some embodiments, the ionizable lipid is of formula (IV-A):

Chemical formula

Chemical formula

[0165] In some embodiments, the ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains of formula (IV-A): [Chemical formula] or a pharmaceutically acceptable salt thereof, and in the formula , Y is [Chemical formula] , n is an integer of 2, 3 or 4, R 22 is a polyene hydrocarbon chain of formula A, formula A', formula A'' or formula A''' or formula B, and each of R 10 and R 12 is independently (C1-C4) alkyl optionally substituted with hydroxyl. In some embodiments, R in formula (IV-A) 10 and R 12 are independently selected from methyl , ethyl, -(CH2)(CH2)OH and -(CH2)2(CH2)OH .

[0166] In some embodiments, the ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains of formula (IV-A): [Chemical formula] or a pharmaceutically acceptable salt thereof, and in the formula , Y is [Chemical formula] ​and n is an integer of 2, 3 or 4, and R 22 is a polyene hydrocarbon chain of formula A, formula A’, formula A’’ or formula A’’’ or formula B, and each of R 10 and R 12 is independently (C1-C4) alkyl optionally substituted with hydroxyl. In some embodiments, R and R 10 in formula (IV-A) are independently selected from methyl 12 , ethyl, -(CH2)(CH2)OH and -(CH2)2(CH2)OH. In some embodiments, the compound has the structure of the compounds listed in Table 1 or Table 2. Table 1A shows examples of cationic lipids. Table 2 shows examples of bioreducible cationic lipids.

[0167] In some embodiments, the compound has the structure of the compounds listed in Table 1 or Table 2. Table 1A shows examples of cationic lipids. Table 2 shows examples of bioreducible cationic lipids.

[0168] Table 1A shows examples of cationic lipids. Table 2 shows examples of bioreducible cationic lipids. .

[0169] JPEG2025106257000098.jpg180170

[0170] JPEG2025106257000099.jpg162170

[0171] JPEG2025106257000100.jpg151170

[0172] JPEG2025106257000101.jpg163170

[0173] JPEG2025106257000102.jpg184170

[0174] JPEG2025106257000103.jpg141170

[0175] JPEG2025106257000104.jpg110170

[0176] In some embodiments, the ionizable lipid encapsulates the nucleic acid. In some embodiments, the ionizable lipid encapsulates the nucleic acid in an LNP formulation. In some embodiments, the nucleic acid is an siRNA molecule. In some embodiments, the nucleic acid is an m RNA molecule. In some embodiments, the nucleic acid is a DNA molecule.

[0177] In some embodiments, a composition is provided that further comprises a ligand, such as an antibody conjugate to a cell surface receptor, to target lipid nanoparticles to dendritic cells in a highly specific manner. In some embodiments, the composition further comprises a target ligand that is oriented on the outside of the nanoparticle. In some embodiments, the target ligand is an antibody. In some embodiments, the lipid nanoparticles are in an aqueous medium. In some embodiments, the nucleic acid is encapsulated in the lipid nanoparticles together with a compound disclosed herein, including compounds of Formula I, II, III, IV, or combinations thereof, and the nucleic acid is either RNA or DNA. In some embodiments, the nucleic acid is encapsulated in the lipid nanoparticles together with a compound disclosed herein, including compounds of Formula

[0178] I, I-A, II, II-A, II-B, III, III-A, IV, IV-A, IV-

[0179] B, V, V-A, VI-A, VII, VIII, or combinations thereof, and the nucleic acid is either RNA or D NA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is I, I-A, II, II-A, II-B, III, III-A, IV, IV-A, IV- B, V, V-A, VI-A, VII, VIII, or combinations thereof, and the nucleic acid is either RNA or D NA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is mRNA. In some In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid is D NA.

[0180] In some embodiments, the lipid nanoparticle comprises a membrane comprising phosphatidylcholine and sterol. In some embodiments, the sterol is cholesterol . In some embodiments, the lipid nanoparticle comprises a membrane comprising phosphatidylcholine and ionizable cationic lipid (ICL). In some embodiments, the ICL has the structure of Formula I , II, III or IV, and cholesterol, and the membrane separates the inside of the lipid nanoparticle from the aqueous medium. In some embodiments, the ICL has the structures shown in Tables 1A and 2. In some embodiments, the ICL has the structure shown in Table 1B . In some embodiments, the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC). In some embodiments, the molar ratio of ionizable cationic lipid to cholesterol is about 65:35 to 40:60. In some embodiments, the molar ratio of ICL to cholesterol is about 60:40 to about 45:55. In some embodiments, the molar ratio of phosphatidylcholine to cholesterol is about 1:5 to about 1:2. In some embodiments, the membrane further comprises a polymeric complex lipid.

[0181] In some embodiments, the lipid nanoparticle comprises ICL, DSPC, cholesterol .

[0182] In some embodiments, the membrane further comprises a polymeric complex lipid.

[0183] In some embodiments, the lipid nanoparticle comprises ICL, DSPC, cholesterol and polymer-conjugated lipid in a molar ratio of about 49.5:10.3:39.6:2.5.

[0184] In some embodiments, the polymer conjugated lipid is PEG(2000)-dimyristoyl Dimyristylglycerol (PEG-DMG) or PEG (molecular weight 2,000)-dimyristylglycerol The compound is PEG-DMPE.

[0185] In some embodiments, the percentage of oxidative degradation products of ionized lipids is <50% of DLin-KC2-DMA or DLin-MC3-DMA control formulations .

[0186] In some embodiments, the composition is a liquid pharmaceutical formulation for parenteral administration.

[0187] In some embodiments, the composition is a liquid pharmaceutical for subcutaneous, intramuscular, or intradermal administration. It is a formulation.

[0188] In some embodiments, the composition is in the form of a lyophilized powder and is subsequently and reconstituted with an aqueous medium.

[0189] Another aspect of the present disclosure is a method for preventing bacterial or viral infection, comprising administering to a subject in need thereof administering to a subject an effective amount of a composition provided herein to generate an immune response. Some embodiments relate to a method for vaccinating a subject in need thereof, comprising: A method comprising administering a composition comprising a nucleic acid encoding an antigenic protein. to provide.

[0190] In some embodiments, the compositions are administered subcutaneously, intramuscularly, or intradermally.

[0191] In some embodiments, the bacterial infection is Mycobacterium tuberculosis infection. In some embodiments, the bacterial infection is , in the form of nontuberculosis mycobact erium.

[0192] In some embodiments, the viral infection is a coronavirus. In some embodiments, the coronavirus is SARS-CoV, MERS-CoV or SA RS-CoV-2.

[0193] In some embodiments, the viral infection is HIV / AIDs.

[0194] In some embodiments, the lipid nanoparticles are administered parenterally.

[0195] In some embodiments, the lipid nanoparticle composition is administered as part of a single dose thereof.

[0196] The present disclosure features lipid nanoparticles comprising a nucleic acid, such as DNA, mRNA, siRNA, an antisense oligonucleotide, a CRISPR component, such as a guide RNA (gRNA or sgRNA) and a CRISPR-associated endonuclease (Cas protein), and a lipid. Exemplary lipids include ionizable cationic lipids (ICLs), phospholipids, sterol lipids, alkylene glycol lipids (e.g., polyethylene glycol lipids), sphingolipids, glycerol lipids, glycerophospholipids, prenol lipids, glycolipids, fatty acids, and polypetides. In some embodiments, the LNP comprises a single type of lipid and includes sphingolipids, glycerol lipids, glycerophospholipids, prenol lipids, glycolipids, fatty acids, and In some embodiments, the LNP comprises multiple (e.g., two or more) lipids. LNPs are composed of ionizable cationic lipids, phospholipids, sterols, or alkylene glycosyltransferases. The lipid composition may include one or more glycol lipids (eg, polyethylene glycol lipids).

[0197] In one embodiment, the LNP comprises an ionizable cationic lipid. In this case, the terms "ionized cationic lipid", "ionized lipid" and "ICL" are used interchangeably. ICLs are used to measure the activity of a compound under specific conditions (e.g., physiological conditions, e.g., in a specific pH range). ions that can carry a charge (e.g., positive charge, e.g., cationic lipids) under The ionizable moiety may comprise an amine, preferably a substituted amine. The ionizable lipid may be a cationic lipid or an anionic lipid. In addition to the ionizable moiety, the ionizable lipids can be, for example, more than 6 carbon atoms in length (e.g., more than about 8 carbon atoms in length). of carbon, 10 carbon, 12 carbon, 14 carbon, 16 carbon, 18 carbon, 2 The alkyl or alkenyl groups may be 0 or more carbons in length. Additional ionizable lipids that can be included in LNPs are described by Jayaraman et al. gew.Chem.Int.Ed.51:8529-8533(2012)),Semp le et al.Nature Biotechnol.28:172-176(20 10)) and U.S. Pat. Nos. 8,710,200 and 8,754,062. No. 6,393,945, each of which is incorporated herein by reference.

[0198] In some embodiments, the LNP has formula (IV): [ka] comprising an ionizable lipid having the structure or a pharmaceutically acceptable salt thereof, wherein Y is

Chemical formula

Chemical formula

[0199] In some embodiments, Y is

Chemical formula

[0200] In some embodiments, the LNP comprises an ionizable lipid having the structure of formula (IV-A) or a pharmaceutically acceptable salt thereof.

[0201]

Chemical formula

Chemical formula

[0202] In some embodiments, v of the compound of formula (IV-B) is equal to 0. In some embodiments, v of the compound of formula (IV-B) is equal to 1. In some embodiments wherein, v of the compound of formula (IV-A) is equal to 1, and q1 is equal to 1. In some embodiments wherein, v of the compound of formula (IV-B) is equal to 1, and q1 is equal to 2.

[0203] In some embodiments, R of the compound of formula (IV-B) 22 wherein the sum of a and c is 6, 7, 8 or 9. In some embodiments, R of the compound of formula (IV-B) R 22 wherein the sum of a and c is 6. In some embodiments, R of the compound of formula (IV-B) R 22 wherein the sum of a and c is 7. In some embodiments, for the compound of formula ( IV-B), R 22 wherein the sum of a and c is 9.

[0204] In some embodiments, R of the compound of formula (IV-B) 22 wherein v is equal to 0, and the sum of a and c is 6, 7, 8 or 9. In some embodiments, for the compound of formula (IV -B), R 22 wherein v is equal to 0, and the sum of a and c is 6. In some embodiments, R of the compound of formula (IV-B) 22 wherein v is equal to 0, and a and c wherein the sum is 7. In some embodiments, R of the compound of formula (IV-B) 22 wherein v is equal to 0, and the sum of a and c is 9.

[0205] In some embodiments, for the compound of formula (IV-B), R 10 and R 12 are independently selected from methyl, ethyl, -(CH2)(CH2)OH, and -(CH2)2(CH 2)OH. In some embodiments, for the compound of formula (IV-B), R 2 2 in, R 10 and R 12 are each methyl, and the sum of a and c is 6, 7, 8 or 9. In some embodiments, for the compound of formula (IV-B), R 22 in, R1 0 and R 12 are each methyl, v is 0, and the sum of a and c is 6, 7, 8 or 9.

[0206] In some embodiments, for the compound of formula (IV-B), v is equal to 0, R 22 is

Chemical formula

Chemical formula

Chemical formula

Chem.

[0207] In some embodiments, for the compound of formula (IV-B), v is equal to 1, and R 22 is

Chem.

Chem.

Chem.

[0208] In some embodiments, for the compound of formula (IV-B), v is equal to 0, and R 22 is

Chem.

Chem.

Chem.

Chemical formula

Chemical formula

[0209] In some embodiments, for the compound of formula (IV-B), v is equal to 0 and R 22 is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0210] The LNPs may contain ionized lipids, e.g., at a concentration greater than about 0.1 molar % of the total lipid content of the LNP. In one embodiment, the LNPs may comprise, for example, about 1 mol % of the total lipid content of the LNP. , about 2 mol%, about 4 mol%, about 8 mol%, about 20 mol%, about 40 mol%, about 50 mol%, In one embodiment, the LN P comprises an ionized lipid at a concentration of greater than about 20 mol%, about 40 mol%, or about 50 mol%. In one embodiment, the LNPs may comprise, for example, about 1 mol % to about 95 mol % of the total lipid content of the LNP. In one embodiment, the LNP comprises an ionized lipid at a concentration of, for example, 100% of the total lipid content of the LNP. About 2 mol % to about 90 mol %, about 4 mol % to about 80 mol %, about 10 mol % to about 70 mol % of the molecular weight Ionization concentrations of 0 mol%, about 20 mol% to about 60 mol%, and about 40 mol% to about 55 mol% In one embodiment, the LNPs contain ionized water at a concentration of about 20 mol % to about 60 mol %. In one embodiment, the LNP comprises a methylated lipid at a concentration of about 40 mol % to about 55 mol %. Contains ionized lipids.

[0211] In one embodiment, the LNP comprises a phospholipid. A phospholipid is a lipid that contains a phosphate group and a small amount of A lipid containing at least one alkyl, alkenyl, or heteroalkyl chain. The lipids may be natural or non-natural (e.g., synthetic phospholipids). It may contain amine, amide, ester, carboxyl, choline, hydroxyl, acetal, ether , carbohydrate, sterol, or glycerol. In some embodiments , the phospholipid may contain phosphocholine, phosphosphingolipid, or plasmalogen . Exemplary phospholipids include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) , hydrogenated soy phosphatidylcholine (HSPC), 1,2-dilauroyl-sn-glycero -3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phospho choline (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-phosphatidyl choline (PLPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), bis(monoacylglycerol) phosphate (BMP), L- α-phosphatidylcholine, 1,2-diheptadecanoyl-sn-glycero-3-phospho rilcholine (DHDPC), and 1-stearoyl-2-arachidonoyl-sn-gly Examples include ceramide-3-phosphate (SAPC). Additional phospholipids that can be included in the LNPs described herein are incorporated by reference in their entirety from Li, J. et al. (Asian J. Pharm. Sci. 10:81-98 (2015)). which is disclosed therein.

[0212] In some embodiments, the LNP comprises a phospholipid having the structure of formula (V):

Chemical formula

[0213] In some embodiments, each R 23 is independently alkyl (e.g., C2-C3 2-alkyl, C4-C 28 alkyl, C8-C 24 alkyl, C 12 -C 22 alkyl, or C 16 -C 20 alkyl). In some embodiments, each R 23 is independently alkenyl (e.g., C2-C alkenyl, C4-C 32 alkenyl​​​​28 Alkenyl, C8~ C 24 Alkenyl, C 12 ~C 22 Alkenyl, or C 16 ~C 20 Alkenyl). In some embodiments, each R is. In some embodiments, each R 23 is independently heteroalkyl (e.g., C 4~C 28 Heteroalkyl, C8~C 24 Heteroalkyl, C 12 ~C 22 Heteroalkyl l, C 16 ~C 20 Heteroalkyl). In some embodiments, each R 23 is , independently, C 16 ~C 20 Alkyl. In some embodiments, each R 23 is , independently, C 17 Alkyl. In some embodiments, each R 23 is, independently tridecyl. In some embodiments, each R 23 is the same. In some embodiments, each R 23 is different. In some embodiments, each R 23 is, optionally substituted by R C . In some embodiments, R C is, independently, Alkyl, halo, hydroxy, amino, cycloalkyl, or heterocyclyl.

[0214] In some embodiments, one of the Rs 25 is hydrogen. In some embodiments, one of the Rs 25 is alkyl. In some embodiments, one of the Rs 25 is meth is. In some embodiments, each R 25 is independently alkyl. In some embodiments, each R 25 is independently methyl. In some embodiments each R 25 is independently methyl and u is 2. In some embodiments, each R 25 is independently methyl and u is 3.

[0215] In some embodiments, R 24 is absent and the oxygen to which it is attached bears a negative charge. In some embodiments, R is hydrogen. 24 is hydrogen.

[0216] In some embodiments, m is an integer from 1 to 10, 1 to 8, 1 to 6, 1 to 4 . In some embodiments, m is 1, 2, 3 or 4. In some embodiments m is 1. In some embodiments, m is 2. In some embodiments m is 3.

[0217] In some embodiments, there is provided a composition comprising targeting moieties of both a cationic ionizable lipid and an anionic phospholipid. In some embodiments, the anionic phospholipid is a composition of formula (V-A). is a composition of formula (V-A).

[0218]

Chemical formula

Chemical formula

[0219] 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-phos phocholine (DPPC). In some embodiments, the phospholipid is 1,2-dio leoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0220] Uptake of phosphatidylserine LNP (e.g., as described herein) comprises the following components: (i) C1 6 alkyl or C16 alkenyl group or C18 alkyl or C18 alkenyl group-containing ionizable cationic lipid (ICL) at a concentration of about 1 mol% to about 95 mol % (or any value therebetween, e.g., about 20 mol% to about 80 mol%); (ii) a phospholipid containing a C 16 or C18 alkyl or alkenyl group at a concentration of 0.1 mol% to about 20 mol % (or any value therebetween, e.g., about 2.5 mol% to about 10 mol%); (iii) about 1 mol % to about 95 mol% (or any value therebetween, e.g., about 20 mol% to about 80 mol% ) cholesterol concentration; (iv) 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 concentration of phosphatidylserine (PS) or phospho added to the LNP lipid formulation fatidylglycerol (PG); (v) about 0.1 mol% to about 5 mol% (any value between these, for example about 1 mol% to about 2.5 mol%) concentration of polyethylene glycol (PEG )-2000-containing lipid (e.g., DPG-PEG2000, DPPE-PEG2000, DMPE-PEG2000, DMG-PEG2000) may include one or more. In one embodiment, the LNP includes two of (i)-(v). In one embodiment, the LNP includes three of (i)-(v). In one embodiment, the LNP is (i) - includes four of (v). In one embodiment, the LNP includes each of (i)-(v). In some embodiments, the LNP includes (i) and (ii). In some embodiments, the LNP includes (i) and (iii). In some embodiments the LNP includes (i) and (v). In some embodiments, LN P includes (ii) and (iii). In some embodiments, LNP is (i i) and (v). In some embodiments, the LNP includes (iii) and ( iv). In some embodiments, the LNP includes (iii) and (v) . In some embodiments, the LNP includes (i), (ii) and (iii). In some embodiments, the LNP includes (i), (ii) and (v). In some embodiments, the LNP includes (ii), (iii) and (v). In some In an embodiment, the LNP comprises (ii), (iii), (iv), and (v). In one embodiment, the LNP consists of, or consists essentially of, 4 out of (i)-(v). In one embodiment, the LNP consists of, or consists essentially of, each of (i)-(v). In some embodiments, the LNP consists of, or consists essentially of, (i) and (ii). In some embodiments, the LNP consists of, or consists essentially of, (i) and (iii). In some embodiments, the LNP consists of, or consists essentially of, (i) and , (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), (i i), and (v). In some embodiments, the L NP comprises (ii), (iii), and (v). In some embodiments, the LN P consists of, or consists essentially of, (ii), (iii), (iv), and (v). .

[0221] The LNP may contain, for example, phospholipids at a concentration of more than about 0.1 mol% of the total lipid content of the LNP. In one embodiment, the LNP contains, 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% , and contains phospholipids at a concentration of more than about 50 mol%. In one embodiment, the LNP is about 1 mol% , about 5 mol%, or contains phospholipids at a concentration of more than about 10 mol%. In one embodiment, the L NP contains phospholipids, for example, at a concentration of about 0.1 mol% to about 50 mol% of the total lipid content of the LNP . In one embodiment, the LNP contains phospholipids, for example, at about 0.5 mol of the total lipid content of the LNP % 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 phospholipids. In one embodiment, the LNP contains phospholipids at a concentration of about 5 mol% to about 25 mol% . In one embodiment, the LNP contains phospholipids at a concentration of about 10 mol% to 20 mol% .

[0222] In one embodiment, the LNP contains a sterol or ionized sterol molecule. The ster ol is a lipid containing a polycyclic structure and an optional hydroxyl or ether substituent, which may be a natural or non-natural (e.g., synthetic sterol) lipid. The sterol may or may not contain double bonds, may contain a single double bond, or may contain multiple double bonds . The sterol may further contain an alkyl, alkenyl, halo, ester, ketone, hydroxyl, ami ne, polyether, carbohydrate, or cyclic moiety. The sterol may further contain a bioreducible disulfide bond between the di alkylamino group and the polycyclic moiety of the molecule (see Table 2, compounds 35 - 38). An exemplary list of sterols includes chol esterol (see reference), etc. Sterol, dehydroergosterol, ergosterol, campesterol, β-sitosterol, stigmasterol, lanosterol, dihydrolanosterol, desmosterol, brassicasterol, lasasterol, thymosterol, 7-dehydrodesmosterol, avenasterol, campestanol, lupeol, and cycloartenol are included. In some embodiments, the sterol includes cholesterol, dehydroergosterol, ergosterol, campesterol, β-sitosterol, or stigmasterol. Additional sterols that can be included in the LNPs described herein are disclosed in Fahy, E. et al. (J. Lipid Res. 46:839-862 (2005)).

[0223] Ionized sterol In some embodiments, the LNP includes a sterol having the structure of formula (VI): or a pharmaceutically acceptable salt thereof, wherein R is hydrogen, alkyl, heteroalkyl, or -C(O)R, R is hydrogen, alkyl, or -OR, and each of R and R is independently hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with alkyl, halo, or carbonyl. 26 Hydrogen D 27 E D E Alkenyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl ​​​​​​​​​​​​​​​​​​​ [Chem.] is either a single bond or a double bond, and each carbon atom involved in the single bond or double bond is bonded to 0, 1, or 2 hydrogens and has a valency that is acceptable.

[0224] In some embodiments, [Chem.] one of is a single bond. In some embodiments, [Chem.] one of is a double bond. In some embodiments, [Chem.] two of are single bonds. In some embodiments, [Chem.] two of are double bonds. In some embodiments, each [Chem.] is a single bond. In some embodiments, each [Chem.] is a double bond.

[0225] In some embodiments, the sterol is cholesterol. In some embodiments the sterol is dehydroergosterol. In some embodiments the sterol is ergosterol. In some embodiments, the sterol is campesterol. In some embodiments, the sterol is β-sitosterol It is. In some embodiments, the sterol is stigmasterol. Several In some embodiments, the sterol is a corticosteroid (e.g., cortisone , hydrocortisone, cortisol, or aldosterone).

[0226] In some embodiments, the LNP is of formula (VI-A):

Chemical formula

Chemical formula

[0227] Another aspect of the present disclosure provides a composition comprising an anionic phospholipid of formula (V-A) and a branched ionizable lipid of formula (VIII).

[0228]

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0229] In some embodiments, the ionizable lipid may be a branched ionizable lipid selected from ALC-0315 and SM-102

[0230]

Chemical formula

[0231] ​ The LNP may contain a sterol at a concentration of more than about 0.1 mol% of the total lipid content of the LNP, for example. In one embodiment, the LNP contains a sterol at a concentration of, for example, about 0.5 mol%, about 1 mol%, about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, or more than about 70 mol% of the total lipid content of the LNP. In one embodiment the LNP contains a sterol at a concentration of more than about 10 mol%, about 15 mol%, about 20 mol%, or about 25 mol %. In one embodiment, the LNP contains a sterol at a concentration of, for example, about 1 mol% to about 95 mol% of the total lipid content of the LNP. In one embodiment, the LNP contains a sterol at a concentration of, for example, about 5 mol% to about 90 mol%, about 10 mol% to about 85 mol%, about 20 mol% to about 80 mol%, about 20 mol% to about 60 mol%, about 20 mol % to about 50 mol%, or about 20 mol% to 40 mol% of the total lipid content of the LNP. In one embodiment the LNP contains a sterol at a concentration of about 20 mol% to about 50 mol%. In one embodiment the LNP contains a sterol at a concentration of about 30 mol% to about 60 mol%.

[0232] In some embodiments, the LNP contains an alkylene glycol-containing lipid. The alkylene glycol-containing lipid is a lipid containing at least one alkylene glycol moiety, for example, a methylene glycol or ethylene glycol moiety. In some embodiments the alkylene glycol-containing lipid contains polyethylene glycol (PEG). The alkylene glycol-containing lipid may be a PEG-containing lipid. The polymeric complex lipid is a poly(ethylene glycol) conjugated (PEGylated) lipid (PEG-lipid), such as PE G (molecular weight 2,000) methoxy-poly(ethylene glycol)-1,2-distearoyl -sn-glycerol (PEG-DSG), PEG (molecular weight 2,000) methoxy-p oly(ethylene glycol)-1,2-palmitoyl-sn-glycerol (PEG-DP G), PEG (molecular weight 2,000) 1,2-distearoyl-sn-glycerol-3-phos phoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG -DSPE) or N-palmitoyl-sphingosine-1-{succinyl[methoxy( polyethylene glycol)2000]}(PEG-ceramide) may be included. The molecular weight of the PEG moiety in the PEG-lipid component can also vary from 500 to 10,000 g / mol, 1,500 to 6 000 g / mol, but is preferably about 2,000 MW. Other polymers used for conjugation to the lipid anchor include poly(2-methyl-2-oxazoline) (PMO Z), poly(2-ethyl-2-oxazoline) (PEOZ), poly-N-vinylpyrrolid ine (PVP), polyglycerol, poly(hydroxyethyl L-asparagine) (PHE A), and poly(hydroxyethyl L-glutamine) (PHEG) may be included. The PEG-containing lipid may further contain an amine, amide, ester, carboxyl, phosphate, choline

[0233] , hydroxyl, acetal, ether, heterocycle, or carbohydrate. The PEG-containing lipid may, for example, in addition to the PEG moiety, have a length of more than 6 carbon atoms ( for example, more than about 8 carbons, 10 carbons, 12 carbons, 14 carbons, 16 carbons, . The PEG-containing lipid may, for example, in addition to the PEG moiety, have a length of more than 6 carbon atoms ( for example, more than about 8 carbons, 10 carbons, 12 carbons, 14 carbons, 16 carbons, It may contain at least one alkyl group or alkenyl group having 18 carbons or a length of 20 or more carbons. In one embodiment, the PEG-containing lipid contains at least 20 PEG monomers, for example, at least 30 PEG monomers, 40 PEG monomers, 45 PEG monomers, 50 PEG monomers, 100 PEG monomers, 200 PEG monomers, 300 PEG monomers, 500 PEG monomers, 1000 PEG monomers, or 2000 PEG monomers, and contains a PEG moiety. Exemplary PEG-containing lipids include PEG-DMG (e.g., DMG-PEG2k), PEG- c-DMG, PEG-DSG, PEG-DPG, PEG-DSPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, and PEG-DLPE. In some embodiments, the PEG-lipids include PEG-DMG (e.g., DMG-PEG 2k), PEG-c-DMG, PEG-DSG, and PEG-DPG. Additional PEG-lipids that can be included in the LNPs described in this specification are incorporated herein by reference and are those disclosed in Fahy, E. et al. (J. Lipid Res. 46 :839-862(2005).

[0234] In some embodiments, the LNP has the formula (VII):

Chemical formula

[0235] In some embodiments, each R 28 is independently alkyl. In some embodiments, each R is independently heteroalkyl. In some embodiments, each R 28 is independently alkenyl. In some embodiments, A is O or NH. In some embodiments 28 A is CH2. In some embodiments, A is carbonyl. In some

[0236] embodiments, A is absent. In some embodiments, E is alkyl. In some embodiments, E is heteroalkyl. In some embodiments, both A and E are absent.

[0237] In some embodiments, A is absent. In some embodiments, E is absent. In some embodiments, either A or E is absent. In some embodiments, both A and E are independently absent. In some embodiments, z is from 10 to 200 (e.g., 20 to 180, 20 to 1

[0238] ​​an integer in the range of 60, 20 to 120, 20 to 100, 40 to 80, 40 to 60, 40 to 50 In some embodiments, z is 45.

[0239] In some embodiments, the PEG-lipid is PEG-DMG (e.g., DMG-PEG2k). In some embodiments, the PEG-lipid is α-(3’-{[1,2-bis(myristyloxy)propanoxy]carbonylamino}propyl)-ω-methoxy, polyoxyethylene (PEG-c-DMG). xy, polyoxyethylene (PEG-c-DMG). In some embodiments the PEG-lipid is PEG-DSG. In some embodiments, the PEG-lipid is PEG-DPG.

[0240] The LNP may include, for example, an alkylene glycol-containing lipid at a concentration of more than about 0.1 mol% of the total lipid content of the LNP. In one embodiment, the LNP includes, for example, an alkylene glycol-containing lipid at a concentration of about 0.5 mol%, about 1 mol%, about 1.5 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 8 mol%, about 10 mol%, about 12 mol%, about 15 mol%, about 20 mol%, more than about 50 mol% of the total lipid content of the LNP. In one embodiment, the LNP includes an alkylene glycol-containing lipid at a concentration of more than about 1 mol%, about 4 mol%, or about 6 mol%. In one embodiment, the LNP includes, for example, an alkylene glycol-containing lipid at a concentration of about 0.1 mol% to about 50 mol% of the total lipid content of the LNP In one embodiment, the LNP includes, for example, an alkylene glycol-containing lipid at a concentration of 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 mol%, about 3 mol% to about 15 mol%, about 3 mol% to about 10 mol%, about 4 mol% to about 8 mol%, or about 6 mol% to about 8 mol% of the total lipid content of the LNP. In one embodiment, the LNP includes an alkylene glycol-containing lipid at a concentration of about 1 mol%, about 4 mol%, or about 6 mol% or more of the total lipid content of the LNP. In one embodiment, the LNP includes, for example, an alkylene glycol-containing lipid at a concentration of about 0.1 mol% to about 50 mol% of the total lipid content of the LNP. In one embodiment, the LNP includes, for example, an alkylene glycol-containing lipid at a concentration of 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 mol%, about 3 mol% to about 15 mol%, about 3 mol% to about 10 mol%, about 4 mol% to about 8 mol%, or about 6 mol% to about 8 mol% of the total lipid content of the LNP. In one embodiment, the LNP includes an alkylene glycol-containing lipid at a concentration of about 1 mol%, about 4 mol%, or about 6 mol% or more of the total lipid content of the LNP. In one embodiment, the LNP includes, for example, an alkylene glycol-containing lipid at a concentration of about 0.1 mol% to about 50 mol% of the total lipid content of the LNP. In one embodiment, the LNP includes, for example, an alkylene glycol-containing lipid at a concentration of about 0.5 mol 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 mol%, about 3 mol% to about 15 mol%, about 3​ An alkylene glycol-containing lipid having a concentration of 0.5 mol% to about 10 mol%, or about 4 mol% to about 9 mol%. In one embodiment, the LNP contains an alkylene glycol-containing lipid having a concentration of about 4 mol% to 9 mol%. In some embodiments, the LNP contains at least two types of lipids. In one embodiment, the LNP contains two of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP contains at least three types of lipids. In one embodiment, the LNP contains three of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP contains at least four types of lipids. In one embodiment, the LNP contains each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid.

[0241] In some embodiments, the LNP contains at least two types of lipids. In one embodiment, the LNP contains two of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP contains at least three types of lipids. In one embodiment, the LNP contains three of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP contains at least four types of lipids. In one embodiment, the LNP contains each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In one embodiment, the LNP contains two of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP contains at least three types of lipids. In one embodiment, the LNP contains three of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP contains at least four types of lipids. In one embodiment, the LNP contains each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP contains at least three types of lipids. In one embodiment, the LNP contains three of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP contains at least four types of lipids. In one embodiment, the LNP contains each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In one embodiment, the LNP contains three of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP contains at least four types of lipids. In one embodiment, the LNP contains each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP contains at least four types of lipids. In one embodiment, the LNP contains each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP contains at least four types of lipids. In one embodiment, the LNP contains each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In one embodiment, the LNP contains each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In one embodiment, the LNP contains each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid.

[0242] The LNP (e.g., as described herein) contains the following components: (i) an ionizable cationic lipid having a concentration of about 1 mol% to about 95 mol% (e.g., about 20 mol% to about 80 mol%); (ii) a phospholipid having a concentration of 0.1 mol% to about 50 mol% (e.g., about 2.5 mol% to about 20 mol%); (iii) a sterol having a concentration of about 1 mol% to about 95 mol% (e.g., about 20 mol% to about 80 mol%); and (iv) one or more of a PEG-containing lipid having a concentration of about 0.1 mol% to about 50 mol% (e.g., about 2.5 mol% to about 20 mol%). In one embodiment, the LNP contains one of (i) to (iv). In one embodiment, the LNP contains two of (i) to (iv). In one embodiment, the LNP contains three of (i) to (iv). In one embodiment, the LNP contains one of (i) to (iv). In one embodiment, the LNP contains two of (i) to (iv). In one embodiment, the LNP contains three of (i) to (iv). In one embodiment, the LNP contains two of (i) to (iv). In one embodiment, the LNP contains three of (i) to (iv). In one embodiment, the LNP contains three of (i) to (iv). In one embodiment, the LNP contains three of (i) to (iv). In one embodiment, the LNP contains three of (i) to (iv). In one embodiment, the LNP contains three of (i) to (iv). In one embodiment, the LNP contains three of (i) to (iv). comprises. In one embodiment, the LNP comprises each of (i)-(iv). In some embodiments, the LNP comprises (i) and (ii). In some embodiments, the LNP comprises (i) and (iii). In some embodiments, the LNP comprises (i) and (iv). In some embodiments, the LNP comprises (ii) and (iii). In some embodiments, the LNP comprises (ii) and (iv) . In some embodiments, the LNP comprises (iii) and (iv). In some embodiments, the LNP comprises (i), (ii) and (iii). In some embodiments, the LNP comprises (i), (ii) and (iv). In some embodiments, the LNP comprises (ii), (iii) and (iv).

[0243] The LNP (e.g., as described herein) comprises the following components: (i) an ionizable cationic lipid at a concentration of about 1 mol% to about 95 mol% (e.g., about 20 mol% to about 80 mol%); (ii) DSPC at a concentration of 0.1 mol% to about 50 mol% (e.g., about 2.5 mol% to about 20 mol%); (iii) cholesterol at a concentration of about 1 mol% to about 95 mol% (e.g., about 20 mol% to about 80 mol%); 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%) and may comprise one or more of them. In one embodiment, the LNP comprises two of (i)-(iv). In one embodiment, the LNP Well. In some embodiments, the LNP comprises (i) and (iii). Some embodiments, the LNP comprises (i) and (iv). In some embodiments the LNP comprises (ii) and (iii). In some embodiments, the LN P comprises (ii) and (iv). In some embodiments, the LNP comprises (ii i) and (iv). In some embodiments, the LNP comprises (iii) and (iv). In some embodiments, the LNP comprises (i), (ii) and (i ii). In some embodiments, the LNP comprises (i), (ii) and (iv ). In some embodiments, the LNP comprises (ii), (iii) and (iv ).

[0244] In one embodiment, the LNP has a ratio of ionizable lipid to phospholipid of about 50:1 to about 1:1 (e.g., 40:1, 32:3 , 6:1, 7:1, 5:1, 24:5, 26:5, 10:3, 15:2, 16:7, 18 :1, 3:1, 3:2, or 1:1). In one embodiment, the LNP has a ratio of ionizable lipid to phospholipid of about 15:2. In one embodiment, the LNP has a ratio of ionizable lipid to phospholipid of about 5:1. In one embodiment the LNP has a ratio of ionizable lipid to phospholipid of about 10:1 to about 1:10 (e.g., 9:1, 8:1, 8:7, 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3:1, 2:1, 1:1, 1:2, 1:3, 3:4, 1:4, 3:5, 1:5, 4:5, 1:6, 5:6, 7:6, 7:8, or 8:9) of ionizable lipid to sterol. In one embodiment, the LNP has a ratio of ionizable lipid to sterol of about 1:10 to about 10:1 (e.g., 1:9, 1:8, 7 :1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3:1, : 8, 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3 : 1, 2:1, 1:1, 1:2, 1:3, 3:4, 1:4, 3:5, 1:5, 4:5, 1 : 6, 5:6, 7:6, 7:8, or an ionizable lipid and an alkylene-containing lipid of 8:9) having a ratio of. In one embodiment, the LNP is about 10:1 to about 1:10 (e.g., 9: 1, 8:1, 8:7, 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4: 1, 4:3, 3:1, 2:1, 1:1, 1:2, 1:3, 3:4, 1:4, 3:5, 1: 5, 4:5, 1:6, 5:6, 7:6, 7:8, or an alkylene of 8:9) having a ratio of phospholipid to containing lipid. In one embodiment, the LNP is about 50:1 to about 1:1 (e.g., 40:1, 32:3, 6:1, 7:1, 5:1, 24:1, 22:1, 20:1, 2 2:5, 24:5, 26:5, 10:3, 15:2, 16:7, 18:1, 3:1, 3: 2, or 1:1) having a ratio of sterol to alkylene-containing lipid.

[0245] In one embodiment, the LNP (e.g., as described herein) is an ionizable lipid, a phospholipid quality, a sterol, and an alkylene glycol-containing lipid (e.g., a PEG-containing lipid) among including two. In another embodiment, the LNP (e.g., as described herein) is an ion nized lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid (e.g., PE G-containing lipid) including three of them. In one embodiment, the LNP (e.g., as described herein) is an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid described herein) includes each of an alkylene glycol-containing lipid (e.g., a PEG-containing lipid). (e.g., a PEG-containing lipid).

[0246] In some embodiments, the LNPs described herein are 5 to 500 nm, for example, 10 to 400 nm, 20 to 350 nm, 25 to 325 nm, 30 to 300 nm, 50 to 2 50 nm, 60 to 200 nm, 75 to 190 nm, 80 to 180 nm, 100 to 200 n m, 200 to 300 nm, and 150 to 250 nm in diameter. The diameter of the LNPs is determined by any method known in the art, for example, dynamic light scattering, transmission electron microscopy (TEM ) or scanning electron microscopy (SEM). In some embodiments the LNPs are 50 to 100 nm, 70 to 100 nm, and 80 to 100 nm in diameter. In one embodiment, the LNP has a diameter of about 90 nm. In some embodiments, the LNPs described herein have a diameter greater than about 30 nm. In some embodiments, the LNPs have a diameter of 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 14 0 nm, about 160 nm, about 180 nm, about 200 nm, about 225 nm, about 250 nm, about 275 nm or greater than about 300 nm. In one embodiment, the LNP has a diameter greater than about 7 0 nm. In one embodiment, the LNP has a diameter greater than about 90 nm. . In one embodiment, the LNP has a diameter greater than about 180 nm.

[0247] In some embodiments, the plurality of LNPs described herein have an average diameter in the range of about 40 nm to about 18 0 nm. In some embodiments, the plurality of LNPs described herein have an average diameter of about 50 nm to about 150 nm. In some embodiments Furthermore, the plurality of LNPs described in this specification have an average diameter of about 50 nm to about 120 nm. In some embodiments, the plurality of LNPs described in this specification have an average diameter of about 60 nm to about 120 nm. In some embodiments, the plurality of LNPs have an average diameter of about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm.

[0248] In some embodiments, the one nanoparticle or plurality of nanoparticles described in this specification have an average surface charge that is negative to neutral, less than -100 mV, for example, -90 mV, -80 mV, -70 mV, -60 mV, -50 mV, -40 mV, -30 mV, and less than -20 mV. In some embodiments, the one nanoparticle or plurality of nanoparticles have a neutral to negative surface charge from -100 m V to 100 mV, from -75 mV to 0, or from -50 mV to -10 mV. In some embodiments, at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 3

[0249] 0%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the plurality of nanoparticles have a neutral to negative surface charge with an average less than -100 mV. In some embodiments, the one nanoparticle or plurality of nanoparticles have an average surface charge of -20 mV to +20, -10 mV to +10 mV, or -5 mV to +5 mV at pH 7.4. Neutral In some embodiments, at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the plurality of nanoparticles have a neutral to negative surface charge with an average less than -100 mV. In some embodiments, the one nanoparticle or plurality of nanoparticles have an average surface charge of -20 mV to +20, -10 mV to +10 mV, or -5 mV to +5 mV at pH 7.4. Neutral Furthermore, the plurality of LNPs described in this specification have an average diameter of about 50 nm to about 120 nm. In some embodiments, the plurality of LNPs described in this specification have an average diameter of about 60 nm to about 120 nm. In some embodiments, the plurality of LNPs have an average diameter of about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm. In some embodiments, the one nanoparticle or plurality of nanoparticles described in this specification have an average surface charge that is negative to neutral, less than -100 mV, for example, -90 mV, -80 mV, -70 mV, -60 mV, -50 mV, -40 mV, -30 mV, and less than -20 mV. In some embodiments, the one nanoparticle or plurality of nanoparticles have a neutral to negative surface charge from -100 mV to 100 mV, from -75 mV to 0, or from -50 mV to -10 mV. In some embodiments, at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the plurality of nanoparticles have a neutral to negative surface charge with an average less than -100 mV. In some embodiments, the one nanoparticle or plurality of nanoparticles have an average surface charge of -20 mV to +20, -10 mV to +10 mV, or -5 mV to +5 mV at pH 7.4. Neutral In some embodiments, the one nanoparticle or plurality of nanoparticles have an average surface charge of -20 mV to +20, -10 mV to +10 mV, or -5 mV to +5 mV at pH 7.4. Neutral The LNP of the charge has improved pharmacokinetics and biological performance compared to cationic LNP. It has.

[0250] Generation of lipid nanoparticles (LNP) The method for generating LNP can include mixing a first solution and a second solution. The mixing can be achieved using standard solution mixing techniques such as propeller mixing, vortexing of the solution, or preferably microfluidic mixing or high-efficiency T mixing. In some embodiments, the first solution contains one lipid or a plurality of lipids and nucleic acids, and all components are solubilized in a water / solvent system. The solvent can be any water-miscible solvent (e.g., ethanol , methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, or tetrahydrofuran). In some embodiments, the first solution contains a small proportion of water or pH-buffered water. The first solution contains water up to at least 60% by volume, for example, at least about 0.05% by volume, 0.1% by volume , 0.5% by volume, 1% by volume, 2% by volume, 3% by volume, 4% by volume, 5% by volume, 10% by volume, 15% by volume, 20% by volume, 25% by volume, 30% by volume, 35% by volume, 40% by volume, 45% by volume , 50% by volume, 55% by volume, or 60% by volume of water. In one embodiment, the first solution contains water in the range of about 0.05% to 60% by volume, for example, about 0.05% to 50% by volume, about 0.05% to 40% by volume, or about 5% to 20% by volume of water. .

[0251] In some embodiments, the first solution contains a single type of lipid, such as ionized lipid, phospholipid, sterol, or PEG-containing lipid. In some embodiments, and, the first solution contains a plurality of lipids. In some embodiments, the plurality includes ionized lipids, phospholipids, sterols, or PEG-containing lipids. In some embodiments the plurality of lipids includes cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DMG-PEG2k), or α-(3’-{[1,2-di (myristyloxy)propanoxy]carbonylamino}propyl)-ω-methoxy, polyoxyethylene (PEG2000-C-DMG), and ionizable lipids. The plurality of lipids can be present in any ratio. In one embodiment, the plurality of lipids includes ionizable lipids or sterols, phospholipids, sterols, PEG-containing lipids of the above lipids, or combinations thereof, in a specific ratio (e.g., the ratio described herein).

[0252] 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 may contain a loading component, such as a nucleic acid (e.g., mRNA). The second solution may contain a small ratio of a water-miscible organic solvent. The second solution contains at least one water-miscible organic solvent up to at least 60% by volume, e.g., at least about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or any volume % therebetween of at least one organic solvent (e.g., water-miscible It may contain an organic solvent. In one embodiment, the second solution is from about 0.05% by volume to 60% by volume of an organic solvent, for example, from about 0.05% by volume to 50% by volume, from about 0.05% by volume to 4 0% by volume, or from about 5% by volume to 20% by volume of an organic solvent (e.g., a water-miscible organic solvent). The aqueous buffer solution may be an aqueous solution of a citrate buffer. In some embodiments the aqueous buffer solution is a citrate buffer having a pH of 4 to 6 (e.g., a pH of about 4, about 5, or about 6). In one embodiment, the aqueous buffer solution is a citrate buffer having a pH of about 6 .. .. ..

[0253] In some embodiments, a solution containing a mixture of the first and second solutions containing the LNP suspension may be diluted. In some embodiments, the pH of a solution containing a mixture of the first and second solutions containing the LNP suspension can be adjusted. Dilution of the LNP suspension or adjustment of the pH can be achieved by adding water, an acid, a base, or an aqueous buffer. In some embodiments, neither dilution of the LNP suspension nor adjustment of the pH is performed .. .. .. In some embodiments, both dilution of the LNP suspension and adjustment of the pH are performed.

[0254] In some embodiments, excess reagents, solvents, and non-encapsulated nucleic acids can be removed from the LNP suspension by tangential flow filtration (TFF) (e.g., hemodiafiltration). Organic solvents (e.g., ethanol) and buffers can also be removed from the LNP suspension by TFF. In some embodiments, the LNP suspension is dialyzed and not subjected to TFF. In some embodiments, the LNP suspension is subjected to TF .. .. .. .. Receives F and does not undergo dialysis. In some embodiments, the LNP suspension undergoes both dialysis and TFF.

[0255] In one aspect, the present disclosure provides an LNP sample containing a nucleic acid for a period suitable for degrading the lipid layer to encapsulate and / or release the encapsulated nucleic acid, and treating the sample with a fluid containing a detergent (e.g., , Triton X-100, or an anionic detergent (including, but not limited to, sodium dodecyl sulfate (SDS), etc.), or a non-ionic detergent (including, but not limited to, , β-octyl glucoside, etc.), or an amphoteric detergent 3-14), etc. The method is characterized by the above. In one embodiment, the method further includes analyzing the sample for the presence, absence, and / or the amount of the released nucleic acid.

[0256] LNP containing a ligand Some aspects of the present disclosure relate to an LNP containing a ligand having binding specificity for a cell surface antigen (also referred to as a target ligand in the present specification), wherein the binding of the ligand to the antigen induces internalization of the ligand. Some embodiments relate to a composition comprising an LNP containing the ligand described herein.

[0257] In some embodiments, the target ligand is coupled to a lipid complex. For example, the lipid complex may be a hydrophilic polymer lipid complex such as, but not limited to, PEG(2000)-DSPE or PEG(2000)-DSG. The coupling can be achieved by various chemistries known in the art (e.g., , Bioconjugates Techniques (Greg T. Hermans (see, 3rd Edition, 2013, Elsevier). In some embodiments, the target ligand is coupled to the lipid complex by a linker. In some embodiments, the target ligand is coupled to the lipid complex by a linker. Linker molecules generally contain a hydrophilic polymer chain such as a lipid domain (phospholipid or cholesterol) to which a PEG terminus is linked, and contain a thiol-reactive functional group such as maleimide at the terminus. Linker molecules generally contain a hydrophilic polymer chain such as a lipid domain (phospholipid or cholesterol) to which a PEG terminus is linked, and contain a thiol-reactive functional group such as maleimide at the terminus. Sizes, phosphatidylethanolamine (PE) lipid anchors with various hydrocarbon chain lengths, and linkers containing PEG spacers with terminal maleimide or iodoacetate groups are currently commercially available from Avanti Polar Lipids (Alabama, USA) and NOF Corporation (Japan). One strategy commonly used is to couple the protein to a thiol-reactive lipopolymmeric linker such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] ( Mal-PEG-DSPE). Preferably, the protein of interest is engineered to contain one cysteine at the C-terminus to ensure site-specific conjugation. Alternatively, F(ab)2 or Fab’ can be enzymatically generated from IgG by reduction of disulfide bonds using reducing agents such as dithiothreitol (DTT), mercaptoethylamine, tris(2-carboxyethyl)phosphine) TCEP-HCL, which are reactive with cysteine thiol groups for coupling to Mal-PEG-DSPE. The reaction of Mal-PEG-DSPE with reduced cysteine occurs in an aqueous buffer at pH 5.5 - 7.5, for example, pH 5.5, 6, 6.5, 7, 7. (Mal-PEG-DSPE). Preferably, the protein of interest is engineered to contain one cysteine at the C-terminus to ensure site-specific conjugation. Alternatively, F(ab)2 or Fab’ can be enzymatically generated from IgG by reduction of disulfide bonds using reducing agents such as dithiothreitol (DTT), mercaptoethylamine, tris(2-carboxyethyl)phosphine) TCEP-HCL, which are reactive with cysteine thiol groups for coupling to Mal-PEG-DSPE. The reaction of Mal-PEG-DSPE with reduced cysteine occurs in an aqueous buffer at pH 5.5 - 7.5, for example, pH 5.5, 6, 6.5, 7, 7. (Mal-PEG-DSPE). Preferably, the protein of interest is engineered to contain one cysteine at the C-terminus to ensure site-specific conjugation. Alternatively, F(ab)2 or Fab’ can be enzymatically generated from IgG by reduction of disulfide bonds using reducing agents such as dithiothreitol (DTT), mercaptoethylamine, tris(2-carboxyethyl)phosphine) TCEP-HCL, which are reactive with cysteine thiol groups for coupling to Mal-PEG-DSPE. The reaction of Mal-PEG-DSPE with reduced cysteine occurs in an aqueous buffer at pH 5.5 - 7.5, for example, pH 5.5, 6, 6.5, 7, 7. (2-carboxyethyl)phosphine) TCEP-HCL, etc. The reaction of Mal-PEG-DSPE with reduced cysteine occurs in an aqueous buffer at pH 5.5 - 7.5, for example, pH 5.5, 6, 6.5, 7, 7. groups for coupling to Mal-PEG-DSPE. The reaction of Mal-PEG-DSPE with reduced cysteine occurs in an aqueous buffer at pH 5.5 - 7.5, for example, pH 5.5, 6, 6.5, 7, 7. 5, preferably pH 6.0. The reaction typically occurs within 4 hours. 5, preferably pH 6.0. The reaction typically occurs within 4 hours. It is completed. A small amount of cysteine or mercaptoethanol is added to react with the unreacted maleimide groups to deactivate the coupling reaction. Prior to the subsequent in-membrane insertion step, it is not necessary to remove the uncoupled protein, but it is useful to purify the complex for storage purposes and to enable more precise characterization evaluation. Due to the large size of the lipopolymers micelles (equal molecular weight 850k Da, Nellis et al., 2005a), size exclusion chromatography (SEC) is a convenient method. The characterization of such protein complexes is achieved by various techniques. For example, purity is determined by SEC, molecular weight is determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the melting point of the protein is determined by differential scanning calorimetry (DSC), the determination of the isoelectric point is by capillary electrophoresis, and the target binding affinity is quantified by surface plasmon resonance (BIAcore ) and biolayer interferometry (ForteBio).

[0258] Examples of target ligands include the Her2 receptor, epidermal growth factor receptor (EGFR), Ephrin A 2 receptor, CLEC9A receptor, DEC205 receptor, CLEC4A receptor, XCR1 receptor, CD141 receptor, HLA-DR receptor, transferrin receptor type 1, transferrin receptor type 2, VEGF receptor, PDGF receptor, integrin, NGF receptor, CD19, CD20, CD22, CD33, CD43, CD38, CD56, C D69, prostate-specific membrane antigen (PSMA) or various other cell surface receptors, or complex carbohydrates, proteoglycans, glycoproteins, and asialoglycoprotein receptors (ASG receptors), Complex carbohydrates that bind to PR), such as N-acetylgalactosamine (GalNAc) ligands, including glycolipids, or small molecule complexes such as folic acid PEG-DSPE that target folate receptors However, it may also be an antibody or antibody fragment against a cell surface receptor.

[0259] In one embodiment, the target ligand is an anti-DEC205 antibody. DEC205 (C D205) is a type I cell surface protein mainly expressed by dendritic cells (DCs) . This is found on finger-like interdigitating DCs, bone marrow-derived DCs, Langerhans cells within the T cell area of lymphoid tissues, as well as on macrophages and T cells at low levels, and is significantly upregulated during DC maturation. A positive correlation is observed between the expression of DEC-205 and that of CD8a , both of which are found at high levels on lymphoid DCs and at low levels on bone marrow DCs. DEC -205 is also expressed by B cells at moderate levels and is upregulated during the transition from pre-B cells to B cells . Recombinant anti-human DEC205 antibodies are commercially available from Creative Bi olabs.

[0260] In one embodiment, antigen-specific targeting on the LNP is achieved by co-incubating the LNP with a target ligand-lipid complex to prepare ligand-targeted LNP. The target ligand-lipid complex may be prepared before preparing the LNP (Nelli s et al. Biotechnol Prog. 2005 Jan-Feb;21( 1):205-20 reference). 1):205-20 reference).

[0261] In one embodiment, the LNP is an antibody or fragment-PEG-phospholipid micelle Or co-incubate with other ligand complexes and heat overnight at 37°C to promote the insertion of the antibody complex into the LNP outer membrane (Nellis et al. Biotechnol Prog. 2005 Jan-Feb;21(1):221-32). In another aspect, the insertion can be achieved by heating at an elevated temperature for a shorter time, e.g., 0.5-8 hours, at 37°C, or preferably 0.5-2 hours, at 37°C. Micelle insertion can be stopped by placing the LNP on ice (and then storing it in the refrigerator at 4°C) and rapidly lowering the temperature. The total amount of lipid complex added can be 0.02%-2% of the total lipid, or preferably 0.1%-1%, or preferably 0.1%-0 .5%. The uptake efficiency of the antibody-lipid complex can be measured by SDS-PAGE after dissociation of the LNP by SDS or other detergents (comparing with the standard curve of the same protein) (Nellis et al. Biotechnol Prog. 2005 Jan-Feb;21(1):205-20). The uptake efficiency of other target ligands can be measured by ultra-performance liquid chromatography equipped with an evaporative light scattering detector (UPLC-EL SD) (Gauthier et al., J Mol Sci. 2019 Nov 12;20(22):5669).

[0262] Figure 2 shows the reaction of the reduced C-terminal cysteine of the Fab’ antibody fragment with the maleimide-terminated poly(ethylene glycol)2000-derivatized distearoyl phosphatidylethanolamine. R1 and R2 are stearic acid. The final antibody-lipopolymersome complex is and is an intermediate that is subsequently inserted into the outer lipid layer of the lipid nanoparticles for active targeting.

[0263] Targeting of LNP can also be achieved by adding lipids to the formulation. For example, phosphatidylserine is known to redistribute to the outer surface of the plasma membrane during apoptosis and is a molecular stimulus for the cellular attraction of phagocytosis (Fadok et al. Curr Biol. 2003 Aug 19;13(16):R655-7). Phosphatidyl serine (PS) and phosphatidylglycerol (PG) can be recognized by dendritic cells and can induce the uptake and activation of dendritic cells. Targeting of LNP can also be achieved by adding specific anionic phospholipids to the formulation (Table 3) . For example, phosphatidylserine is known to redistribute to the outer surface of the plasma membrane during apoptosis and is a molecular stimulus for the cellular attraction of phagocytosis (Fadok et al. Cu rr Biol. 2003 Aug 19;13(16):R655-7). Phosphatidyl serine (PS) and phosphatidylglycerol (PG) can be recognized by dendritic cells and can induce the uptake and activation of dendritic cells (Caronni e t al., Nat Comm. 2021 April 14;12:2237-225 3; Ischihashi et al., PLOS One 2013). Anionic phospholipids have been used previously in the context of liposomes, but inclusion in lipid nanoparticles containing condensed nucleic acids can inhibit intracellular escape by altering the surface charge, where the anionic head groups can compete for the binding sites of ionizable lipids with the phosphate backbone of mRNA (Caronni et al., Nat Comm. 2021 April 14;12:2237-2253; Ischihashi et al., PLOS One 2013). Anionic phospholipids have been used previously in the context of liposomes, but inclusion in lipid nanoparticles containing condensed nucleic acids can inhibit intracellular escape by altering the surface charge, where the anionic head groups can compete for the binding sites of ionizable lipids with the phosphate backbone of mRNA (Caronni et al., Nat Comm. 2021 April 14;12:2237-2253; Ischihashi et al., PLOS One 2013). Anionic phospholipids have been used previously in the context of liposomes, but inclusion in lipid nanoparticles containing condensed nucleic acids can inhibit intracellular escape by altering the surface charge, where the anionic head groups can compete for the binding sites of ionizable lipids with the phosphate backbone of mRNA (Caronni et al., Nat Comm. 2021 April 14;12:2237-2253; Ischihashi et al., PLOS One 2013). Anionic phospholipids have been used previously in the context of liposomes, but inclusion in lipid nanoparticles containing condensed nucleic acids can inhibit intracellular escape by altering the surface charge, where the anionic head groups can compete for the binding sites of ionizable lipids with the phosphate backbone of mRNA This is unexpected as it may result in aggregation of the LNPs during formation or storage.

[0264] JPEG2025106257000148.jpg195170

[0265] In one embodiment, the anionic targeting ligand is phosphatidylserine (PS), Sphatidylglycerol (PG), N-glutaryl-phosphatidylethanolamine (N-glu-PE), or N-succinyl-phosphatidylethanolamine (N- In one embodiment, the anionic The phospholipid is phosphatidylserine. In another embodiment, phosphatidylserine In another embodiment, the dimyristoyl phosphatase contains the L-isomer of serine. Dipalmitoyl-L-serine (DMPS), Dipalmitoylphosphatidyl-L-serine (DPPS ), or distearoylphosphatidyl-L-serine (DSPS), The acyl chains of phatidylserine are fully saturated. The PS used is either the L-isomer of DPPS or DSPS. Serine, for example, is a saccharide in which one acyl chain is stearic acid and the other is palmitic acid. , may also contain asymmetric acyl chain compositions.

[0266] In one embodiment, PS or PG is present in an amount ranging from about 0.1 mol % to about 1 mol % of the total lipid content of the LNP. 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 % at a concentration and added to the LNP lipid formulation. In one embodiment, PS is added to the LNP lipid formulation at a concentration of about 1 mol% to about 20 mol%, about 2.5 mol% to about 10 mol%, about 3 mol% to about 9 mol%, or about 4 mol% to about 8 mol% of the total lipid content of the LNP. In one embodiment, PS is added to the LNP lipid formulation at a concentration of about 1 mol% to about 20 mol%, about 2.5 mol% to about 10 mol%, about 3 mol% to about 9 mol% %, or about 4 mol% to about 8 mol% of the total lipid content of the LNP.

[0267] In one embodiment, the PS lipid is included in an LNP composition containing ionizable cationic lipids known in the art, including DODAP, AKG-OA-DM2, O-1176 9, DLin-MC3-DMA, DLin-KC2-DMA, DLin-KC3-DMA , ALC-0315, and SM-102. In another embodiment, the PS lipid is included in an LNP composition containing ICLs of Formula I, II, III, combinations thereof

[0268] or pharmaceutically acceptable salts thereof. In another embodiment, the PS lipid is included in the LNP composition using an N / P ratio of 3-8, 4-7, or 5-6. In another embodiment, the PS lipid is included in an LNP composition containing ICLs of Formula I, II, III, combinations thereof or pharmaceutically acceptable salts thereof. In another embodiment, the PS lipid is included in the LNP composition using an N / P ratio of 3-8, 4-7, or 5-6. In some embodiments, provided is a method of delivering a nucleic acid to a cell, the method comprising contacting the cell with a composition comprising an LNP comprising a ligand having binding specificity for a cell surface antigen (also referred to herein as a target ligand), wherein binding of the ligand to the antigen induces internalization of the ligand. In some embodiments, the target ligand may be, but is not limited to, an internalizing antibody or fragment thereof, a small molecule conjugate, or a glycoconjugate. In some embodiments, binding of the target ligand to a specific cell surface antigen induces internalization of the LNP, contacts the cell under internalization conditions, and incubates with the cell

[0269] In some embodiments, provided is a method of delivering a nucleic acid to a cell, the method comprising contacting the cell with a composition comprising an LNP comprising a ligand having binding specificity for a cell surface antigen (also referred to herein as a target ligand), wherein binding of the ligand to the antigen induces internalization of the ligand. In some embodiments, the target ligand may be, but is not limited to, an internalizing antibody or fragment thereof, a small molecule conjugate, or a glycoconjugate. In some embodiments, binding of the target ligand to a specific cell surface antigen induces internalization of the LNP, contacts the cell under internalization conditions, and incubates with the cell In some embodiments, provided is a method of delivering a nucleic acid to a cell, the method comprising contacting the cell with a composition comprising an LNP comprising a ligand having binding specificity for a cell surface antigen (also referred to herein as a target ligand), wherein binding of the ligand to the antigen induces internalization of the ligand. In some embodiments, the target ligand may be, but is not limited to, an internalizing antibody or fragment thereof, a small molecule conjugate, or a glycoconjugate. In some embodiments, binding of the target ligand to a specific cell surface antigen induces internalization of the LNP, contacts the cell under internalization conditions, and incubates with the cell In some embodiments, provided is a method of delivering a nucleic acid to a cell, the method comprising contacting the cell with a composition comprising an LNP comprising a ligand having binding specificity for a cell surface antigen (also referred to herein as a target ligand), wherein binding of the ligand to the antigen induces internalization of the ligand. In some embodiments, the target ligand may be, but is not limited to, an internalizing antibody or fragment thereof, a small molecule conjugate, or a glycoconjugate. In some embodiments, binding of the target ligand to a specific cell surface antigen induces internalization of the LNP, contacts the cell under internalization conditions, and incubates with the cell In some embodiments, the target ligand may be, but is not limited to, an internalizing antibody or fragment thereof, a small molecule conjugate, or a glycoconjugate. In some embodiments, binding of the target ligand to a specific cell surface antigen induces internalization of the LNP, contacts the cell under internalization conditions, and incubates with the cell In some embodiments, the target ligand may be, but is not limited to, an internalizing antibody or fragment thereof, a small molecule conjugate, or a glycoconjugate. In some embodiments, binding of the target ligand to a specific cell surface antigen induces internalization of the LNP, contacts the cell under internalization conditions, and incubates with the cell In some embodiments, binding of the target ligand to a specific cell surface antigen induces internalization of the LNP, contacts the cell under internalization conditions, and incubates with the cell In some embodiments, binding of the target ligand to a specific cell surface antigen induces internalization of the LNP, contacts the cell under internalization conditions, and incubates with the cell When it occurs, the target ligand linked to the cell expresses at least 100,000 or at least 1,000,000 antigen molecules.

[0270] JPEG2025106257000149.jpg212170

[0271] JPEG2025106257000150.jpg78170

[0272] Composition In some embodiments, the lipid nanoparticle composition comprises a lipid and a nucleic acid, and the lipid nanoparticles comprise a compound of formula I, II, III, a combination thereof or a pharmaceutically acceptable salt thereof.

[0273] In some embodiments, the LNP comprises an ionizable lipid having the structure of formula (IV).

[0274] In some embodiments, the composition further comprises a pharmaceutical excipient.

[0275] In some embodiments, the lipid nanoparticles are in an aqueous medium.

[0276] In some embodiments, the nucleic acid is encapsulated in the lipid nanoparticles together with a compound of formula I, II, III, IV or a combination thereof, and the nucleic acid is either RNA or DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid is DNA.

[0277] In some embodiments, the lipid nanoparticles comprise a membrane comprising phosphatidylcholine and sterol. In some embodiments, the sterol is cholesterol. . In some embodiments, the lipid nanoparticles comprise a membrane containing phosphatidylcholine, ionizable cationic lipid (ICL). In some embodiments, the ICL has a structure of Formula I, II, III or IV and cholesterol, and the membrane separates the interior of the lipid nanoparticles from the aqueous medium. In some embodiments, the ICL has the structures shown in Table 1A and Table 2. In some embodiments, the ICL has the structure shown in Table 1B. In some embodiments, the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC). In some embodiments, the molar ratio of the ionizable cationic lipid to cholesterol is about 65:35 to 40:60. In some embodiments, the molar ratio of the ICL to cholesterol is about 60:40 to about 45:55. . In some embodiments, the lipid nanoparticles comprise a membrane containing phosphatidylcholine, ionizable cationic lipid (ICL). . In some embodiments, the ICL has a structure of Formula I, II, III or IV and cholesterol, and the membrane separates the interior of the lipid nanoparticles from the aqueous medium. . In some embodiments, the ICL has the structures shown in Table 1A and Table . In some embodiments, the ICL has the structure shown in Table 1B. . In some embodiments, the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC). . In some embodiments, the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC). . In some embodiments, the molar ratio of the ionizable cationic lipid to cholesterol is about 65:35 to 40:60. . In some embodiments, the molar ratio of the ICL to cholesterol is about 60:40 to about 45:55.

[0278] . In some embodiments, the molar ratio of phosphatidylcholine to cholesterol is . about 1:5 to about 1:2.

[0279] . In some embodiments, the membrane further comprises a polymer composite lipid.

[0280] . In some embodiments, the lipid nanoparticles comprise ICL, DSPC, cholesterol . and polymer composite lipid in a molar ratio of about 49.5:10.3:39.6:2.5.

[0281] . In some embodiments, the polymer composite lipid is PEG(2000)-dimyristoyl glycerol (PEG-DMG) or PEG (molecular weight 2000)-dimyristoyl phosphatidylethanolamine (PEG-DMPE). . In some embodiments, the polymer composite lipid is PEG(2000)-dimyristoyl glycerol (PEG-DMG) or PEG (molecular weight 2000)-dimyristoyl phosphatidylethanolamine (PEG-DMPE). . In some embodiments, the polymer composite lipid is PEG(2000)-dimyristoyl glycerol (PEG-DMG) or PEG (molecular weight 2000)-dimyristoyl phosphatidylethanolamine (PEG-DMPE).

[0282] The compositions of the present disclosure can be administered by various routes, for example, intravenously, parenterally, intraperitoneally, or via a topical route for systemic delivery. The compositions can be administered to a subject intravenously, subcutaneously, or intraperitoneally. In some embodiments, the present disclosure provides a method for performing in vivo delivery of nucleic acids to a subject.

[0283] In some embodiments, the composition is a liquid pharmaceutical formulation for oral administration.

[0284] In some embodiments, the composition is a liquid pharmaceutical formulation for subcutaneous, intramuscular, or intradermal administration.

[0285] In some embodiments, the composition is in the form of a lyophilized powder, which is reconstituted with an aqueous medium subsequent to administration.

[0286] Method of Use Targeting Dendritic Cells Dendritic cells (DCs) are specialized antigen-presenting cells that play a central role in initiating and regulating adaptive immunity. Their potent antigen (Ag)-presenting ability and unique ability to generate T cell responses make efficient and specific delivery of Ag to DCs the basis for generating Ag-specific effectors and memory cells against tumors or pathogens.

[0287] Dendritic cells can be generated from human blood monocytes by adding granulocyte macrophage colony-stimulating factor (GM-CSF), IL-4, and IFN-gamma and differentiating monocyte-derived DCs in vitro. The cells under culture show both dendritic and veiled shapes, with the former being adherent and the latter being suspended. Phenotypically, they are CD1a− / d ​im, CD11a+, CD11b++, CD11c+, CD14dim / -, CD16a - / dim, CD18+, CD32dim / -, CD33+, CD40+, CD45R0 +, CD50+, CD54+, CD64- / dim, CD68+, CD71+, CD80 dim, CD86+ / ++, MHC class I++, HLA-DR++, HLA-DP +, and HLA-DQ (Geiseler et al. Dev Immunol. 1998;6(1-2):25-39).

[0288] Alternatively, human primary blood dendritic cell lines have been developed and are commercially available from Creative Biolabs.

[0289] CD8+ T cells are known to have important functions during Mycobacterium tuberculosis infection and can produce cytokines IL2, IFN-γ, and TNF. Importantly, CD8+ T cells have cytolytic functions to kill Mycobacterium tuberculosis-infected cells via granule-mediated functions (through perforin, granzyme, and granulysin) or Fas-Fas ligand interactions to induce apoptosis. In humans, CD8+ T cells can produce granulysin, which can directly kill Mycobacterium tuberculosis. Therefore, antigen-producing mRNA LNPs delivered to DCs are expected to stimulate CD8+ T cell responses to fight Mycobacterium tuberculosis infection.

[0290] CD8+ T cells can recognize Mycobacterium tuberculosis-specific antigens (as peptides) presented by classical and non-classical MHC molecules. Antigen presentation by antigen-presenting cells in the context of classical MHC Ia (HLA-A, B, C) molecules ​​​​​​​​​​ Classically restricted CD8+ T cells that recognize the antigen were identified. Non-classically restricted CD8+ T cells include CD8+ T cells that can recognize the antigen in the context of MHC I-related molecules (MR1), such as HLA-E molecules (non-MHC Ia), group 1 CD1 molecules, glycolipids associated with them, and mucosal associated invariant T cells (MAIT). Finally, γδ T cells represent distinct populations of CD8 (and CD4) T cells that have both innate and adaptive functions in response to Mycobacterium tuberculosis infection. CD8+ T cells have been shown to function directly in response to Mycobacterium tuberculosis infection, but also play an important role in regulating many different functions (e.g., interactions to provide optimal CD4 T cell function) in the overall host immune response.

[0291] In one embodiment, the LNP may be added to cultured human dendritic cells at a suitable concentration (e.g., 1 - 5 μg / mL mRNA). After allowing some time for cell uptake and antigen expression, human T cells (HemaCare) can be added, and the cell culture medium can be sampled at various time points for INF-γ by ELISA (R&D Systems, DIF50C). Alternatively, the cells can be analyzed by flow cytometry for the CD8+ marker or intracellular INFγ production (PE anti-human IFN-γ antibody, Biolegend).

[0292] In one embodiment, the LNP can be administered to a subject by any route of administration at a dose of 0.01 - 5 mg / kg mRNA. According to some embodiments, a proportion of the LNP is taken up by DC cells, but most accumulates in the liver and spleen. It does. DC cells express antigen peptides and process them for MHC I presentation and present them to naive T cells that induce the education of memory T cells against the antigen, and can migrate to the lymph nodes .

[0293] In one embodiment, LNP modified with a target ligand such as anti-DEC205-PEG-DSPE can be administered to the subject at a dose of 0.01-5 mg / kg of mRNA. According to some embodiments, a higher percentage of LNP can be taken up by DC cells, increasing the production of antigen peptides compared to un-targeted LNP and enabling a more effective vaccination against pathogens. Additional target ligands for dendritic cells include, but are not limited to, CLE C9A, CLEC4A, XCR1, CD141, and HLD-DR. For example, the evaluation of the CD8+ reactivity against antigens generated in vivo can be achieved by measuring the concentration of INFγ in plasma by species-specific IFN- gamma Quantikine ELISA Kits (R&D Systems).

[0294] In some embodiments, the LNP composition provides desired pharmacokinetic properties such as an extended plasma concentration half-life and encapsulation of mRNA . The plasma concentration half-life can be measured as the percentage of the injected volume (ID) remaining in the blood 6 or 24 hours after intravenous injection into immunocompetent mice. The stability of mRNA encapsulation in plasma over 24 hours can be determined by the change in the ratio of mRNA to lipid (mRNA / L ratio) after intravenous administration of the mice. In some embodiments, in the blood The percentage of remaining encapsulated mRNA is more than 20% of the injected amount at 6 hours, preferably more than 30%, most preferably more than 40%. The percentage retained in the blood after 24 hours is more than, preferably more than 10%, more preferably more than 20% of the injected amount. This specification discloses methods for preventing mycobacterial infections such as Mycobacterium tuberculosis, or Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Additional mycobacteria and Gram-positive bacteria include, but are not limited to, Mycobacterium avium complex, Mycobacterium leprae, Mycobacterium gordonae, Mycobacterium abscessus, Mycobacterium abscessus, Mycobacterium mucogenicum, streptococci, vancomycin-resistant enterococci (VRE), Staphylococcus pneumoniae, Enterococcus faecium, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes (Streptococcus pyogenes), viridans streptococci

[0295] In this specification, methods for preventing mycobacterial infections such as Mycobacterium tuberculosis, or Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are disclosed. Additional mycobacteria and Gram-positive bacteria include, but are not limited to, Mycobacterium avium complex, Mycobacterium leprae, Mycobacterium gordonae, Mycobacterium abscessus, Mycobacterium abscessus, Mycobacterium mucogenicum, streptococci, vancomycin-resistant enterococci (VRE), Staphylococcus pneumoniae, Enterococcus faecium, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes (Streptococcus pyogenes), viridans streptococci p streptococci), Listeria monocytogenes, Nocardia, and Corynebacterium monocytogenes), Nocardia, and Corynebacterium are included.

[0296] Administration of a vaccine to induce a second immune response can induce a CD4+ helper T cell response against cells expressing an antigen that induces an MHC-presented epitope, resulting in an MHC class II-presented epitope. Alternatively, or in addition, administration of a vaccine to induce a second immune response can induce a CD8+ T cell response against cells expressing an antigen that induces an MHC-presented epitope, resulting in an MHC class I-presented epitope. Further, administration of a vaccine to induce a second immune response can result in one or more neo-epitopes (including known neo-epitopes) and cancer-specific somatic mutations that do not contain cancer-specific somatic mutations but are expressed by cancer cells and preferably induce an immune response against cancer cells, preferably a cancer-specific immune response. In one embodiment, administration of a vaccine to induce a second immune response is an MHC class II-presented epitope and / or a neo-epitope that can induce a CD4+ helper T cell response against cells expressing an antigen that induces an MHC-presented epitope, as well as an MHC class I-presented epitope and / or a cancer-specific somatic mutation that does not contain cancer-specific somatic mutations but is expressed by cancer cells and preferably induces a CD8+ T cell response against cells expressing an antigen that induces an MHC-presented epitope, resulting in an epitope. In one embodiment, the epitope does not contain cancer-specific somatic mutations. mutations that do not contain cancer-specific somatic mutations but are expressed by cancer cells and preferably induce an immune response against cancer cells, preferably a cancer-specific immune response. In one embodiment, administration of a vaccine to induce a second immune response is an MHC class II-presented epitope and / or a neo-epitope that can induce a CD4+ helper T cell response against cells expressing an antigen that induces an MHC-presented epitope, as well as an MHC class I-presented epitope and / or a cancer-specific somatic mutation that does not contain cancer-specific somatic mutations but is expressed by cancer cells and preferably induces a CD8+ T cell response against cells expressing an antigen that induces an MHC-presented epitope, resulting in an epitope. In one embodiment, the epitope does not contain cancer-specific somatic mutations. In one embodiment, administration of a vaccine to induce a second immune response is an MHC class II-presented epitope and / or a neo-epitope that can induce a CD4+ helper T cell response against cells expressing an antigen that induces an MHC-presented epitope, as well as an MHC class I-presented epitope and / or a cancer-specific somatic mutation that does not contain cancer-specific somatic mutations but is expressed by cancer cells and preferably induces a CD8+ T cell response against cells expressing an antigen that induces an MHC-presented epitope, resulting in an epitope. In one embodiment, the epitope does not contain cancer-specific somatic mutations. resulting in a neo-epitope and an epitope that does not contain cancer-specific somatic mutations but is expressed by cancer cells and preferably induces a CD8+ T cell response against cells expressing an antigen that induces an MHC-presented epitope, resulting in an epitope. In one embodiment, the epitope does not contain cancer-specific somatic mutations. resulting in an epitope. In one embodiment, the epitope does not contain cancer-specific somatic mutations. In one embodiment, the epitope does not contain cancer-specific somatic mutations. does not contain cancer-specific somatic mutations.

[0297] "Cellular immune response", "cellular response", "cellular response to an antigen", or synonyms thereof, means a cellular response characterized by the presentation of an antigen having MHC of class I or class II directed towards cells. The cellular response relates to cells called T cells or T-lymphocytes that act as either "helper cells" or "killer cells". Helper T cells (also referred to as CD4+ T cells) play a central role by regulating the immune response, and killer cells (also referred to as cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill abnormal cells such as cancer cells and prevent the production of more abnormal cells. In a preferred embodiment, the present disclosure requires stimulation of an anti-Mycobacterium tuberculosis CTL response against Mycobacterium that expresses one or more expressed antigens and preferably presents the expressed antigen having MHC of class I.

[0298] "Antigen" according to the present disclosure encompasses any substance that induces an immune response. In particular, an "antigen" is any substance that specifically reacts with an antibody or a T-lymphocyte (T cell), preferably related to a peptide or a protein. As used herein, the term "antigen" includes any molecule that contains at least one epitope. Preferably, an antigen in the context of the present disclosure is, optionally, after processing, preferably a molecule that induces an immune reaction, specific for the antigen (including cells expressing the antigen). According to the present disclosure, any suitable antigen that is a candidate for an immune reaction (preferably a cellular immune reaction) may be used. In the context of the embodiments of the present disclosure, the antigen is preferably An immune response to the antigen is elicited by antigen-presenting cells comprising it, in the context of MHC molecules. The antigen is preferably a product corresponding to or derived from a native antigen. Such native antigens can include tumor antigens.

[0299] As used herein, "antigenic peptide" relates to a part or fragment of an antigen capable of stimulating an immune response, preferably a cellular response, to the antigen, characterized by the expression of the antigen, preferably abnormal cells, in particular antigens such as cancer cells, or cells. Preferably, the antigenic peptide can stimulate a cellular response to cells characterized by the presentation of an antigen having class I MHC, preferably, antigen-responsive cytotoxic T lymphocytes (CTLs). Preferably, the antigenic peptides according to the present disclosure are MHC class I and / or class II presenting peptides, or can be processed to generate MHC class I and / or class II presenting peptides. Preferably, the antigenic peptide comprises an amino acid sequence substantially corresponding to the amino acid sequence of a fragment of the antigen. Preferably, the fragment of the antigen is an MHC class I and / or class II presenting peptide. Preferably, the antigenic peptides according to the present disclosure comprise an amino acid sequence substantially corresponding to the amino acid sequence of the fragment, which fragment, i.e., the MHC class I and / or class II presenting peptide derived from the antigen, is processed to generate the same. When the peptide is presented directly, i.e., without processing, especially without cleavage, it has a length suitable for binding to MHC molecules, especially class I MHC molecules. Preferably, the antigenic peptides according to the present disclosure are MHC class I and / or class II presenting peptides, or can be processed to generate MHC class I and / or class II presenting peptides. Preferably, the antigenic peptide comprises an amino acid sequence substantially corresponding to the amino acid sequence of a fragment of the antigen. Preferably, the fragment of the antigen is an MHC class I and / or class II presenting peptide. Preferably, the antigenic peptides according to the present disclosure comprise an amino acid sequence substantially corresponding to the amino acid sequence of the fragment, which fragment, i.e., the MHC class I and / or class II presenting peptide derived from the antigen, is processed to generate the same. When the peptide is presented directly, i.e., without processing, especially without cleavage, it has a length suitable for binding to MHC molecules, especially class I MHC molecules. Preferably, the antigenic peptides according to the present disclosure comprise an amino acid sequence substantially corresponding to the amino acid sequence of the fragment, which fragment, i.e., the MHC class I and / or class II presenting peptide derived from the antigen, is processed to generate the same. When the peptide is presented directly, i.e., without processing, especially without cleavage, it has a length suitable for binding to MHC molecules, especially class I MHC molecules. i.e., the MHC class I and / or class II presenting peptide derived from the antigen, is processed to generate the same. When the peptide is presented directly, i.e., without processing, especially without cleavage, it has a length suitable for binding to MHC molecules, especially class I MHC molecules. When the peptide is presented directly, i.e., without processing, especially without cleavage, it has a length suitable for binding to MHC molecules, especially class I MHC molecules. When the peptide is presented directly, i.e., without processing, especially without cleavage, it has a length suitable for binding to MHC molecules, especially class I MHC molecules. and preferably has a length of 7 to 20 amino acids, more preferably a length of 7 to 12 amino acids even more preferably a length of 8 to 11 amino acids, particularly a length of 9 or 10 amino acids is.

[0300] The main types of professional antigen-presenting cells are dendritic cells, which have the broadest range of antigen presentation, perhaps the most important antigen-presenting cells, macrophages, B cells, and certain activated epithelial cells are. Dendritic cells (DCs) are a population of leukocytes that present antigens captured in peripheral tissues to T cells via the MHC class II and I antigen presentation pathways. Dendritic cells are a powerful inducer of the immune response, and activation of these cells is a well-known important step for the induction of antitumor immunity. Dendritic cells can be classified, for convenience, into "immature" cells and "mature" cells, and can be used as a simple way to distinguish two well-characterized phenotypes. 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 expression of Fcγ receptors and mannose receptors and a high capacity for antigen uptake and processing. The mature phenotype is typically characterized by low expression of these markers, but high expression of cell surface molecules

[0301] involved in T cell activation, such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB). Dendritic cell maturation leads to T cell priming by antigen-presenting dendritic cells, whereas antigen presentation by immature dendritic cells leads to tolerance. The mature phenotype is typically characterized by low expression of these markers, but high expression of cell surface molecules involved in T cell activation, such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB). Dendritic cell maturation leads to T cell priming by antigen-presenting dendritic cells, whereas antigen presentation by immature dendritic cells leads to tolerance. whereas antigen presentation by immature dendritic cells leads to tolerance, while antigen-presenting dendritic cells lead to T cell priming. It is called the state of dendritic cell activation. Dendritic cell maturation is mainly caused by innate receptors (such as bacterial DNA, viral RNA, endotoxin, etc.), inflammatory cytokines (TNF, IL-1, IFN), ligation of CD40 on the surface of dendritic cells by CD40L, and biomolecules with characteristics of microorganisms detected by substances released from cells that have undergone stressful cell death. Dendritic cells can be induced by culturing bone marrow cells with cytokines such as granulocyte macrophage colony-stimulating factor (GM CSF) and tumor necrosis factor alpha in vitro. Non-professional antigen-presenting cells do not constitutively express MHC class II proteins required for interaction with naive T cells, and these are expressed only by stimulation of non-professional antigen-presenting cells by certain specific cytokines. "Antigen-presenting cells" can carry MHC class I-presented peptides by transducing cells with nucleic acids encoding peptides or polypeptides containing the presented peptides, preferably mRNA, for example, nucleic acids encoding antigens. irus RNA, endotoxin, etc.), inflammatory cytokines (TNF, IL-1, IFN), CD 40L-mediated ligation of CD40 on the surface of dendritic cells, and biomolecules with characteristics of microorganisms detected by substances released from cells that have undergone stressful cell death. elicited by biomolecules with characteristics of microorganisms. Dendritic cells can be induced by culturing bone marrow cells with cytokines such as granulocyte macrophage colony-stimulating factor ( GM CSF) and tumor necrosis factor alpha in vitro. Non-professional antigen-presenting cells do not constitutively express MHC class II proteins required for interaction with naive T cells, and these are expressed only by stimulation of non-professional antigen-presenting cells by any specific cytokine. "Antigen -presenting cells" can carry MHC class I-presented peptides by transducing cells with nucleic acids encoding peptides or polypeptides containing the presented peptides, preferably mRNA, for example, nucleic acids encoding antigens. Non-professional antigen-presenting cells do not constitutively express the MHC class II proteins required for interaction with naive T cells, and these are expressed only by stimulation of non-professional antigen-presenting cells by any specific cytokine. "Antigen -presenting cells" can carry MHC class I-presented peptides by transducing cells with nucleic acids encoding peptides or polypeptides containing the presented peptides, preferably mRNA, for example, nucleic acids encoding antigens. elicited by biomolecules with characteristics of microorganisms. Dendritic cells can be induced by culturing bone marrow cells with cytokines such as granulocyte macrophage colony-stimulating factor ( GM CSF) and tumor necrosis factor alpha in vitro. Non-professional antigen-presenting cells do not constitutively express MHC class II proteins required for interaction with naive T cells, and these are expressed only by stimulation of non-professional antigen-presenting cells by any specific cytokine. "Antigen -presenting cells" can carry MHC class I-presented peptides by transducing cells with nucleic acids encoding peptides or polypeptides containing the presented peptides, preferably mRNA, for example, nucleic acids encoding antigens. elicited by biomolecules with characteristics of microorganisms. Dendritic cells can be induced by culturing bone marrow cells with cytokines such as granulocyte macrophage colony-stimulating factor (

[0302] In some embodiments, a pharmaceutical composition comprising a gene delivery vehicle that targets dendritic cells or other antigen-presenting cells can be administered to a patient to effect transfection that occurs in vivo. As used herein, "nucleic acid" is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, most preferably RNA transcribed in vitro (IVT RNA) or synthetic RNA. Nucleic acids include genomic DNA, cDNA, mRNA, molecules generated by recombinant techniques, and chemically synthesized molecules according to the present disclosure. According to the present disclosure, nucleic acids can be single-stranded or double-stranded linear chains. According to the present disclosure, nucleic acids include genomic DNA, cDNA, mRNA, molecules generated by recombinant techniques, and chemically synthesized molecules. According to the present disclosure, nucleic acids can be single-stranded or double-stranded linear or may exist as a covalently closed circular molecule. According to the present disclosure, nucleic acids can be isolated. The term "isolated nucleic acid" according to the present disclosure means that the nucleic acid is (i) amplified in vitro, for example, via polymerase chain reaction (PCR), (ii) generated by recombinant techniques such as cloning, (iii) purified by cleavage and separation, for example, by gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis. Nucleic acids can be used, in particular, for introduction into cells, i.e., transfection of cells, in the form of RNA prepared by in vitro transcription from a DNA template. Moreover, RNA can be modified prior to application by sequence stabilization, capping, and polyadenylation. Furthermore, RNA can be modified prior to application by sequence stabilization, capping, and polyadenylation.

[0303] As used herein, the term "RNA" relates to molecules containing ribonucleotide residues, preferably or consisting entirely or substantially of ribonucleotide residues. "Ribonucleotide" relates to a nucleotide having a hydroxyl group at the 2'-position of the B-D-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA (such as partially or entirely purified RNA), substantially pure RNA, synthetic RNA, and RNA generated by recombinant techniques (such as modified RNA that is different from natural RNA by addition, deletion, substitution, and / or modification of one or more nucleotides). Such modifications can include, for example, addition of non-nucleotide material at the end or within the RNA, for example, at one or more nucleotides of the RNA. Nucleotides in an RNA molecule can include non-natural nucleotides or non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. / or modification). Such modifications can include, for example, addition of non-nucleotide material at the end or within the RNA, for example, at one or more nucleotides of the RNA. Nucleotides in an RNA molecule can include non-natural nucleotides or non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. as well as non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. can be termed analogs or analogs of natural RNAs. These modified RNAs .

[0304] As used herein, the term "RNA" includes "mRNA" and preferably relates to " m RNA". The term "mRNA" means "messenger RNA" and relates to a "transcript" produced using a DNA template and encodes a peptide or polypeptide. Typic ally, mRNA includes a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA has a limited half-life both intracellularly and in vitro. In the context of the present disclosure, mRNA can be produced by in vitro transcription from a DNA template. The term "modification" in the context of RNA as used in the present disclosure includes any modification of RNA th at is not naturally present in said RNA. In one embodiment of the present disclosure, the RNA used according to the present disclosure does not have an uncapped 5'-triphosphate. Removal of such uncapped 5'-triphosphate can be achieved by treating the RNA with phosphatase. The RNA according to th e present disclosure can have modified ribonucleotides for improving its stability and / or reducing its cytotoxicity. For example, in one embodiment, in the RNA used according to the present disclosure, in the case of cytid ine, 5-methylcytidine is partially or completely substituted, preferably

[0305] In one embodiment, the term "modification" relates to generating an RNA having a 5-cap or 5'-cap analog. The term "5-cap" refers to the cap structure present at the 5'-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via an unusual 5'-5 triphosphate bond. In one embodiment, this guanosine is methylated at the 7-position. The term "conventional 5'-cap" refers to a native RNA 5'-cap, preferably a 7-methylguanosine cap (m'G). As used herein, the term "5'-cap" is similar to an RNA cap structure and preferably has the ability to stabilize RNA in vivo and / or intracellularly and / or is modified to enhance the translation of the RNA (when bound to the RNA), and includes 5'-cap analogs. According to the present disclosure, the stability and translation efficiency of RNA may be modified as needed. For example, one or more modifications having a stabilizing effect on the RNA and / or an improved translation efficiency can stabilize the RNA and increase its translation. Such modifications are described, for example, in PCT / EP20 06 / 009448, which is incorporated herein by reference in its entirety. To increase the expression of the RNA used in accordance with the present disclosure, preferably, the GC content is increased to improve mRNA stability, codon optimization is performed, and thus the sequence of the expressed peptide or protein is modified without

[0306] modifying the coding region, i.e., the sequence encoding the expressed peptide or protein, to enhance translation in the cell. For example, one or more modifications having a stabilizing effect on the RNA and / or an improved translation efficiency can stabilize the RNA and increase its translation. Such modifications are described, for example, in PCT / EP20 06 / 009448, which is incorporated herein by reference in its entirety. To increase the expression of the RNA used in accordance with the present disclosure, preferably, the GC content is increased to improve mRNA stability, codon optimization is performed, and thus the sequence of the expressed peptide or protein is modified without modifying the coding region, i.e., the sequence encoding the expressed peptide or protein, to enhance translation in the cell. For example, to increase the expression of the RNA used in accordance with the present disclosure, preferably, the GC content is increased to improve mRNA stability, codon optimization is performed, and thus the sequence of the expressed peptide or protein is modified without modifying the coding region, i.e., the sequence encoding the expressed peptide or protein, to enhance translation in the cell.

[0307] Aspects of the present disclosure are methods of preventing bacterial or viral infections, comprising administering to a subject in need thereof an effective amount of a composition produced herein to induce an immune response.

[0308] Aspects of the present disclosure provide a method of vaccinating a subject, comprising administering to the subject in a single dose an effective amount of a nucleic acid (e.g., mRNA) encoding a polypeptide, contained in a composition described herein. In some embodiments, the nucleic acid is formulated in cationic lipid nanoparticles. In some embodiments, the lipid nanoparticle composition is administered by a single injection method.

[0309] In some embodiments, the bacterial infection is Mycobacterium tuberculosis infection.

[0310] In some embodiments, the viral infection is a coronavirus. In some embodiments, the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2.

[0311] In some embodiments, the viral infection is HIV / AIDS.

[0312] In some embodiments, the lipid nanoparticles are administered parenterally.

[0313] Generally, administration to patients by intradermal injection is possible. However, the injection may also be made into lymph nodes via intramuscular (Maloy et al. (2001), Proc Natl Acad Sci USA 98:3299-3033). As a result The resulting cells are given the target complex and are recognized by autologous cytotoxic T lymphocytes, which then proliferate.

[0314] In some embodiments, the composition is administered by inhalation. In some embodiments, the composition is formulated as a nasal spray and / or an aerosol.

[0315] The actual dosage level of the active agent in the pharmaceutical compositions disclosed herein may vary to obtain an amount of the active agent that is effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration, without undue toxicity to the patient.

[0316] As used herein in the context of administration, "parenteral" means a method of administration other than enteral and topical administration and typically includes, but is not limited to, administration by injection, including intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, epidural, and intrasternal injection and infusion.

[0317] As used herein, the phrases "parenteral administration" and "administered parenterally" typically refer to a method of administration other than enteral (i.e., via the digestive tract) and topical administration, by injection or infusion, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, inhalation, subcapsular, subarachnoid, respiratory mucosa, intraspinal, epidural, and intrasternal injection and infusion. Intravenous injection and infusion are often (but not exclusively) used for the administration of liposomal drugs.

[0318] ​​​​​​The dosing regimen can be adjusted to obtain an optimal desired response (e.g., a therapeutic response). For example, a single or multiple doses may be administered over time, or the dose may be proportionally decreased or increased as indicated by the requirements of the treatment situation.

[0319] In some embodiments, the dose comprises 0.01 - 5 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 - 5 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 - 3 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 - 3 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 - 1 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 - 1 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 - 0.5 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 - 0.5 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 - 1 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 - 0.1 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 - 0.05 mg / kg of mRNA. In some embodiments, the dose comprises 0.01 - 0.1 mg / kg of nucleic acid. In some embodiments, the dose comprises 0.01 - 0.05 mg / kg of mRNA.

[0320] The dosage of the LNP comprising the compound and / or its pharmaceutically acceptable salt or the compound and / or its pharmaceutically acceptable salt can vary within a wide range, and each specific ​In such cases, it should be necessarily adjusted according to the individual conditions and the pathogens to be controlled.

[0321] Additional Embodiments 1. A compound of formula I or a pharmaceutically acceptable salt thereof.

[0322] 2. A compound of formula II or a pharmaceutically acceptable salt thereof.

[0323] 3. A compound of formula III or a pharmaceutically acceptable salt thereof.

[0324] 4. A compound of formula IV or a pharmaceutically acceptable salt thereof.

[0325] 5. A compound having the structure in Table 1A.

[0326] 6. A bioreductive compound having the structure in Table 2.

[0327] 7. A compound having a pKa of 6 - 7 and being any one of the compounds of Embodiments 1 - 6 above.

[0328] 8. A lipidic nanoparticle composition comprising an ionizable lipid of formula I or a pharmaceutically acceptable salt thereof, and a nucleic acid.

[0329] 9. A lipidic nanoparticle composition comprising an ionizable lipid of formula II or a pharmaceutically acceptable salt thereof, and a nucleic acid.

[0330] 10. A lipidic nanoparticle composition comprising an ionizable lipid of formula III or a pharmaceutically acceptable salt thereof, and a nucleic acid.

[0331] 11. A lipidic nanoparticle composition comprising an ionizable lipid of formula IV or a pharmaceutically acceptable salt thereof, and a nucleic acid.

[0332] ​​​​​​12. A composition according to any one of embodiments 8 to 11 above, wherein the ionizable lipid encapsulates the nucleic acid. Two compositions.

[0333] 13. A composition according to any one of embodiments 8 to 11, wherein the nucleic acid is siRNA.

[0334] 14. A composition according to any one of embodiments 8 to 11, wherein the nucleic acid is DNA.

[0335] 15. A composition according to any one of embodiments 8 to 11, wherein the nucleic acid is mRNA.

[0336] 16. A composition according to any one of embodiments 8 to 11, further comprising a sterol, phosphatidylcholine, or a combination thereof. Two compositions.

[0337] 17. The composition of embodiment 16, wherein the sterol is cholesterol.

[0338] 18. The composition of embodiment 17, wherein the molar ratio of the ionizable lipid to cholesterol is from about 65:35 to about 40:60. Two compositions.

[0339] 19. The composition of embodiment 17, wherein the molar ratio of the ionizable lipid to cholesterol is from about 60:40 to about 45:55. Two compositions.

[0340] 20. The composition of embodiment 17, wherein the molar ratio of phosphatidylcholine to cholesterol is from about 1:5 to about 1:2. Two compositions.

[0341] 21. The composition of embodiment 17, further comprising a polymeric complex lipid.

[0342] 22. The polymeric complex lipid is PEG(2000)-dimyristoyl glycerol (PEG-DMG) or PEG (molecular weight 2,000)-dimyristoyl phosphatidylethanol EG-DMG) or PEG (molecular weight 2,000)-dimyristoyl phosphatidylethanol The composition of embodiment 21 containing noramine (PEG-DMPE).

[0343] 23. A composition according to any one of embodiments 8-11, further comprising a target ligand, wherein the target ligand is oriented on the outside of the nanoparticles.

[0344] 24. The composition of embodiment 23, wherein the target ligand is an antibody.

[0345] 25. A composition according to any one of embodiments 8-11, which is a liquid pharmaceutical formulation.

[0346] 26. The percentage of oxidative degradation products in the case of ionizable lipids is less than 50% of the oxidative degradation products in the case of the DLin-KC2-DMA or DLin-MC3-DMA control formulation. A composition according to any one of embodiments 8-11.

[0347] 27. A method comprising administering to a subject in need thereof an effective amount of a composition according to any one of embodiments 9-26 and a pharmaceutical excipient, wherein the administration induces an immune response, a method for preventing a bacterial or viral infection.

[0348] 28. The method of embodiment 27, wherein the composition is administered subcutaneously, intramuscularly, or intradermally.

[0349] 29. The method of embodiment 27, wherein the bacterial infection is a Mycobacterium tuberculosis infection.

[0350] 30. The method of embodiment 27, wherein the viral infection is a SARS-CoV, MERS-CoV or SARS-CoV-2 infection.

[0351] 31. The method of embodiment 27, wherein the viral infection is an HIV infection.

[0352] ​​​​​​32. The method of embodiment 27, wherein the infection is in a non-tuberculous form.

[0353] 33. An ionizable lipid nanoparticle (LNP) composition comprising a chemical structure consisting of a pair of 16 or 18 carbon linear polyunsaturated lipid tails covalently attached to a head group containing a dialkylamino group with a pKa of 6 - 7, wherein the head group contains a heterocyclyl or alkyl moiety covalently attached to the dialkylamino group and optionally further contains a phosphate group, and each polyunsaturated lipid tail is unsaturated except for at least two olefins separated by at least two methylene groups along the length of the lipid tail, and optionally, the composition contains a single acyl group at the terminus covalently attached to the head group.

[0354]

[0355] 34. The composition of embodiment 33, wherein each lipid tail is identical and each lipid tail has a total of two olefins separated only by unsubstituted ethylene, n-propyl, or n-butyl.

[0355] 35. The composition of embodiment 34, wherein each lipid tail further contains an acyl group that binds to the oxygen of the head group to form an ester.

[0356] 36. Each lipid tail is of formula A:

Chemical formula

Chemical formula

[0357] 37. The composition of embodiment 36, wherein b is 4.

[0358] 38. The ionizable lipid is

Chem.

Chem.

[0359] 39. The dialkylamino moiety of the head group has the formula (IV-A):

Chem.

[0360] 40. R in formula (IV-A) 10 and R 12 are each independently methyl, ethyl l, -(CH2)(CH2)OH or -(CH2)2(CH2)OH, embodiment 39 of the composition.

[0361] 41. The ionizable lipid has the formula (I-A): [Chemical formula] having the chemical structure of, wherein a is 1, 2, 3, 4, 5 or 6, b is 2, 3 or 4 and c is 3, 4, 5, 6 or 7, and the sum of a, b and c is 10 or 12 and R 10 and R 12 each independently is (C1-C4) alkyl optionally substituted with one or more hydroxy groups, and L is [Chemical formula] as defined in formula, wherein v is 0 or 1 and q2 is 1 or 2, the composition of embodiment 33 .

[0362] 42. The composition of claim 41, wherein v is 0.

[0363] 43. The composition of claim 41, wherein v is 1.

[0364] 44. The ionizable lipid has the formula II-A: [Chemical formula] having the chemical structure of, wherein a is 1, 2, 3, 4, 5 or 6, b is 2, 3 or 4 and c is 4, 5, 6, 7 or 8, and R2 is [Chemical formula] as defined in formula, wherein q and q' are each independently 1 or 2, and R 10 and R 12 are each independently (C1-C4) alkyl optionally substituted with hydroxyl, the composition of embodiment 33.

[0365] 45. The ionizable lipid has the formula II-A: [Chemical formula] having the chemical structure, wherein a is 5, 6 or 7, c is 3, 4 or 5, and R2 is [Chemical formula] where q and q' are each independently 1 or 2, and R 10 and R 12 are each, independently, (C1-C4) alkyl optionally substituted with hydroxyl, Example form 33 of the composition. [Example] [Examples]

[0366] The present disclosure has been described in connection with specific embodiments and has set forth numerous details for purposes of illustration but it will be apparent to those skilled in the art that the present disclosure includes additional embodiments and that some of the details described herein may vary significantly without departing from the present disclosure. The present disclosure includes such additional embodiments, modifications and equivalents. In particular, the present disclosure includes any combination of various exemplary components and features of the examples, terms, or elements. Unless otherwise expressly specified, the isomeric form of phosphatidylserine lipid used in the examples is phosphatidyl-L-serine.

[0367] Unless otherwise expressly specified, the isomeric form of phosphatidylserine lipid used in the examples is phosphatidyl-L-serine. is phosphatidyl-L-serine.

[0368] Specific examples are provided below to illustrate various embodiments of the embodiments disclosed herein. Those skilled in the art will appreciate that the various embodiments disclosed herein are not limited to these specific exemplary examples. examples.

[0369] [Example 1A] Synthesis of Ionizable Lipids Synthesis of Acid Intermediates for Schemes 1 AKG-UO-1 to AKG-UO-3

[0370] [Chemical formula]

[0371] The following acid intermediates (6Z,12Z)-6,12-octadecadienoic acid and (6Z, 12Z)-6,12-hexadecadienoic acid were prepared by general synthesis. (S)-4-(Dimethylamino)butane-1,2-diyl (6Z,6’Z,12Z,12 ’Z)-bis(octadeca-6,12-dienoate) (AKG-UO-1, O-119 56) (S)-4-(Diethylamino)butane-1,2-diyl (6Z,6’Z,12Z,12 ’Z)-bis(octadeca-6,12-dienoate) (AKG-UO-1A, O-11 955) (S)-4-(Dimethylamino)butane-1,2-diyl (6Z,6’Z,12Z,12 ’Z)-bis(hexadeca-6,12-dienoate, AKG-UO-4, O-1240 1) (S)-4-(Diethylamino)butane-1,2-diyl (6Z,6’Z,12Z,12 ’Z)-bis(hexadeca-6,12-dienoate, AKG-UO-4A, O-124 02) (S)-4-(Dimethylamino)butane-1,2-diyl (6Z,6’Z,11Z,11 ’Z)-bis(octadeca-6,11-dienoate) (AKG-UO-1a)

[0372] [Chemical formula] Experimental Procedure Synthesis of 2-((5-Bromopentyl)oxy)tetrahydro-2H-pyran 2

[0373]

Chem.

[0374]

Chem.

[0375]

Chem.

[0376]

Chemical Structure

[0377]

Chemical Structure

[0378]

Chem.

[0379]

Chem.

[0380]

Chemical formula

[0381]

Chemical formula

[0382]

Chemical formula

[0383]

Chemical formula

[0384] [Chemical formula] (S)-2-(2,2-Dimethyl-1,3-dioxolan-4-yl)ethyl 4-methyl benzenesulfonate 16 (10 g, 33.3 mmol) and dimethylamine solution 1 7 (166 mL, 333.3 mmol) (2 M in THF) was stirred at room temperature for 2 days The mixture was concentrated and the crude residue was diluted with CH2Cl2 (500 mL) and washed with saturated NaH It was washed with CO3, water and brine. The organic layer was dehydrated with anhydrous Na2SO4. The solvent was evaporated to give a crude residue which was purified by flash chromatography (SiO2: CH2Cl2 = 1% N MeOH in CH2Cl2 containing 100% to 10% of CH4OH), to give the colorless oily product 19 (2.1 g, 37%). 1H NMR (300 MHz, CDCl3): δ ppm 4.15 - 4.01 (m, 2H), 3.52 (dd, J = 7.4, 7.4 Hz, 1H ), 2.41 - 2.23 (m, 2H), 2.21 (s, 6H), 1.82 - 1.62 (m, 2H), 1.39 (s, 3H), 1.33 (s, 3H ). MS (APCI + ): 174.1 (M+1) (S)-2-(2,2-Diethyl-1,3-dioxolan-4-yl)-N,N-dimethyl ethan-1-amine 20

[0385]

Chemical formula

[0386]

Chemical formula

[0387]

Chemical formula

[0388] [Chemical formula] Oxalyl chloride (0.33 mL, 3.9 mmol) was added dropwise to a solution of (6Z,12Z)-octa deca-6,12-dienoic acid, 14 (0.36 g, 1.3 mmol) in dichloromethane / DMF (15 mL, 25 mL) at 0 °C, and the reaction mixture was warmed to room temperature and stirred for 1 hour. After 1 hour, the reaction mixture was concentrated to dryness under vacuum. The residue was redissolved in dichloromethane (10 mL), and N,N-diisopropylethylamine (2.3 mL, 10 mmol), 4- dimethylaminopyridine (317 mg, 2.6 mmol), and (S)-4-(dimethyl amino)butane-1,2-diol hydrochloride, 21 (101 mg, 0.6 mmol) were added to the mixture. The resulting solution was stirred for 24 hours. After 24 hours, the reaction mixture was cooled to 0 °C and quenched with water (10 mL). The reaction mixture was extracted with dichloromethane (2 × 100 mL), and the organic layer was washed with water and brine (2 × 100 mL). The organic layer was dried over magnesium sulfate, filtered, and the filtrate was concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 2% methanol in dichloromethane as the eluent, yielding (S)-4-(dimethylamino)butane-1,2-diyl (6Z,6’Z ,12Z,12’Z)-bis(octadeca-6,12-dienoate), AKG-UO- ,1 (0.12 g, 30%) as a yellow oil. 1H NMR (300 MHz, CDCl3): 5.40-5.29 (m, 8H), 5.14-5.12 (m, 1H), 4.25 (dd, J = 11 ​.8, 3.3 Hz, 1H), 4.05 (dd, J = 12.1, 6.3 Hz, 1H), 2.32 - 2.26 (m, 6H), 2.20 (s, 6H ), 2.06 - 1.99 (m, 16H), 1.78 - 1.70 (m, 2H), 1.65 - 1.58 (m, 4H), 1.42 - 1.25 (m, 24H), 0.90 - 0.85 (m, 6H). MS (APCI+): 658.5 (M+1) (S)-4-(Diethylamino)butane-1,2-diyl(6Z,6’Z,12Z,12 ’Z)-bis(octadeca-6,12-dienoate) AKG-UO-1A (O-119 55)

[0389]

Chem.

[0390] [Chemical] 1H NMR (300 MHz, CDCl3): 5.39 - 5.29 (m, 8H), 5.13 - 5.12 (m, 1H), 4.24 (dd, J = 11 .8, 3.3 Hz, 1H), 4.05 (dd, J = 11.8, 6.3 Hz, 1H), 2.32 - 2.27 (m, 6H), 2.19 (s, 6H ), 2.01 - 1.99 (m, 16H), 1.75 - 1.72 (m, 2H), 1.65 - 1.58 (m, 4H), 1.36 - 1.31 (m, 16H), 0.91 - 0.86 (m, 6H). MS (APCI+): 602.5 (M+1) (S)-4-(Diethylamino)butane-1,2-diyl (6Z,6’Z,12Z,12 ’Z)-bis(hexadec-6,12-dienoate) AKG-UO-4A(O-124 02) Synthesis

[0391] [Chemical] 1H NMR (300 MHz, CDCl3): 5.40 - 5.29 (m, 8H), 5.12 - 5.11 (m, 1H), 4.25 (dd, J = 11 .8, 3.3 Hz, 1H), 4.05 (dd, J = 11.8, 6.3 Hz, 1H), 2.54 - 2.43 (m, 6H), 2.29 (t, J = 7.4 Hz, 4H), 2.11 - 1.96 (m, 16H), 1.74 - 1.65 (m, 2H), 1.65 - 1.59 (m, 4H), 1.39 - 1. 31 (m, 16H), 0.99 (t, J = 7.1 Hz, 6H), 0.91 - 0.89 (m, 6H). MS(APCI+): 630.5 (M+1), i) 5-Bromopentanol and corresponding The first Wittig reaction of triphenylphosphonium ylide prepared from the aldehyde to be used , ii) conversion of terminal alcohol to bromide by mesylation and substitution, iii) repetition of the sequence of ylide formation and Wittig reaction, and finally iv) hyperiodic acid oxidation of the terminal alcohol. The resulting acid intermediate was utilized for the synthesis of AKG-UO-1 to AKG-UO-4 described below. Scheme 2 Synthesis of the acid intermediate for AKG-UO-5

[0392]

Chem.

[0393]

Chem.

[0394]

Chem.

[0395]

Chem.

[0396]

Chem.

[0397]

Chemical formula

[0398]

Chemical Structure

[0399]

Chem.

[0400]

Chem.

[0401]

Chem.

[0402]

Chemical formula

[0403]

Chemical formula

[0404]

Chem.

[0405]

Chem.

[0406]

Chem.

[0407]

Chemical Structure

[0408] [Chemical formula] 2-(Trideca-6,12-diyn-1-yloxy)tetrahydro-2H-pyran, 4 (7.14 g, 25.86 mmol) and hexamethylphosphoramide (18 mL, 103.4 mmol) in tetrahydrofuran (100 mL) was added dropwise with [n- butyllithium 2.5 M in n-hexane] (41.3 mL, 103.4 mmol) at -78 °C. When the addition was complete, the solution was stirred at -78 °C for 1 hour and then warmed to -20 °C for an additional 1 hour. The resulting solution was cooled to -78 °C again, and at that point 1-iodo propane 5 (9.9 mL, 103.4 mmol) in tetrahydrofuran (20 mL) was added. The resulting solution was warmed to room temperature and stirred for 12 hours. After 12 hours, the reaction was cooled to 0 °C and quenched with water (100 mL). The reaction mixture was then concentrated under Tetrahydrofuran was removed and then diluted with n-hexane. The organic matter was washed with water and brine (2 × 100 mL). The organic layer was dried over magnesium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 9 g of a crude oil. The crude oil was purified by chromatography on silica using 5% ethyl acetate in n-hex ane as the eluent to give 2- (hexadeca-6,12-diyn-1-yloxy)tetrahydro-2H-pyran, 7( 5.9 g, 72%) as a clear oil. 1H NMR (300 MHz, CDCl3): 4.57 - 4.55 (m, 1H), 3.86 - 3.74 (m, 1H), 3.73 - 3.71 (m, 1H) , 3.50 - 3.39 (m, 1H), 3.37 - 3.36 (m, 1H), 2.16 - 2.11 (m, 8H), 1.59 - 1.56 (m, 2H), 1. 55 - 1.47 (m, 16H), 0.98 - 0.93 (t, J = 1.6 Hz, 3H). 2-(octadeca-6,12-diyn-1-yloxy)tetrahydro-2H-pyran 8

[0409]

Chemical formula

[0410]

Chemical formula

[0411]

Chem.

[0412] [Chemical formula] 2-(((6Z,12Z)-Hexadeca-6,12-dien-1-yl)oxy)tet rahydro-2H-pyran, 9 (4.67 g, 14.5 mmol) in methanol (20 mL ) was added p-toluenesulfonic acid monohydrate (300 mg, 1.58 mmol) at room temperature. The resulting solution was stirred at room temperature for 3 hours and then quenched with water. The mixture was extracted with ethyl acetate (2 × 50 mL). The combined organics were washed with water and then dried over magnesium sulfate, filtered, and the filtrate was concentrated under vacuum to give a crude oil weighing 4 g. The crude oil was purified by chromatography on silica using 5-10% diethyl ether in n-hexane as the eluent to give (6Z,12Z)-hexadeca-6,12-dien-1-ol, 11 (2.5 g, 72%) as a clear oil. 1H NMR (300 MHz, CDCl3): 5.34-5.33 (m, 4H), 3.65-3.61 (m, 2H), 2.02-2.00 (m, 8H ), 1.36-1.34 (m, 2H), 1.34-1.25 (m, 10H), 0.89-0.86 (t, J = 0.82 Hz, 3H). (6Z,12Z)-Octadeca-6,12-dien-1-ol 12 1H NMR (300 MHz, CDCl3): 5.34-5.33 (m, 4H), 3.65-3.61 (m, 2H), 2.02-2.00 (m, 8H ), 1.36-1.34 (m, 2H), 1.34-1.25 (m, 10H), 0.89-0.86 (t, J = 0.82 Hz, 3H). (6Z,12Z)-Octadeca-6,12-dien-1-ol 12

[0413] [Chemical formula] 1H NMR (300 MHz, CDCl3): 5.36 - 5.33 (m, 4H), 3.65 - 3.61 (m, 2H), 2.02 - 2.01 (m, 8H ), 1.36 - 1.35 (m, 2H), 1.34 - 1.25 (m, 14H), 0.88 - 0.85 (t, J = 0.76 Hz, 3H). Representative procedure for the oxidation of alcohols to carboxylic acids using Jones reagent Synthesis of (6Z,12Z)-hexadeca-6,12-dienoic acid 13

[0414]

Chemical formula

[0415] [Chemistry] 1H NMR (300 MHz, CDCl3): 5.36 - 5.32 (m, 4H), 2.35 - 2.33 (t, 2H), 2.06 - 2.01 (m, 8H ), 1.64 - 1.42 (m, 2H), 1.34 - 1.28 (m, 12H), 0.90 - 0.85 (t, 3H). (S)-2-(2,2-Dimethyl-1,3-dioxolan-4-yl)ethyl 4-methyl Synthesis of benzenesulfonate 16

[0416] [Chemistry] (S)-2-(2,2-Dimethyl-1,3-dioxolan-4-yl)ethan-1-ol 15 (25 g, 171.1 mmol) in a mixture of pyridine (30 mL) was added with p- toluene sulfonyl chloride (35.8 g, 188.2 mmol) and DMAP (1 40 mg, 1.14 mmol) at 0 °C, and the reaction mixture was stirred overnight at room temperature. The mixture was diluted with CH2Cl2 (500 mL) and washed with saturated NH4Cl, water and brine. The organic layer was dried over anhydrous Na2SO4. The solvent was evaporated and the crude residue was used in the next step without purification. (43.8 g, 85%). 1H NMR (300 MHz, CDCl3): δ ppm 7.77 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2 H), 4.15 - 4.01 (m, 3H), 3.65 - 3.47 (m, 2H), 2.43 (s, 3H), 1.82 - 1.62 (m, 2H), 1.32 (s, 3H), 1.27 (s, 3H). Typical procedure for dialkylamine substitution (S)-2-(2,2-Dimethyl-1,3-dioxolan-4-yl)-N,N-dimethyl synthesis of ethan-1-amine 19

[0417]

Chem.

[0418]

Chem.

[0419]

Chemical Structure

[0420] [Chemical formula] 1H NMR (300 MHz, D2O): δ ppm 3.77 - 3.72 (m, 1H), 3.54 - 3.46 (m, 2H), 3.22 - 3.15 (m , 6H), 1.92 - 1.74 (m, 2H), 1.24 (t, J = 7.4 Hz, 6H). MS (APCI+): 162.1 (M+1) Typical procedure for diesterification (S)-4-(Dimethylamino)butane-1,2-diyl(6Z,6’Z,12Z,12 ’Z)-bis(octadeca-6,12-dienoate) AKG-UO-1(O-1195 6) synthesis

[0421] [Chemical formula] Oxalyl chloride (0.33 mL, 3.9 mmol) was added dropwise to a solution of (6Z,12Z)-oct tadeca-6,12-dienoic acid, 14 (0.36 g, 1.3 mmol) in dichloromethane / DMF (15 mL, 25 mL) at 0 °C, and the reaction mixture was warmed to room temperature and stirred for 1 hour . After 1 hour, the reaction mixture was concentrated to dryness under vacuum. The residue was redissolved in dichloromethane (10 mL) and added to a mixture of N,N-diisopropylethylamine (2.3 mL, 10 mmol), 4- dimethylaminopyridine (317 mg, 2.6 mmol) and (S)-4-(dimethyl amino)butane-1,2-diol hydrochloride, 21 (101 mg, 0.6 mmol). The resulting solution was stirred for 24 hours. After 24 hours, the reaction mixture was cooled to 0 °C and quenched with water (10 mL). The reaction mixture was extracted with dichloromethane (2 × 100 mL) , and the organic layer was washed with water and brine (2 × 100 mL). The organic layer was dried over magnesium sulfate It was dehydrated, filtered, and the filtrate was concentrated under vacuum to obtain a crude oily substance. The crude oily substance was chromatographed on silica using 2% methanol in dichloromethane as the eluent to give (S)-4-(dimethylamino)butane-1,2-diyl(6Z,6’Z ,12Z,12’Z)-bis(octadeca-6,12-dienoate), AKG-UO- ,12Z,12’Z)-bis(octadeca-6,12-dienoate), AKG-UO- 1 (0.12 g, 30%) as a yellow oily substance. 1H NMR (300 MHz, CDCl3): 5.40 - 5.29 (m, 8H), 5.14 - 5.12 (m, 1H), 4.25 (dd, J = 11 .8, 3.3 Hz, 1H), 4.05 (dd, J = 12.1, 6.3 Hz, 1H), 2.32 - 2.26 (m, 6H), 2.20 (s, 6H ), 2.06 - 1.99 (m, 16H), 1.78 - 1.70 (m, 2H), 1.65 - 1.58 (m, 4H), 1.42 - 1.25 (m, 24H), 0.90 - 0.85 (m, 6H). MS (APCI+): 658.5 (M+1) (S)-4-(diethylamino)butane-1,2-diyl(6Z,6’Z,12Z,12 ’Z)-bis(octadeca-6,12-dienoate) AKG-UO-1A(O-119 55)

[0422]

Chemical Structure

[0423]

Chem.

[0424]

Chem.

[0425] [Chemical Structure] Alternatively, the acid intermediate having two methylene groups between the double bond positions in the hydrocarbon chain is as described in Caballeira et al., Chem. Phys. Lipids, vol .100, p.33 - 40, 1999 or as described by D’yakon ov et al. (D’yakonov et al., Med. Chem. Res. , 2016, vol.25, p.30 - 39; D’yakonov et al., Ch em. Commun. 2013, vol.49, p 8401 - 8403; D’yako nov et al., 2020, Phytochem. Rev.) and synthesized as described therein.

[0426] [Example 1B] Synthesis of Ionizable Lipids ​(S)-4-(Dimethylamino)butane-1,2-diyl (6Z,6’Z,12Z,12 ’Z)-bis(octadeca-6,12-dienoate) (AKG-UO-1, O-119 56) (S)-4-(Diethylamino)butane-1,2-diyl (6Z,6’Z,12Z,12 ’Z)-bis(octadeca-6,12-dienoate) (AKG-UO-1A, O-11 955) (S)-4-(Dimethylamino)butane-1,2-diyl (6Z,6’Z,12Z,12 ’Z)-bis(hexadeca-6,12-dienoate, AKG-UO-4, O-1240 1) (S)-4-(Diethylamino)butane-1,2-diyl (6Z,6’Z,12Z,12 ’Z)-bis(hexadeca-6,12-dienoate, AKG-UO-4A, O-124 02) (S)-4-(Dimethylamino)butane-1,2-diyl (6Z,6’Z,11Z,11 ’Z)-bis(octadeca-6,11-dienoate) (AKG-UO-1a)

[0427]

Chem.

[0428]

Chem.

[0429]

Chemical Structure

[0430] Synthesis of 2-(hexadeca-6,12-diyn-1-yloxy)tetrahydro-2H-pyran 7

Chemical Structure

[0431]

Chemical Structure

[0432]

Chemical Structure

[0433]

Chemical Structure

[0434]

Chemical Structure

[0435]

Chemical Structure

[0436]

Chemical Structure

[0437]

Chemical formula

[0438]

Chemical formula

[0439]

Chemical formula

[0440]

Chem.

[0441]

Chem.

[0442]

Chemical formula

[0443]

Chemical formula

[0444]

Chem.

[0445]

Chem.

[0446]

Chem.

[0447] [Chemical Structure] Experimental Procedure Synthesis of 2-((5-Bromopentyl)oxy)tetrahydro-2H-pyran 2

[0448] [Chemical Structure] To a solution of 5-bromo-1-pentanol 1 (3.6 g, 21.6 mmol) in dichloromethane ( 100 mL) and pyridinium p-toluenesulfonate (40 mg, 0.16 mmol l), 3,4-dihydro-2H-pyran (6.54 mL, 71.8 mmol l) was added at 0 °C. The resulting solution was stirred at room temperature for 1 hour and then quenched with water. The mixture was extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with brine and then dried over magnesium sulfate, filtered, and the filtrate was concentrated under vacuum to afford a crude oil. The crude oil was purified by chromatography on silica using 5 - 10% ethyl acetate in n-hexane as the eluent to give 2-((5-bromopentyl)oxy)tetrahydro -2H-pyran, 2 (4.5 g, 83%) as a clear oil. 1 H NMR (300 MHz, CDCl3): δ ppm 4.55 - 4.54 (d, J = 4.3 Hz, 1H), 3.92 - 3.72 (m, 2H) , 3.42 - 3.38 (m, 3H), 1.88 - 1.55 (m, 3H), 1.52 - 1.50 (m, 10H). 2-(dodeca-6,11-diyn-1-yloxy)tetrahydro-2H-pyran 4a synthesis

[0449] [ka] 1,6-Heptadiyne 3a (5 g, 54.3 mmol) and hexamethylphosphorazine To a solution of 19 mL (108 mmol) of -7 [2.5 M n-butyllithium in n-hexane] (21.7 mL, 54.3 mm ol) was added dropwise. Upon completion of the addition, the solution was stirred at -78°C for 1 h and then The resulting solution was cooled again to -78°C at which point it was cooled to -20°C for an additional hour. 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran, 2 (6.8 g, 2 A solution of 7.1 mmol) in tetrahydrofuran (10 mL) was added. Warm to room temperature and stir for 12 h. After 12 h, the reaction is cooled to 0° C. and washed with water (100 mL). The reaction mixture was then concentrated in vacuo to remove tetrahydrofuran, followed by The organics were washed with water and brine (2 x 100 mL). The organic layer was dried over magnesium sulfate, filtered, and the filtrate was concentrated in vacuo to give a crude oil. The crude oil was purified by column chromatography using 5-10% ethyl acetate in n-hexane as the eluent. The product was purified by chromatography on a silica gel column to give 2-(dodeca-6,11-diyne-1- (yloxy)tetrahydro-2H-pyran, 4a (4.1 g, 58%) was obtained as a clear oil. I got it. 1 H NMR (300 MHz, CDCl3): δ ppm 4.57-4.56 (m, 1H), 3.96-3.82 (m, 1H), 3.77-3.69 (m, 1H), 3.50-3.41 (m, 1H), 3.39-3.34 (m, 1H), 2.29-2.25 (m, 4H), 2.15-2.12 (m, 2H), 1.95-1.94 (t, J = 5.8 Hz, 1H), 1.73-1.43 (m, 14H). 2-(Octadeca-6,11-diyn-1-yloxy)tetrahydro-2H-pyran 6 Synthesis of a

[0450] [ka] 2-(dodeca-6,11-diyn-1-yloxy)tetrahydro-2H-pyran, 4 a (4.1 g, 15.64 mmol) and hexamethylphosphoramide (11 mL, 6 A solution of 2.6 mmol) of [n-hexane] in tetrahydrofuran (100 mL) was added at -78°C. Add 2.5 M n-butyllithium in toluene (12.5 mL, 31.3 mmol) dropwise. Once the addition was complete, the solution was stirred at -78 °C for 1 h and then at -20 °C for an additional 1 h. The resulting solution was cooled again to -78°C, at which point 1-iodohexa A solution of 5a (9.5 mL, 62.6 mmol) in tetrahydrofuran (20 mL) was added. The resulting solution was allowed to warm to room temperature and stirred for 12 hours. After 12 hours, the reaction was cooled to 0°C. The reaction mixture was then concentrated in vacuo to give tetrahydrofuran. The furan was removed and then diluted with n-hexane. The organics were washed with water and brine (2x The organic layer was dried over magnesium sulfate, filtered, and the filtrate was concentrated under vacuum. Concentration gave a crude oil, which was purified with 5% ethyl acetate in n-hexane as eluent. The compound was purified by chromatography on silica gel using ethyl acetate to give 2-(octadeca-6 , 11 - diyn - 1 - yloxy)tetrahydro - 2H - pyran, 6a (3.1 g, 57 %) was obtained as a transparent oil. 1 H NMR (300 MHz, CDCl3): 4.58 - 4.55 (m, 1H), 3.86 - 3.82 (m, 1H), 3.77 - 3.69 (m, 1H) , 3.51 - 3.47 (m, 1H), 3.41 - 3.34 (m, 1H), 2.26 - 2.21 (m, 6H), 2.14 - 2.12 (m, 6H), 1. 66 - 1.26 (m, 18H), 0.93 - 0.85 (t, J = 6.5 Hz, 3H). 2 - (((6Z,11Z) - octadeca - 6,11 - diene - 1 - yl)oxy)tetra hydro - 2H - pyran 7a synthesis

[0451]

Chemical Structure

[0452]

Chemical formula

[0453]

Chemical formula

[0454]

Chem.

[0455] [Example 1C] Synthesis of KC-01 series of ionizable lipids 2-((S)-2,2-bis((6Z,12Z)-octadeca-6,12-dien-1-yl )-1,3-dioxolan-4-yl)-N,N-dimethylethane-1-amine(AK G-KC2-01, O-12095) synthesis 3-((S)-2,2-di((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine (A KG-KC3-01, O-12096)

[0456]

Chem.

[0457]

Chem.

[0458] [Chemical formula] (6Z,12Z)-1-bromooctadeca-6,12-diene, 2 (2 g, 6.08 m mol) in ether (10 mL) was added to a mixture of magnesium turnings (16 2 mg, 6.69 mmol) and iodine in ether (2 mL) under argon at room temperature. The mixture was stirred at room temperature for 90 minutes (the magnesium turnings were consumed), at which point ethyl formate (0.24 mL, 3.04 mmol) was added. After stirring at room temperature for 1 hour, the reaction was quenched with 1 N HCl solution. The mixture was extracted with ethyl acetate (2 × 100 mL), and the combined organic matter was washed with water and then brine. The organic matter was 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 m L) and added to a solution of potassium hydroxide (260 mg) in water (3 mL). For 12 hours After stirring, the pH of the mixture was adjusted to 4 with 2N HCl. The aqueous solution was extracted with dichloromethane ( 2×), combined. The organic matter was washed with brine and then dried over magnesium sulfate and filtered. The filtrate was concentrated under vacuum to obtain a crude oil. The crude oil was purified on silica using 10 - 30% ethyl acetate in n - hexane as the eluent to give (6Z, 12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraene- 19-ol, 3 (0.29 g, 0.55 mmol, 18%) as a clear oil. 19 - ol, 3 (0.29 g, 0.55 mmol, 18%) was obtained as a clear oil. 1 1H NMR (300 MHz, CDCl3): 5.36 - 5.32 (m, 8H), 3.57 (bs, 1H), 3.33 - 3.32, (m, 2H), 2.13 - 1.97 (m, 16H), 1.36 - 1.29 (m, 34H), 0.90 - 0.86 (t, J = 6.6 Hz, 6H). (6Z,12Z,25Z,31Z)-Heptatriaconta-6,12,25,31-tetraene- 19 - one, 4 synthesis

[0459]

Chemical formula

[0460] [Chemical formula] (6Z,12Z,25Z,31Z)-Heptatriaconta-6,12,25,31-tetraene-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 (9 mg) in toluene (10 mL) were heated under reflux under a positive pressure of nitrogen. After 12 h, the mixture was concentrated in vacuo to give a crude oil. The resulting crude oil was purified by chromatography on silica gel using 20 - 40% ethyl acetate in n-hexane as the eluent to give 2-( (S)-2,2-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)- (S)-2,2-bi...

Claims

1. A lipid nanoparticle (LNP) composition comprising an ionizable lipid having a chemical structure consisting of a pair of linear polyunsaturated lipid tails covalently attached to a head group, wherein the head group comprises a dialkylamino group having a pKa of 6-7, the head group comprises a heterocyclyl or alkyl moiety covalently attached to the dialkylamino group, and optionally further comprises a phosphate group, each polyunsaturated lipid tail is unsaturated except for at least two olefins separated by at least two methylene groups along the length of the lipid tail, and optionally, the composition comprises a single acyl group at the terminus of the lipid tail covalently attached to the head group.

2. The composition of claim 1, wherein each lipid tail is identical and each lipid tail has a total of two olefins separated only by unsubstituted ethylene, n-propyl, or n-butyl.

3. The composition of claim 2, wherein each lipid tail further comprises an acyl group that binds to the oxygen of the head group to form an ester, and the acyl group has a total of 16 or 18 carbon atoms.

4. a. The dialkylamino moiety of the head group has a chemical structure of formula (IV-A) [wherein, n in formula (IV-A) is 2, 3 or 4, the alkyl is optionally substituted with one or more hydroxyls], b. The ionizable lipid further comprises a chemical structure comprising an acyl group of each lipid tail covalently attached to a part of the head group distal to the dialkylamino moiety of formula (IV-A), wherein, each part of each lipid tail is shown, and has a chemical structure of formula A: wherein, in formula A, a is 4, 1, 2, or 3, b is 4, 2, or 3, 【Chemical 1】 c is 4, 3, 5, 6, or 7, provided that the sum of a, b and c in formula A is 12, 10, 11, or 13. R in formula (IV-A) １０ and R １２ are each independently methyl, ethyl, and selected from the group consisting of alkyl groups selected from propyl, R １０ and R １２ in The composition of claim 2.

5. The composition of... 【Chemical 2】

6. 【Chemical Formula 3】 shows the linkage to formula IV-A within the head group, R ２２ is covalently attached to the acyl group The composition of claim 5. 【Chemical Formula 4】

7. [Chemical Formula 5] represents the linkage between Formula A and R within each lipid tail ２２ and shows The composition of claim 1, wherein the ionizable lipid has a chemical structure of formula (I-A) [wherein, a is 4, 1, 2, 3, 5 or 6, b is 4, 3 or 2, c is 4, 3, 5, 6, or 7, the sum of a, b and c is 12 or 10, R in formula (IV-A) １０ and R １２ are each independently methyl, ethyl, -( CH ２ )(CH ２ )(OH, or -(CH ２ )) ２ (CH ２ )OH as described in claim 4 q is 1, 2, 3 or 4, L is b is 4 and R in formula (IV-A) １０ and R １２ are each methyl, claim where v is 0 or 1 and q2 is 2 or 1]. The composition of claim 1.

8. 【Chemical Formula 6】 ​ ​ ​ ​ ​ ​ R １０ and R １２ are each independently optionally substituted with one or more hydroxyl groups is replaced (C １ ~C ４ ) alkyl, and ​ [Chemical Formula 7] ​ ​ ​ The composition according to claim 7, wherein v is 0 and q is 1, 2 or 3.

9. The ionizable lipid is selected from the group consisting of AKG-UO-1, AKG-UO-1A, AKG-UO-1B, AKG-UO-2, AKG-UO-4, AKG-UO-4A, AKG-UO-5, AKG -UO-6, AKG-UO-7, AKG-UO-7, AKG-UO-8, AKG-UO- 9, and AKG-UO-10: 【Chemical Formula 8】 【Chem.】 The composition according to claim 8, selected from the group consisting of.

10. a. Nucleic acid, b. The ionizable lipid according to any one of claims 1 to 9, c. Sterol, d. One or more phospholipids including phosphatidylserine (PS) lipid, e. Optionally further comprising a complex lipid, The composition according to any one of compositions 1 to 9.

11. The composition according to claim 10, wherein the nucleic acid is mRNA.

12. The composition according to claim 11, wherein the sterol is cholesterol.

13. The one or more phospholipids are a. One or more phospholipids selected from the group consisting of DSPC, DPPC and DOPC, and b. PS lipids selected from the group consisting of DPPs, DSPS and DOPS The composition according to claim 12, consisting of.

14. The one or more phospholipids are a. DSPS, and b. One or more PS lipids selected from the group consisting of (L-serine) DPPs and (L-serine) DSPS The composition according to claim 13, consisting of.

15. The composition according to claim 13, comprising the PS lipid in a total amount of 2.5 to 10 mol% of the total lipids in the composition.

16. The composition according to claim 15, wherein the complex lipid contains PEG.

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

18.

19.

20.

21.

22.

23. The composition according to claim 17, wherein the nucleic acid is mRNA.

19. The composition according to claim 18, wherein the sterol is cholesterol.

20. The composition according to claim 19, wherein the one or more phospholipids consist of DSPC and L-serine PS.

21. The composition according to claim 20, comprising the PS in a total amount of 5.0 to 7.5 mol% of the total lipids in the composition.

22. The composition according to any one of claims 17 to 21, wherein the complex lipid contains PEG.

23. The composition according to claim 22, wherein the complex lipid is PEG-DMG.

24. The composition according to claim 23, wherein the LNP contains the complex lipid in a total amount of 0.5 to 1.5 mol% of the total lipid content of the LNP composition.

25. The composition according to claim 24, wherein the LNP contains the complex lipid in a total amount of less than 1 mol% of the total lipid content of the LNP composition.

26. a. The nucleic acid is mRNA, b. The total amount of the ionizable cationic lipid is 45 to 55 mol% of the total lipid content of the LNP composition, c. The sterol is cholesterol in a total amount of 35 to 45 mol% of the total lipid content of the LNP composition, d. The total amount of phospholipids is 7 to 15 mol% of the total lipid content of the LNP composition, e. The one or more phospholipids consist of DSPC, and the PS lipid is one or more lipids selected from the group consisting of DPPPS and the L-serine form of DSPS, f. The total amount of the PS lipid is 3 to 9 mol% of the total lipid content of the LNP composition.

27. The composition according to claim 26, containing the 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 of the LNP composition.

28. a. A nucleic acid that is mRNA, b. An ionizable cationic lipid in a total amount of 45 to 55 mol% of the total lipid content of the LNP composition, c. A sterol that is cholesterol in a total amount of 35 to 45 mol% of the total lipid content of the LNP composition, d. The total amount of phospholipids is 10 mol% of the total lipid content of the LNP composition, and the LNP composition contains phosphatidylserine (PS) in a total amount of 3 to 9 mol% of the total lipid content of the LNP composition. e. One or more phospholipids, f. The complex lipid in a total amount of 0.5 to 1.5 mol% of the total lipid content of the LNP composition. ​ ​ A nucleic acid-lipid nanoparticle (LNP) composition comprising

29. The PS lipid is selected from the group consisting of DSPS (L-isomer), DPPS (L-isomer), DMPPS (L-isomer ), DOPPS (L-isomer), DSPS (D-isomer), DSPG, DPPG, N-Glu- DSPE, and N-Suc-DSPE, according to any one of claims 17 to 28 The composition according to any one of the above.

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

31. The ionizable cationic lipid is selected from the group consisting of Compounds 1-28 (Table 1), Compounds 29-38 (Table 2) , AKG-UO-1, AKG-UO-1A, AKG-UO-1B, AKG-UO-1B, , AKG-UO-2, AKG-UO-3, AKG-UO-4, AKG-UO-4A, AKG -UO-5, AKG-BDG-01, AKG-BDG-02, AKG-UO-6, AKG -UO-7, AKG-UO-8, AKG-UO-9, and AKG-UO-10, according to any one of claims 17 to 28 The composition according to any one of the above.

32. The ionizable cationic lipid is selected from the group consisting of Compounds 1-3, 5-8, 9-12, 14-28 The composition according to any one of claims 17 to 28, which is one or more compounds selected from the group consisting of The composition according to any one of the above.

33. The ionizable cationic lipid is one or more compounds selected from the group consisting of Compounds 29-38 The composition according to any one of claims 17 to 28, which is one or more compounds selected from the group consisting of The composition according to any one of the above.

34. The ionizable cationic lipid is selected from the group consisting of KC2-OA, KC3-OA, Dlin-KC2-D MA, DlinKC3-DMA, KC2-PA, DODAP, AKG-OA-DM2, A KG-OA-DM3, O-11769, Dlin-MC3-DMA, ALC-0315 and SM-102, according to any one of claims 17 to 2 8 The composition according to any one of the above.

35. a. Nucleic acid, b. An ionizable cationic lipid in an amount of 50 mol% of the total lipid content of the LNP composition 、 c. Cholesterol in an amount of 38.5 mol% of the total lipid content of the LNP composition, d. One or more phospholipids in an amount of 7-15 mol% of the total lipid content of the LNP composition comprising phosphatidylserine (PS) lipid in a total amount of 3 to 9 mol% of the total lipid content of the LNP composition, one or more phospholipids, and e. PEG-containing lipid in a total amount of 0.5 to 1.5 mol% of the total lipid content of the LNP composition The nucleic acid lipid nanoparticle (LNP) composition according to claim 17, comprising lipid.

36. The composition according to claim 34, wherein the phospholipid consists of one or more phospholipids selected from the group consisting of DSPC, DPPC, and DOPC.

37. The composition according to claim 36, wherein the PS lipid is one or more L-serine phospholipids selected from the group consisting of DPPS and DSPS.

38. The composition according to claim 17, wherein the one or more phospholipids comprise at least two (L-serine) PS lipids having incompatible acyl chain lengths.

39. The composition according to claim 38, wherein the PS lipids are DPPC and DSPS.

40. The composition according to claim 39, wherein the DPPC and DSPS are each present in the LNP in a total amount of 5 mol% based on the total lipid content of the LNP composition.

41. A nucleic acid lipid nanoparticle (LNP) composition comprising a nucleic acid, an ionizable cationic lipid AKG-UO-1, and a (L-serine) PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition.

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

43. The composition according to claim 42, wherein the LNP composition further comprises PEG-DMG or PEG-DSG in an amount of 0.5 to 1.5 mol% based on the total lipid content of the LNP composition.

44. A nucleic acid lipid nanoparticle (LNP) composition comprising a nucleic acid, an ionizable cationic lipid selected from KC2OA, KC2, KC2-01, ALC0315, and SM102, and a (L-serine) PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition.

45. The composition according to claim 44, wherein the LNP composition has an N / P ratio of 3 to 8.

46. The composition according to claim 45, having an N / P ratio of 5 to 7.

47. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The composition according to claim 45, having an N / P ratio of 5.

48. The composition according to any one of claims 44 to 47, wherein the ionizable cationic lipid is KC2OA.

49. The composition according to any one of claims 44 to 47, wherein the ionizable cationic lipid is KC2.

50. The composition according to any one of claims 44 to 47, wherein the ionizable cationic lipid is KC2-01.

51. The composition according to any one of claims 44 to 47, wherein the ionizable cationic lipid is ALC0315.

52. The composition according to any one of claims 44 to 47, wherein the ionizable cationic lipid is SM102.

53. A nucleic acid, an ionizable cationic lipid selected from AKG-UO-6 and AKG-UO-7, and a (L-serine)PS lipid, in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition, a nucleic acid-lipid nanoparticle (LNP) composition.

54. The composition according to any one of claims 17 to 21, 26 to 28, 35 to 44, or 53, having an N / P ratio of 3 to 8.

55. The composition according to claim 54, having an N / P ratio of 5 to 7.

56. The composition according to claim 55, having an N / P ratio of 5.

57. The composition according to claim 55, having an N / P ratio of 7.

58. The composition according to any one of claims 17 to 21, 26 to 28, 35 to 47, or 53, wherein the nucleic acid is an mRNA encoding the SARS-CoV-2 spike protein.

59. a. An mRNA nucleic acid having an N / P ratio of 3 to 8, b. An ALC-0315 ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition, c. Cholesterol in a total amount of 25 to 40 mol% of the total lipid content of the LNP composition, d. A (L-serine)PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition, e. A DSPC phospholipid in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition, and f. A PEG-DMG in a total amount of 0 to 2.5 mol% of the total lipid content of the LNP composition, a nucleic acid-lipid nanoparticle (LNP) vaccine composition.

60. a. An mRNA nucleic acid having an N / P ratio of 3 to 8, b. A Dlin-KC2-DMA ionizable cationic lipid in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition, ​ c. Cholesterol in an amount of 25 to 40 mol% of the total lipid content of the LNP composition, d. (L-serine)PS lipid in an amount of 2.5 to 10 mol% of the total lipid content of the LNP composition PS lipid, e. DSPC phospholipid in an amount of 5 to 25 mol% of the total lipid content of the LNP composition, and f. PEG-DMG in an amount of 0 to 2.5 mol% of the total lipid content of the LNP composition A nucleic acid-lipid nanoparticle (LNP) vaccine composition comprising.

61. a. mRNA nucleic acid with an N / P ratio of 3 to 8, b. Ionized KC3-OA cationic lipid in an amount of 40 to 65 mol% of the total lipid content of the LNP composition Cationic lipid, c. Cholesterol in an amount of 25 to 40 mol% of the total lipid content of the LNP composition, d. (L-serine)PS lipid in an amount of 2.5 to 10 mol% of the total lipid content of the LNP composition PS lipid, e. DSPC phospholipid in an amount of 5 to 25 mol% of the total lipid content of the LNP composition, and f. PEG-DMG in an amount of 0 to 2.5 mol% of the total lipid content of the LNP composition A nucleic acid-lipid nanoparticle (LNP) vaccine composition comprising.

62. a. mRNA nucleic acid with an N / P ratio of 3 to 8, b. Ionized cationic lipid in an amount of 40 to 65 mol% of the total lipid content of the LNP composition Lipid, c. Cholesterol in an amount of 25 to 40 mol% of the total lipid content of the LNP composition, d. (L-serine)PS lipid in an amount of 2.5 to 10 mol% of the total lipid content of the LNP composition PS lipid, e. DSPC phospholipid in an amount of 5 to 25 mol% of the total lipid content of the LNP composition, and f. PEG-DMG in an amount of 0 to 2.5 mol% of the total lipid content of the LNP composition A nucleic acid-lipid nanoparticle (LNP) vaccine composition comprising.

63. The composition according to any one of claims 59 to 62, wherein the nucleic acid is the mRNA of SEQ ID NO: 2 Composition.

64. Use of (L-serine)PS lipid in an amount of 2.5 to 10 mol% of the total lipid content of the LNP composition in the LNP for targeting the LNP to dendritic cells %.

65. Use according to claim 64, wherein the LNP contains mRNA.

66. Use according to claim 64 or 65, wherein the LNP further contains cholesterol.

67. Use according to claim 66, wherein the LNP further contains an ionizable cationic lipid (ICL). Use.

68. The use according to claim 67, wherein the LNP further comprises one or more additional phospholipids including DSPC. **Claim 69** The use according to claim 68, wherein the LNP further comprises a complex lipid. **Claim 70** The LNP is a. mRNA nucleic acid with an N / P ratio of 3 to 8, b. Ionizable cationic lipid (ICL) in a total amount of 40 to 65 mol% of the total lipid content of the LNP composition , c. Cholesterol in a total amount of 25 to 40 mol% of the total lipid content of the LNP composition, d. (L-serine)PS lipid in a total amount of 2.5 to 10 mol% of the total lipid content of the LNP composition , e. DSPC phospholipid in a total amount of 5 to 25 mol% of the total lipid content of the LNP composition, and f. Complex lipid in a total amount of 0 to 2.5 mol% of the total lipid content of the LNP composition The use according to claim 69. **Claim 71** The use according to claim 70, wherein the ICL is selected from the compounds according to any one of claims 1 to 9. ​

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