Stabilized nucleic acid composition and its manufacturing, storage, and use methods

Abstract

This disclosure generally relates to the field of stabilized compositions comprising particles dispersed in an aqueous phase, wherein the aqueous phase comprises a buffer system and has a pH of about 4.0 to 5.5, and the particles comprise (i) nucleic acids (DNA or RNA, particularly mRNA or inhibitory RNA, e.g., siRNA); and (ii) cationic or cationic ionized lipids, and relates to methods for preparing and storing such compositions, as well as the use of such compositions in therapeutics.

Description

[Technical Field]

[0001] This disclosure generally relates to the field of stabilized compositions comprising particles dispersed in an aqueous phase, wherein the aqueous phase comprises a buffer system and has a pH of approximately 4.0 to 5.5, and the particles comprise (i) nucleic acids (DNA or RNA, particularly mRNA or inhibitory RNA, e.g., siRNA); and (ii) cationic or cationic ionized lipids. The disclosure also relates to methods for producing and storing such compositions, as well as the use of such compositions in therapeutic applications. [Background technology]

[0002] The use of recombinant nucleic acids (such as DNA or RNA) to deliver foreign genetic information to target cells is well known. Recombinant nucleic acids may be administered in their naked form to the target that needs them, but they are usually administered using compositions. For example, nucleic acids such as RNA can be delivered to the target using various delivery media, mainly cationic polymers or lipids that form nanoparticles together with the nucleic acid. Nanoparticles are intended to protect nucleic acids such as RNA from degradation, deliver RNA or other nucleic acids to the target site, and facilitate their uptake and processing by target cells. However, nucleic acids in solution or nanoparticles, especially RNA, are known to fragment slowly, and lipid-RNA formulations have limited liquid stability, which places a significant burden on the supply chain and storage. [Overview of the project] [Problems that the invention aims to solve]

[0003] Therefore, in the art, there is still a need for compositions and methods for introducing nucleic acids such as RNA into cells that avoid such drawbacks. Ideally, these compositions and methods should (i) be stable and can be stored in liquid form within a temperature range compliant with the normal techniques in pharmaceutical practice, particularly at temperatures of +2 to +8°C; (ii) be readily available; and / or (iii) the nucleic acids contained in the composition should be stable and not significantly degraded during storage. This disclosure addresses these and other needs. [Means for solving the problem]

[0004] The inventors have surprisingly discovered that the compositions and methods described herein satisfy the above requirements. In particular, it has been demonstrated that by adjusting the pH of the composition to a range of about 4.0 to 5.5, it is possible to prepare compositions that are stable (particularly with respect to the colloidal size of the particles contained in the composition and / or the chemical stability of the nucleic acids), can be stored in a liquid state, contain nucleic acids in a stable form, and maintain high biological efficacy.

[0005] overview In a first embodiment, the Disclosure provides a composition comprising particles dispersed in an aqueous phase, wherein the aqueous phase comprises a buffer system and has a pH of about 4.0 to 5.5, and the particles comprise (i) nucleic acids; and (ii) cationic or cationic ionized lipids.

[0006] As demonstrated in this application, the degradation of nucleic acids (particularly RNA, e.g., mRNA) contained in particles (such as lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), or mixtures thereof) formed from at least nucleic acids (particularly RNA, e.g., mRNA) and cationic or cationic ionized lipids (preferably formed from nucleic acids, cationic or cationic ionized lipids, steroids, and neutral lipids) can be prevented by adjusting the pH of the composition to a range of about 4.0 to 5.5. Thus, the composition according to the claims is stable, can be stored within a temperature range in accordance with the usual techniques in pharmaceutical practice, provides a readily available composition, and / or maintains high biological potency.

[0007] In some embodiments of the first embodiment, the pH of the aqueous phase is less than 5.5 and / or greater than 4.0. In some embodiments of the first embodiment, the pH of the aqueous phase is at least 4.0 to less than 5.5, for example, at least 4.1 to less than 5.5, at least 4.2 to less than 5.5, at least 4.3 to less than 5.5, at least 4.4 to less than 5.5, or at least 4.5 to less than 5.5, for example, at least 5.0 to less than 5.5. In some embodiments of the first embodiment, the pH of the aqueous phase is at least 4.0 to 5.4 or less, for example, at least 4.0 to 5.3 or less, at least 4.0 to 5.2 or less, at least 4.0 to 5.1 or less, at least 4.0 to 5.0 or less. In some embodiments of the first embodiment, the pH of the aqueous phase is at least 4.1 to 5.4, at least 4.2 to 5.3, at least 4.3 to 5.2, at least 4.4 to 5.1, or at least 4.5 to 5.0. In some embodiments of the first embodiment, the pH of the aqueous phase is at least 4.6 to 5.4, for example, at least 4.7 to 5.3, at least 4.8 to 5.2, at least 4.9 to 5.1, for example, about 5.0. In some embodiments of the first embodiment, the pH of the aqueous phase is about 4.0 to about 5.2. In some embodiments of the first embodiment, the pH of the aqueous phase is about 4.5 to about 5.2. In some embodiments of the first embodiment, the pH of the aqueous phase is about 4.8 to about 5.2. In the first embodiment, the pH of the aqueous phase is about 5.0 to about 5.2. In some embodiments of the first embodiment, the pH of the aqueous phase is approximately 4.0 to approximately 5.0, and optionally approximately 4.5 to 5.0. In some embodiments of the first embodiment, the pH of the aqueous phase is at least 4.5 to less than 5.5, and optionally at least 4.5 to less than 5.2. In some embodiments of the first embodiment, the pH of the aqueous phase is at least 5.0 to less than 5.5, for example, at least 5.1 to less than 5.5, at least 5.1 to 5.4 or less, at least 5.2 to 5.5 or less, or at least 5.2 to 5.4 or less. In some embodiments of the first embodiment, the pH of the aqueous phase is approximately 5.3.

[0008] In some embodiments of the first embodiment, the concentration of the buffer system in the aqueous phase is approximately 1 mM to approximately 50 mM. In some embodiments of the first embodiment, the concentration of the buffer system in the aqueous phase is approximately 2 mM to approximately 40 mM, for example, approximately 3 mM to approximately 30 mM, approximately 4 mM to approximately 25 mM, or approximately 5 mM to approximately 20 mM.

[0009] In some embodiments of the first aspect, the buffer system contains histidine or consists essentially of histidine.

[0010] In some embodiments of the first aspect, the buffer system includes HEPES or consists essentially of HEPES.

[0011] In some embodiments of the first aspect, the buffer system includes a combination of HEPES and histidine, or consists essentially of a combination of HEPES and histidine.

[0012] In some embodiments of the first aspect, the aqueous phase further comprises a chelating agent. Suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), salts of EDTA, desferrioxamine B, deferoxamine, sodium dithiocarb, penicillamine, calcium pentetate, sodium salts of pentetate, succimer, trientine, nitrilotriacetic acid, transdiaminocyclohexanetetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), and bis(aminoethyl) glycol ether-N,N,N',N'-tetraacetic acid. In certain embodiments, the chelating agent is EDTA or a salt of EDTA. In some embodiments, the concentration of the chelating agent in the aqueous phase is about 0.1 mM to about 20 mM, for example, about 0.2 mM to about 15 mM, about 0.3 mM to about 12 mM, about 0.4 mM to about 11 mM, or about 0.5 mM to about 10 mM. In some embodiments, the concentration of the chelating agent in the aqueous phase is about 0.5 mM to about 5 mM, for example, about 0.6 mM to about 4 mM, about 0.7 mM to about 3.5 mM, about 0.8 mM to about 3 mM, about 0.9 mM to about 2.5 mM, or about 1 mM to about 2 mM. In some embodiments, the concentration of the chelating agent in the aqueous phase is about 0.5 mM to about 1.5 mM, for example, about 1 mM.

[0013] In some embodiments of the first aspect, the buffer system comprises histidine, and the aqueous phase further comprises a chelating agent, such as EDTA. In some embodiments, the concentration of the chelating agent in the aqueous phase is about 0.1 mM to about 20 mM, for example, about 0.2 mM to about 15 mM, about 0.3 mM to about 12 mM, about 0.4 mM to about 11 mM, or about 0.5 mM to about 10 mM.

[0014] In some embodiments of the first aspect, the buffer system contains histidine, and the aqueous phase is substantially free of EDTA, preferably substantially free of any chelating agent.

[0015] In some embodiments of the first aspect, the buffer system comprises HEPES, and the aqueous phase further comprises a chelating agent such as EDTA. In some embodiments, the concentration of the chelating agent in the aqueous phase is about 0.1 mM to about 20 mM, for example, about 0.2 mM to about 15 mM, about 0.3 mM to about 12 mM, about 0.4 mM to about 11 mM, or about 0.5 mM to about 10 mM.

[0016] In some embodiments of the first aspect, the particles have a size of about 30 nM to about 500 nM. In some embodiments, the particles have a size of about 50 nM to about 150 nM.

[0017] In some embodiments of the first aspect, the particles comprise or are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and two or more combinations thereof. In some embodiments, the particles comprise or are LNPs (particularly when the composition comprises cationic ionized lipids). In some embodiments, the particles comprise or are liposomes. In some embodiments, the particles comprise or are LPXs (particularly when the composition comprises cationic lipids). In some embodiments, the particles comprise or are a mixture thereof of LNPs and liposomes. In some embodiments, the particles comprise or are a mixture thereof of LNPs and LPXs. In some embodiments, the particles comprise or are a mixture thereof of liposomes and LPXs. In some embodiments, the particles comprise or are a mixture thereof of LNPs, liposomes and LPXs.

[0018] In some embodiments of the first aspect, the particles comprise essentially all of the lipids present in the composition (in particular all cationic / cationically ionizable lipids, and, if present, all additional lipids (such as steroids and neutral lipids)).

[0019] In some embodiments of the first aspect, the aqueous phase is substantially free of cationic lipids / cationically ionizable lipids and, if present, additional lipids (such as steroids and neutral lipids), for example, the aqueous phase is substantially lipid-free.

[0020] In some embodiments of the first aspect, the particles contain at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%) of the nucleic acids (in particular, RNA) present in the composition. In some embodiments, the particles contain at least 75%, preferably at least 85%, of the nucleic acids (in particular, RNA) present in the composition.

[0021] In some embodiments of the first aspect, the aqueous phase is substantially free of nucleic acids.

[0022] In some embodiments of the first aspect, nucleic acids (e.g., RNA) are encapsulated within and / or bound to the particles.

[0023] In some embodiments of the first aspect, water is the main component of the composition, and / or the total amount of solvents other than water contained in the composition is less than about 1.0% (v / v), for example, less than about 0.5% (v / v). For example, the amount of water contained in the composition may be at least 50% (w / w), for example, at least 55% (w / w), at least 60% (w / w), at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), at least 90% (w / w), or at least 95% (w / w). In addition, or instead, the total amount of solvents other than water in the composition may be less than about 0.5% (v / v), for example, less than about 0.4% (v / v), less than about 0.3% (v / v), less than about 0.2% (v / v), less than about 0.1% (v / v), less than about 0.05% (v / v), less than about 0.01% (v / v), or less than about 0.005% (v / v). If the composition is substantially free of cryoprotectants, the amount of water in the composition may be at least 95% (w / w). In this regard, cryoprotectants that are liquid under normal conditions are considered cryoprotectants, not solvents other than water. In other words, any of the above limitations that the total amount of solvents other than water in the composition may be less than about 0.5% (v / v), for example, less than about 0.4% (v / v), does not apply to cryoprotectants that are liquid under normal conditions.

[0024] In some embodiments of the first embodiment, the aqueous phase further comprises one or more isotonic agents. In some embodiments, one or more isotonic agents are selected from the group consisting of salts and sugars. In some embodiments, one or more isotonic agents are salts such as sodium chloride. In some embodiments, one or more isotonic agents are sugars such as sucrose or glucose. In some embodiments, the concentration of one or more isotonic agents in the aqueous phase is such that the composition is at most isotonic, in particular, with respect to human blood. For example, the composition may contain sodium chloride at a concentration of at least about 154 mM.

[0025] In some embodiments of the first aspect, the concentration of nucleic acid (particularly RNA) in the composition is about 0.1 mg / l to about 500 mg / l. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is about 0.5 mg / l to about 400 mg / l. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is about 1 mg / l to about 300 mg / l, for example, about 2 mg / l to about 200 mg / l, about 3 mg / l to about 150 mg / l, or about 5 mg / l to about 100 mg / l.

[0026] In some embodiments of the first aspect, the N / P ratio is at least about 2. In some embodiments, the N / P ratio is about 2 to about 12, for example, about 4 to about 10, about 4 to about 8, or about 5 to about 7. In some embodiments, the N / P ratio is about 6.

[0027] In some embodiments of the first aspect, the composition is substantially free of cryoprotectants.

[0028] In some embodiments of the first aspect, the pH of the aqueous phase is at least about 4.0 to less than about 5.5 (e.g., about 4.0 to about 5.0, optionally about 4.5 to 5.0, or the pH is at least 5.0 to less than 5.5, e.g., at least 5.1 to less than 5.5, at least 5.1 to 5.4 or less, at least 5.2 to less than 5.5, or at least 5.2 to 5.4 or less, or the pH of the aqueous phase is about 5.3), and the aqueous phase contains histidine and optionally a chelating agent, preferably in the concentrations shown above or below.

[0029] In some embodiments of the first aspect, the pH of the aqueous phase is at least about 4.0 to less than about 5.5 (e.g., about 4.0 to about 5.0, optionally about 4.5 to 5.0, or the pH is at least 5.0 to less than 5.5, e.g., at least 5.1 to less than 5.5, at least 5.1 to 5.4 or less, at least 5.2 to less than 5.5, or at least 5.2 to 5.4 or less, or the pH of the aqueous phase is about 5.3), and the aqueous phase contains HEPES and optionally a chelating agent, preferably in the concentrations shown above or below.

[0030] In some embodiments of the first aspect, the cationic ionized lipid preferably comprises a head group having at least one tertiary amine moiety that can be protonated under physiological conditions.

[0031] In some embodiments of the first aspect, the cationic ionized lipid is of formula (X): [ka] (X) A compound having the structure shown in the formula, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein L 10 , L 20 G1, G 2 , G 3 , R 35 , R 36 , and R 37 The cationic ionized lipid is as defined herein. In some embodiments, the cationic ionized lipid is selected from: structures X-1 to X-36 (as shown herein); structures A to G (as shown herein); or N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), and 4-((di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutan-1-amine (DPL-14). In some embodiments, the cationic ionized lipid is a lipid having structure X-3. In some embodiments, the cationic ionized lipid is DPL-14 (i.e., a lipid having structure G).

[0032] In some embodiments of the first aspect, the cationic ionized lipid is of formula (XI): [ka] having the structure, wherein R1, R2, R3, R4, L2, G2, and m are as defined herein. In some embodiments, the cationic ionizable lipid is selected from structures (XIV-1), (XIV-2), and (XIV-3) (shown herein). In some embodiments, the cationic ionizable lipid is a lipid having structure XIV-1. In some embodiments, the cationic ionizable lipid is a lipid having structure XIV-2. In some embodiments, the cationic ionizable lipid is a lipid having structure XIV-3.

[0033] In some embodiments of the first aspect, the cationic ionizable lipid is of formula (TL-I):

Chemical formula

[0034] In some embodiments of the first aspect, the cationic ionizable lipid is of formula (AXL-I):

Chemical formula

[0035] In some embodiments of the first aspect, the cationic ionized lipid is completely or partially replaced by a cationic lipid. In some embodiments, the cationic lipid is selected from structures XV-1 to XV-6 (as shown herein).

[0036] In some embodiments of the first aspect, the cationic or cationic ionized lipid is 2,3-dioleyloxy-1-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)- This includes N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), DPL14, or mixtures thereof.

[0037] In some embodiments of the first aspect, cationic or cationically ionizable lipids include [(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-315); 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (D-Lin-MC3-D MA); 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA); Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319); Bis-(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)nonanamide)-nonadecanedioate (A9); (Heptadecan-9-yl8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5); Heptadecan-9-yl8-{ (2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate)(SM-102); O-[N-{(9Z,12Z)-octadeca-9,12-dien-1-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate(HY501); 2-(di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl4-(dimethylamino)butanoate(EA-2); 4-((di-((9Z,12Z)-octadeca-9,12-dien-1-yl) (HYAM-2); (2-(4-(dimethylamino)butanoyl)oxy)ethyl)azandiylbis(octane-8,1-diyl)bis(2-hexyldecanoate)(EA-405); (2-(4-(dimethylamino)butanoyl)oxy)azandiylbis(octane-8,1-diyl)bis(2-hexyldecanoate)(HY-405); palmitoyloleoylorarginine (PONA); guanidino di[(heptadecyl)methyl]carboxylic acid (GUADACA);Selected from the group consisting of 4-methylpyridinium di(heptadecyl)methylcarboxylic acid (MPDACA); 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP); 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA); and mixtures thereof.

[0038] In some embodiments of the first aspect, the cationic ionized lipid is 7,7'-((4-hydroxybutyl)azanejyl)bis(N-hexyl-N-octylheptane-1-sulfonamide)(BNT-51); 7,7'-((4-(3,3-dimethylthioureido)butyl)azanejyl)bis(N-hexyl-N-octylheptane-1-sulfonamide)(BNT-52); BNT-76; [(4-hydroxybutyl)azanejyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate)( ALC-315); 1,2-Dioleoyloxy-3-dimethylaminopropane (DODMA); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-Dioxolane (DLin-KC2-DMA); Heptatriaconta-6,9,28,31-Tetraen-19-yl-4-(dimethylamino)butanoic acid (D-Lin-MC3-DMA); 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA); Di((Z)-non-2-en-1-yl)-9-((4-(dimethylamino Butanoyl)oxy)heptadecanedioate (L319); bis-(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)nonanamide)-nonadecanedioate (A9); (heptadecan-9-yl8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5); heptadecan-9-yl8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102); O- [N-{(9Z,12Z)-octadeca-9,12-dien-1-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (HY501); 2-(di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl4-(dimethylamino)butanoate (EA-2); 4-((di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutan-4-amine (HYAM-2);((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azandiylbis(octane8,1-diyl)bis(2-hexyldecanoate)(EA-405); (2-(4-(dimethylamino)butanoyl)oxy)azandiylbis(octane8,1-diyl)bis(2-hexyldecanoate)(HY-405); di(heptadecan-9-yl)3,3'-((2-(4-methylpiperazine-1-yl)ethyl)azandiyl)dipropionate (BH D-C2C2-PipZ); Bis(2-octyldodecyl)3,3'-((2-(1-methylpyrrolidine-2-yl)ethyl)azandiyl)dipropionate(BODD-C2C2-1Me-Pyr); Bis(2-octyldodecyl)3,3'-((2-(pyrrolidine-1-yl)ethyl)azandiyl)dipropionate(BODD-C2C2-Pyr); Bis(2-octyldodecyl)3,3'-(((1-methylpiperidine-3-yl)methyl)azandiyl )dipropionate (BODD-C2C1-1Me-3PipD); bis(2-octyldodecyl)3,3'-(((1-methylpiperidine-4-yl)methyl)azandiyl)dipropionate (BODD-C2C1-1Me-PipD); bis(2-octyldodecyl)3,3'-((2-(dimethylamino)ethyl)azandiyl)dipropionate (BODD-C2C2-DMA); bis(2-octyldodecyl)3,3'-((4-(4-methylpiperidine Selected from the group consisting of (1-yl)butyl)azandiyl)dipropionate (BODD-C2C4-PipZ); bis(2-octyldodecyl)3,3'-((4-(pyrrolidine-1-yl)butyl)azandiyl)dipropionate (BODD-C2C4-Pyr); bis(2-hexyldecyl)3,3'-((4-(4-methylpiperazine-1-yl)butyl)azandiyl)dipropionate (BHD-C2C4-PipZ); and mixtures thereof.

[0039] In some embodiments of the first aspect, the cationic ionized lipid is 7,7'-((4-hydroxybutyl)azanejyl)bis(N-hexyl-N-octylheptane-1-sulfonamide)(BNT-51); 7,7'-((4-(3,3-dimethylthioureido)butyl)azanejyl)bis(N-hexyl-N-octylheptane-1-sulfonamide)(BNT-52); BNT-76; [(4-hydroxybutyl)azanejyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate)(AL C-315); 1,2-Dioleoyloxy-3-dimethylaminopropane (DODMA); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-Dioxolane (DLin-KC2-DMA); Heptatriaconta-6,9,28,31-Tetraen-19-yl-4-(dimethylamino)butanoic acid (D-Lin-MC3-DMA); 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA); Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutano (Il)oxy)heptadecanedioate (L319); bis-(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)nonanamide)-nonadecanedioate (A9); (heptadecan-9-yl8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5); heptadecan-9-yl8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102); O-[N-{( 9Z,12Z)-Octadeca-9,12-dien-1-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (HY501); ((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azandiylbis(octane-8,1-diyl)bis(2-hexyldecanoate) (EA-405); (2-(4-(dimethylamino)butanoyl)oxy)azandiylbis(octane-8,1-diyl)bis(2-hexyldecanoate) (HY-405);Di(heptadecan-9-yl)3,3'-((2-(4-methylpiperazine-1-yl)ethyl)azandiyl)dipropionate (BHD-C2C2-PipZ); Bis(2-octyldodecyl)3,3'-((2-(1-methylpyrrolidine-2-yl)ethyl)azandiyl)dipropionate (BODD-C2C2-1Me-Pyr); Bis(2-octyldodecyl)3,3' -((2-(pyrroridine-1-yl)ethyl)azandiyl)dipropionate (BODD-C2C2-Pyr); bis(2-octyldodecyl)3,3'-(((1-methylpiperidine-3-yl)methyl)azandiyl)dipropionate (BODD-C2C1-1Me-3PipD); bis(2-octyldodecyl)3,3'-(((1-methylpiperidine-4-yl)methyl) Azandiyl)dipropionate (BODD-C2C1-1Me-PipD); Bis(2-octyldodecyl)3,3'-((2-(dimethylamino)ethyl)azandiyl)dipropionate (BODD-C2C2-DMA); Bis(2-octyldodecyl)3,3'-((4-(4-methylpiperazin-1-yl)butyl)azandiyl)dipropionate (BODD-C2C4- Selected from the group consisting of PipZ; bis(2-octyldodecyl)3,3'-((4-(pyrrolidine-1-yl)butyl)azandiyl)dipropionate (BODD-C2C4-Pyr); bis(2-hexyldecyl)3,3'-((4-(4-methylpiperazin-1-yl)butyl)azandiyl)dipropionate (BHD-C2C4-PipZ); and mixtures thereof.

[0040] In some embodiments of the first aspect, the cationic ionized lipids constitute about 20 mol% to about 80 mol% of the total lipids present in the composition, for example, about 25 mol% to about 65 mol%, about 30 mol% to about 50 mol%, or about 40 mol% to about 50 mol%. In these embodiments, if the cationic ionized lipids are partially or completely replaced by cationic lipids, it is preferable that the same range for cationic ionized lipids as defined above (e.g., about 20 mol% to about 80 mol%) applies to the total of cationic ionized lipids and cationic lipids.

[0041] In some embodiments of the first aspect, the particles comprise a cationic ionizable lipid (particularly when the composition comprises lipid nanoparticles (LNPs)).

[0042] In some embodiments of the first aspect, the particles comprise a cationic lipid (particularly when the composition comprises lipoplexes (LPXs)).

[0043] In some embodiments of the first aspect, the particles further comprise one or more additional lipids. In some embodiments, the one or more additional lipids are selected from the group consisting of polymer-conjugated lipids, neutral lipids, steroids, and combinations thereof.

[0044] In some embodiments of the first aspect, the polymer-conjugated lipid comprises a pegylated lipid. In some embodiments, the pegylated lipid is selected from the group consisting of DSPE-PEG, DOPE-PEG, DPPE-PEG, and DMPE-PEG. In some embodiments, the pegylated lipid has the following structure: [Chemical formula] is a compound having, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R 12 , R 13 , and w are as defined herein.

[0045] In some embodiments of the first aspect, the polymer-conjugated lipid comprises a polysarcosine-lipid conjugate, or a conjugate of polysarcosine and a lipid-like substance. In some embodiments, the polysarcosine-lipid conjugate, or the conjugate of polysarcosine and a lipid-like substance, is a member selected from the group consisting of polysarcosine-diacylglycerol conjugates, polysarcosine-dialkyloxypropyl conjugates, polysarcosine-phospholipid conjugates, polysarcosine-ceramide conjugates, and mixtures thereof.

[0046] In some embodiments of the first aspect, the polymer-bound lipids constitute about 0.5 mol% to about 5 mol% of the total lipids present in the composition. In some embodiments, the polymer-bound lipids constitute about 1 mol% to about 5 mol%, for example, about 1 mol% to about 4.5 mol%, of the total lipids present in the composition.

[0047] In some embodiments of the first aspect, the neutral lipid is a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin. In some embodiments, the phospholipids are distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero- The neutral lipid is selected from the group consisting of 3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 lysoPC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), and diphytanoylphosphatidylethanolamine (DPyPE). In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, and DOPE. In some embodiments, the neutral lipid is DSPC or DOPC. In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutral lipid is DOPC. In some embodiments, the neutral lipid is DOPE.

[0048] In some embodiments of the first aspect, neutral lipids constitute about 5 mol% to about 40 mol% of the total lipids present in the composition, for example, about 5 mol% to about 20 mol% or about 5 mol% to about 15 mol%.

[0049] In some embodiments of the first embodiment, the steroid comprises sterols. In some preferred embodiments of the first embodiment, the steroid comprises cholesterol or is cholesterol.

[0050] In some embodiments of the first aspect, the steroid constitutes about 10 mol% to about 65 mol%, for example, about 20 mol% to about 60 mol%, or about 30 mol% to about 50 mol%, of the total lipids present in the composition.

[0051] In some embodiments of the first aspect, the particles include cationic or cationic ionized lipids, neutral lipids (e.g., phospholipids), and steroids, as well as optionally polymer-bound lipids. In some embodiments, the cationic or cationic ionized lipids constitute about 30 mol% to about 50 mol%, for example, about 40 mol% to about 50 mol%, of the total lipids present in the composition; the neutral lipids (e.g., phospholipids) constitute about 5 mol% to about 15 mol%, of the total lipids present in the composition; the steroids constitute about 30 mol% to about 50 mol%, of the total lipids present in the composition; and, if present, polymer-bound lipids constitute about 1 mol% to about 4.5 mol%, of the total lipids present in the composition.

[0052] In some embodiments of the first aspect, the composition comprises cationic lipids and cationic ionized lipids. In these embodiments, the sum of (1) the amount of cationic ionized lipids and (2) the amount of cationic lipids is used in the calculation. For example, if the amount of cationic ionized lipids in the composition is about 20 mol% to about 80 mol%, and the composition also contains cationic lipids, then the sum of (1) the amount of cationic ionized lipids and (2) the amount of cationic lipids is about 20 mol% to about 80 mol%.

[0053] In some embodiments of the first aspect, the lipids contained in the composition are cationic ionic lipids, steroids, and neutral lipids, and optionally polymer-bound lipids, in particular cationic ionic lipids, steroids, and phospholipids, and optionally polymer-bound lipids.

[0054] In some embodiments of the first aspect, the composition comprises a cationic ionized lipid, a neutral lipid (e.g., phospholipid), a steroid, histidine, and optionally (a) a polymer-bound lipid and / or (b) a chelating agent (e.g., EDTA), wherein the pH of the aqueous phase is at least 4.0 to less than 5.5 (e.g., between 4.0 and 5.0, or the pH is at least 5.0 to less than 5.5, e.g., at least 5.1 to less than 5.5, at least 5.1 to 5.4 or less, at least 5.2 to 5.5 or less, or at least 5.2 to 5.4 or less, or the pH of the aqueous phase is about 5.3). In some embodiments, these compositions are (1) further comprising neither polymer-bound lipids nor chelating agents; (2) comprising a chelating agent; (3) polymer-bound lipids; or (4) a chelating agent and polymer-bound lipids. In some embodiments of these compositions, the neutral lipid is selected from the group consisting of DSPC, DOPC, and DOPE (preferably the neutral lipid is DOPE), and / or the steroid is cholesterol.

[0055] In some embodiments of the first aspect, the composition comprises a cationic ionized lipid, a neutral lipid (e.g., phospholipid), a steroid, HEPES, and optionally (a) a polymer-bound lipid and / or (b) a chelating agent (e.g., EDTA), wherein the pH of the aqueous phase is at least 4.0 to less than 5.5 (e.g., between 4.0 and 5.0, or the pH is at least 5.0 to less than 5.5, e.g., at least 5.1 to less than 5.5, at least 5.1 to 5.4 or less, at least 5.2 to 5.5 or less, or at least 5.2 to 5.4 or less, or the pH of the aqueous phase is about 5.3). In some embodiments, these compositions are (1) further comprising neither polymer-bound lipids nor chelating agents; (2) comprising a chelating agent; (3) polymer-bound lipids; or (4) a chelating agent and polymer-bound lipids. In some embodiments of these compositions, the neutral lipid is selected from the group consisting of DSPC, DOPC, and DOPE (preferably the neutral lipid is DOPE), and / or the steroid is cholesterol.

[0056] In some embodiments of the first aspect, the nucleic acid is DNA.

[0057] In some embodiments of the first aspect, the nucleic acid is RNA, preferably mRNA, inhibitory RNA (e.g., siRNA), or self-replicating RNA.

[0058] In some embodiments of the first aspect, the nucleic acid is RNA (such as mRNA) and (i) comprises a modified nucleoside instead of uridine, (ii) has a codon-optimized coding sequence, and / or (iii) has a coding sequence with increased G / C content compared to a wild-type coding sequence. In some embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0059] In some embodiments of the first aspect, the nucleic acid is RNA (such as mRNA) and comprises at least one of a 5' cap, a 5' UTR, a 3' UTR, and a poly-A sequence. In some embodiments, the RNA (such as mRNA) comprises all of the 5' cap, 5' UTR, 3' UTR, and poly-A sequence. In some embodiments, the poly-A sequence comprises at least 100 A nucleotides, and the poly-A sequence is preferably a suspended sequence of A nucleotides. In some embodiments, the 5' cap is a cap 1 or cap 2 structure.

[0060] In some embodiments of the first aspect, the nucleic acid is RNA (such as mRNA) and encodes one or more polypeptides. In some embodiments, one or more polypeptides are pharmaceutically active peptides or polypeptides and / or contain epitopes for inducing an immune response to an antigen in a subject.

[0061] In some embodiments of the first aspect, the pharmaceutically active polypeptide and / or antigen or epitope is derived from or is derived from a pathogen protein, an immunogenic variant of that protein, or an immunogenic fragment of the protein or its immunogenic variant. In some embodiments, the pathogen is a pathogen that causes an infectious disease.

[0062] In some embodiments of the first aspect, the nucleic acid is an inhibitory RNA (such as siRNA) that selectively hybridizes to and / or is specific to the target mRNA. In some embodiments, the target mRNA includes an ORF encoding a pharmaceutically active peptide or polypeptide, in particular an ORF whose expression (in particular an increased expression compared to, for example, expression in a healthy subject) is associated with a disease. In some embodiments, the target mRNA includes an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (in particular an increased expression compared to, for example, expression in a healthy subject) is associated with cancer.

[0063] In some embodiments of the first aspect, the composition is preferably in a liquid state at a temperature of about 0°C to about 10°C, for example, about 2°C to about 8°C.

[0064] In some embodiments of the first aspect, the nucleic acid integrity (e.g., RNA integrity) of the composition after storage for at least one week, preferably at a temperature of 0°C or higher, for example, about 2°C to about 8°C, is sufficient to achieve a desired effect, such as inducing an immune response. In some embodiments, the nucleic acid integrity (e.g., RNA integrity) of the composition after storage for at least one week (e.g., at least two weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least four months, at least six months, at least nine months, or at least twelve months), preferably at a temperature of 0°C or higher, for example, about 2°C to about 8°C, is at least 50%, optionally at least 80%, preferably at least 90%, compared to the nucleic acid integrity before storage. In some embodiments, the nucleic acid integrity (e.g., RNA integrity) of the composition after storage for at least 4 weeks, preferably at a temperature of 0°C or higher, for example, about 2°C to about 8°C, is at least 50%, optionally at least 80%, and preferably at least 90%, compared to the nucleic acid integrity before storage. In some embodiments, the nucleic acid integrity (e.g., RNA integrity) of the composition after storage for at least 3 months, preferably at a temperature of 0°C or higher, for example, about 2°C to about 8°C, is at least 50%, optionally at least 80%, and preferably at least 90%, compared to the nucleic acid integrity before storage.

[0065] In a second embodiment, the present invention provides a method for delivering nucleic acids to target cells, the method comprising administering the target a composition of the first embodiment. Any embodiment described herein in relation to the first embodiment is understood to be applicable to any embodiment of the second embodiment.

[0066] In a third aspect, the present invention provides a method for delivering a therapeutic peptide or polypeptide, the method comprising administering to a subject a composition of the first aspect, wherein the nucleic acid encodes a therapeutic peptide or polypeptide. It is understood that any embodiment described herein in the context of the first or second aspect is also applicable to any embodiment of the third aspect.

[0067] In a fourth aspect, the present disclosure provides a method for treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a composition of the first aspect, wherein delivering the nucleic acid to the cells of the subject is beneficial for treating or preventing the disease or disorder. In a related aspect, the present disclosure relates to a composition of the first aspect for use in a method for treating or preventing a disease or disorder in a subject, wherein delivering the nucleic acid to the cells of the subject is beneficial for treating or preventing the disease or disorder. It is understood that any embodiment described herein in connection with the first, second, or third aspect is also applicable to any embodiment of the fourth aspect.

[0068] In a fifth aspect, the present disclosure provides a method for treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a composition of the first aspect, wherein the nucleic acid encodes a therapeutic peptide or polypeptide, and wherein delivering the therapeutic peptide or polypeptide to the subject is beneficial for treating or preventing the disease or disorder. In a related aspect, the present disclosure relates to a composition of the first aspect for use in a method for treating or preventing a disease or disorder in a subject, wherein the nucleic acid encodes a therapeutic peptide or polypeptide, and wherein delivering the therapeutic peptide or polypeptide to the subject is beneficial for treating or preventing the disease or disorder. It is understood that any embodiment described herein in the context of the first, second, third, or fourth aspect is also applicable to any embodiment of the fifth aspect.

[0069] In some embodiments of the second to fifth aspects, the subject is a mammal such as a human.

[0070] In a sixth aspect, the present invention provides a composition of the first aspect for use in therapeutic purposes. It will be understood that any embodiment described herein in relation to the first, second, third, fourth, or fifth aspects may also be applicable to any embodiment of the sixth aspect.

[0071] In a seventh embodiment, the Disclosure provides a method for transfecting cells, comprising adding a composition of the first embodiment to cells; and incubating the mixture of the composition and cells for a sufficient amount of time. In particular, in some embodiments where the nucleic acid is DNA or RNA (such as mRNA) and encodes a pharmaceutically active peptide or polypeptide, the mixture of the composition and cells is incubated for a sufficient amount of time to allow expression of the pharmaceutically active peptide or polypeptide. In particular, in some embodiments where the nucleic acid is inhibitory RNA (such as siRNA) against a target mRNA, the mixture of the composition and cells is incubated for a sufficient amount of time to allow inhibition of transcription and / or translation of the target mRNA. In some embodiments, a sufficient amount of time is at least 1 hour (e.g., at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours) and / or up to about 48 hours (e.g., up to about 36 hours or up to about 24 hours).

[0072] In some embodiments of the seventh aspect, the method is performed in vivo (i.e., the cells form part of the organ, tissue and / or organism of interest). In some embodiments of the seventh aspect, the method is performed in vitro (i.e., the cells do not form part of the organ, tissue and / or organism of interest; for example, the cells are an ex vivo cell culture).

[0073] It is understood that any embodiment described herein in relation to the first, second, third, fourth, fifth, or sixth aspect may also be applicable to any embodiment of the seventh aspect.

[0074] In an eighth aspect, the present invention provides a pharmaceutical composition comprising the composition of the first aspect. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and excipients. It is understood that any embodiment described herein in relation to the first, second, third, fourth, fifth, sixth, or seventh aspects may also be applicable to any embodiment of the eighth aspect.

[0075] In a ninth aspect, the present invention provides a kit comprising a composition of the first aspect or a pharmaceutical composition described herein (for example, a pharmaceutical composition of the eighth aspect). In some embodiments, the kit is intended for therapeutic use, such as to induce an immune response. In some embodiments, the kit is intended for use to induce an immune response to a pathogen, such as to treat or prevent an infection. It is understood that any embodiment described herein in relation to the first, second, third, fourth, fifth, sixth, seventh, or eighth aspects may also be applicable to any embodiment of the ninth aspect.

[0076] In a tenth embodiment, the present disclosure provides a method for preparing a composition comprising particles dispersed in a final aqueous phase, wherein the final aqueous phase comprises a final buffer system and has a pH of about 4.0 to 5.5, and the particles comprise (i) at least a portion of nucleic acids and (ii) at least a portion of cationic lipids or cationic ionized lipids, wherein the method comprises: (a) To provide (e.g., prepare) a nucleic acid solution containing water and a first buffer system; (b) To provide (e.g., prepare) an organic solution (e.g., an ethanol solution) containing cationic or cationic ionized lipids and, if present, one or more additional lipids; (c) Mixing the nucleic acid solution provided in (a) and the organic solution provided in (b) to prepare a (first) intermediate formulation containing particles dispersed in a (first) aqueous phase containing a first buffer system; and (d) The (first) intermediate formulation prepared in (c) is filtered (e.g., by dialysis, tangential flow filtration, or diafiltration) and / or diluted using a final aqueous buffer containing the final buffer system, thereby preparing a composition containing particles dispersed in the final aqueous phase.

[0077] In some embodiments of the tenth aspect, the method includes the following steps: (a') Providing (e.g., preparing) an aqueous solution of nucleic acid; (b') To provide (e.g., prepare) a first buffer aqueous solution containing the first buffer system; (c') Mix the nucleic acid aqueous solution provided in (a') with the first buffer aqueous solution provided in (b') to prepare a nucleic acid aqueous solution containing water and the first buffer system; (d') Prepare an organic solution (e.g., an ethanol solution) containing cationic or cationic ionized lipids, and, if present, one or more additional lipids; (e')(c') is mixed with the organic solution provided in (d') to prepare a first intermediate formulation containing particles dispersed in a first aqueous phase including a first buffer system; (f') Optionally, the first intermediate formulation prepared in (e') is filtered using a further aqueous buffer containing a further buffer system, thereby preparing a further intermediate formulation containing particles dispersed in a further aqueous phase containing a further buffer system, where the further aqueous buffer may be the same as or different from the first aqueous buffer; (g') Optionally, repeat step (f') one or more times, wherein the further intermediate formulation obtained after step (f') of one cycle, which contains particles dispersed in a further aqueous phase containing a further buffer system, is used as the first intermediate formulation of the next cycle, wherein in each cycle, the further aqueous buffer solution may be the same as or different from the first aqueous buffer solution; (h') If step (f') is absent, filter the first intermediate formulation obtained in step (e') using the final aqueous buffer containing the final buffer system; or, if step (f') is present but step (g') is absent, filter the further intermediate formulation obtained in step (f') using the final aqueous buffer containing the final buffer system; and (i') If necessary, dilute the formulation obtained in step (h') with the dilution solution; This prepares a composition containing particles dispersed in the final aqueous phase.

[0078] In some embodiments of the 10th aspect, the pH of the final aqueous phase is less than 5.5 and / or greater than 4.0. In some embodiments of the 10th aspect, the pH of the final aqueous phase is at least 4.0 to less than 5.5, for example, at least 4.1 to less than 5.5, at least 4.2 to less than 5.5, at least 4.3 to less than 5.5, at least 4.4 to less than 5.5, or at least 4.5 to less than 5.5, for example, at least 5.0 to less than 5.5. In some embodiments of the 10th aspect, the pH of the final aqueous phase is at least 4.0 to 5.4 or less, for example, at least 4.0 to 5.3 or less, at least 4.0 to 5.2 or less, at least 4.0 to 5.1 or less, at least 4.0 to 5.0 or less. In some embodiments of the 10th aspect, the pH of the final aqueous phase is at least 4.1 to 5.4, at least 4.2 to 5.3, at least 4.3 to 5.2, at least 4.4 to 5.1, or at least 4.5 to 5.0. In some embodiments of the 10th aspect, the pH of the final aqueous phase is at least 4.6 to 5.4, for example, at least 4.7 to 5.3, at least 4.8 to 5.2, at least 4.9 to 5.1, for example, about 5.0. In some embodiments of the 10th aspect, the pH of the aqueous phase is about 4.0 to about 5.2. In some embodiments of the 10th aspect, the pH of the aqueous phase is about 4.5 to about 5.2. In some embodiments of the 10th aspect, the pH of the aqueous phase is about 4.8 to about 5.2. In some embodiments of the 10th aspect, the pH of the aqueous phase is about 5.0 to about 5.2. In some embodiments of the 10th embodiment, the pH of the aqueous phase is at least 5.0 to less than 5.5. In some embodiments of the 10th embodiment, the pH of the aqueous phase is about 4.5 to 5.0. In some embodiments, the pH of the aqueous phase is at least 4.5 to less than 5.5, and optionally at least 4.5 to less than 5.2. In some embodiments of the 10th embodiment, the pH of the aqueous phase is at least 5.0 to less than 5.5, for example, at least 5.1 to less than 5.5, at least 5.1 to 5.4 or less, at least 5.2 to less than 5.5, or at least 5.2 to 5.4 or less. In some embodiments of the 10th embodiment, the pH of the aqueous phase is about 5.3.

[0079] Therefore, the nucleic acid (such as RNA) solution provided / obtained in step (a) or (c') may further contain one or more acids (e.g., inorganic acids (such as hydrochloric acid, hydrobromic acid, nitric acid) and organic acids (single-base, dibasic or polybasic organic acids, e.g., selected from monocarboxylic acids (such as acetic acid, propionic acid, lactic acid), dicarboxylic acids (such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, tartaric acid, malic acid), or polycarboxylic acids (such as citric acid, isocitric acid, trimesic acid)). In some embodiments, the acid is a monobasic acid, for example, a monobasic inorganic acid (such as hydrochloric acid) or a monobasic organic acid (such as acetic acid). In some embodiments, step (d) is carried out under conditions that remove one or more unwanted substances (e.g., an organic solvent (such as ethanol) and / or one or more acids) to obtain a composition containing particles dispersed in a final aqueous phase, the final aqueous phase substantially free of such one or more unwanted substances. For example, under such conditions, the intermediate formulation containing particles dispersed in a second intermediate aqueous phase obtained in step (C) , which may include subjecting the final buffer, which contains a final buffer system (i.e., final buffer substance), to at least one filtration step such as dialysis, tangential flow filtration, or diafiltration, wherein the final buffer system is free of one or more unwanted substances. Alternatively, such conditions include (1) subjecting the intermediate formulation (i.e., first intermediate formulation), which contains particles dispersed in a first intermediate aqueous phase obtained in step (c), to at least one step of diluting it with water or a further aqueous buffer containing a further buffer system, thereby preparing a further intermediate formulation, which contains particles dispersed in a further aqueous phase containing a first or further buffer system, wherein the further buffer system of the further aqueous buffer may be the same as or different from the buffer system used in step (a); and (2) subjecting the further intermediate formulation obtained in step (1), which contains the final aqueous buffer, to at least one filtration step such as dialysis, tangential flow filtration, or diafiltration, wherein at least the final aqueous buffer (preferably the intermediate aqueous buffer and the final aqueous buffer) is free of one or more unwanted substances.

[0080] Similarly, in some embodiments of the tenth embodiment, if the method comprises steps (a') to (e') and (h') (and optionally one or more of steps (f'), (g'), and (i')), the first aqueous buffer (and first aqueous phase) provided in (b') may further comprise one or more acids (e.g., one or more dibasic or polybasic acids). In these embodiments, at least one of steps (f') to (h') is carried out under conditions that remove one or more unwanted substances (e.g., an organic solvent (such as ethanol) and / or one or more dibasic or polybasic acids) from the first intermediate formulation and / or the second intermediate formulation and / or further intermediate formulation, thereby obtaining a further intermediate formulation comprising particles dispersed in a further aqueous phase or final aqueous phase, wherein the further aqueous phase and / or final aqueous phase preferably substantially contains one or more unwanted substances. For example, such conditions may include the use of additional aqueous buffers and / or final buffers, where at least one of the additional aqueous buffers and final buffers (preferably all of the additional aqueous buffers and final buffers) does not contain one or more unwanted substances. In some embodiments, the filtration step may be selected independently from dialysis, tangential flow filtration, and diafiltration, preferably selected from dialysis and tangential flow filtration.

[0081] In some embodiments of the tenth aspect, the concentration of the final buffer system in the final aqueous phase is approximately 1 mM to approximately 50 mM. In some embodiments of the tenth aspect, the concentration of the final buffer system in the final aqueous phase is approximately 2 mM to approximately 40 mM, for example, approximately 3 mM to approximately 30 mM, approximately 4 mM to approximately 25 mM, or approximately 5 mM to approximately 20 mM.

[0082] In some embodiments of the tenth aspect, the final buffer system contains histidine or consists essentially of histidine.

[0083] In some embodiments of the tenth aspect, the final buffer system includes HEPES or consists essentially of HEPES.

[0084] In some embodiments of the tenth aspect, the final buffer system includes a combination of HEPES and histidine, or essentially consists of a combination of HEPES and histidine.

[0085] In some embodiments of the tenth aspect, the final aqueous phase further comprises a chelating agent such as EDTA. In some embodiments, the concentration of the chelating agent in the final aqueous phase is about 0.1 mM to about 20 mM, for example, about 0.2 mM to about 15 mM, about 0.3 mM to about 12 mM, about 0.4 mM to about 11 mM, or about 0.5 mM to about 10 mM.

[0086] In some embodiments of the tenth aspect, the final buffer system comprises histidine, and the final aqueous phase further comprises a chelating agent, such as EDTA. In some embodiments, the concentration of the chelating agent in the final aqueous phase is about 0.1 mM to about 20 mM, for example, about 0.2 mM to about 15 mM, about 0.3 mM to about 12 mM, about 0.4 mM to about 11 mM, or about 0.5 mM to about 10 mM.

[0087] In some embodiments of the tenth aspect, the final buffer system contains histidine, and the final aqueous phase is substantially free of EDTA, preferably substantially free of any chelating agent.

[0088] In some embodiments of the tenth aspect, the final buffer system comprises HEPES, and the final aqueous phase further comprises a chelating agent such as EDTA. In some embodiments, the concentration of the chelating agent in the final aqueous phase is about 0.1 mM to about 20 mM, for example, about 0.2 mM to about 15 mM, about 0.3 mM to about 12 mM, about 0.4 mM to about 11 mM, or about 0.5 mM to about 10 mM.

[0089] In some embodiments of the tenth aspect, the organic solution provided in step (b) and / or (e') comprises an organic solvent selected from lower alcohols, such as alcohols having up to six carbon atoms (particularly aliphatic alcohols), and mixtures thereof (such as mixtures of two or more of these alcohols). In preferred embodiments, the organic solvent is completely miscible with water. In some embodiments, the organic solvent is selected from the group consisting of ethanol, propanol, isopropanol, 1,2-propanediol, and mixtures of two or more of these alcohols. In some embodiments, the organic solvent is ethanol, propanol, or a mixture thereof.

[0090] In some embodiments of the tenth aspect, the composition is substantially free of cryoprotectants.

[0091] In some embodiments of the tenth aspect, the first buffer system used in step (a) includes the final buffer used in step (d), and preferably, the buffer system and pH of the first buffer system used in step (a) are the same as the buffer system and pH of the final aqueous buffer used in step (d). For example, in this embodiment of the tenth aspect, only one aqueous buffer is used.

[0092] Similarly, in some embodiments of the tenth aspect, if the method includes steps (a') to (e') and (h') (and optionally one or more of steps (f'), (g'), and (i')), each of the first buffer systems and all further buffer systems used in steps (b'), (f'), and (g') includes the final buffer used in step (h'), and preferably, the buffer systems and pH of each of the first aqueous buffers and all further aqueous buffers used in steps (b'), (f'), and (g') are identical to the buffer system and pH of the final aqueous buffer. For example, the aqueous buffers used in steps (b'), (f'), (g'), (h'), and (f') are identical in this embodiment of the tenth aspect.

[0093] It is understood that any embodiment described herein in relation to the first, second, third, fourth, fifth, sixth, seventh, eighth, or ninth aspect may also be applicable to any embodiment of the tenth aspect.

[0094] In an eleventh embodiment, the Disclosure provides a method for preserving a composition, which comprises preparing a liquid composition according to the method of the tenth embodiment and preserving the liquid composition at a temperature in the range of about 0°C to about 20°C, for example, about 1°C to about 15°C, about 2°C to about 10°C, or about 2°C to about 8°C. In some embodiments of the eleventh embodiment, preserving the liquid composition is for at least one week, for example, at least two weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least six months, at least nine months, or at least twelve months, preferably at least four weeks. In some embodiments of the eleventh embodiment, preserving the liquid composition is for at least four weeks, preferably at least one month, more preferably at least two months, more preferably at least three months, more preferably at least six months, more preferably at least nine months, and more preferably nine to twelve months, at about 2°C to about 8°C.

[0095] In some embodiments of the eleventh aspect, the composition is substantially free of cryoprotectants.

[0096] It is understood that any embodiment described herein in relation to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspect may also be applicable to any embodiment of the eleventh aspect.

[0097] In a twelfth embodiment, the present invention provides a method for preparing a ready-to-use pharmaceutical composition, the method comprising the step of providing a liquid composition prepared by the method of the tenth or eleventh embodiment, thereby obtaining a ready-to-use pharmaceutical composition.

[0098] It is understood that any embodiment described herein in relation to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh aspect may also be applicable to any embodiment of the twelfth aspect.

[0099] In a thirteenth embodiment, the present invention provides a ready-to-use pharmaceutical composition that can be prepared by the method of the twelfth embodiment.

[0100] It is understood that any embodiment described herein in relation to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or twelfth aspects may also be applicable to any embodiment of the thirteenth aspect. Furthermore, it is understood that a ready-to-use pharmaceutical composition of the thirteenth aspect may be used in any one of the second, third, fourth, fifth, sixth, seventh, eighth, and ninth aspects. [Brief explanation of the drawing]

[0101] [Figure 1] Degradation rates of nucleic acid compositions stored under different environmental conditions. Compositions containing sa-RNA (A: naked sa-RNA, B: sa-RNA / LP-2) and histidine (20 mM) were stored for up to one month at 4°C in the presence of EDTA (1 mM) at different pH values ​​(A, B: pH 4.0-7.0). Degradation rates (% / month) ± standard deviation versus pH value are shown. [Figure 2] Integrity of nucleic acid compositions stored under different environments. RNA / particle compositions were prepared (A: LP-1, B: LP-2) and stored at 4°C and different pH values ​​(pH 5.0, 5.5, 6.0, and 6.5, respectively) for up to 12 months. The percentage of RNA integrity relative to storage time is shown. [Figure 3] Integrity of nucleic acid compositions stored under different environments. RNA / LP-2 compositions were prepared using HEPES (A-D) or histidine (E) as buffers and stored for up to 9 months at 4°C and pH 5.5 (A), 6.0 (B), 6.5 (C), and 7.0 (D), respectively, in the absence (black square) or presence (white triangle) of EDTA (1 mM). The percentage values ​​of RNA integrity relative to storage time are shown. [Figure 4] Integrity of nucleic acid compositions stored under different environmental conditions. RNA / LP-1 compositions were prepared using different phospholipids (DOPE, DOPC, or DSPC) with histidine as a buffer, and stored for up to 9 months at 4°C and pH values ​​of 5.0 (A), 5.5 (B), and 6.0 (C), respectively. RNA integrity percentage values ​​against storage time are shown.

[0102] Array description The following table lists the specific sequences referenced herein. [Table 1] [Modes for carrying out the invention]

[0103] Detailed description of the invention While this disclosure is described in further detail below, it should be understood that this disclosure is not limited to the specific methodologies, protocols, and reagents described herein, as these are subject to change. It should also be understood that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit the scope of this disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0104] The elements of this disclosure are described in more detail below. While these elements are listed with specific embodiments, it should be understood that they can be combined in any way and in any number to create further embodiments. The various examples and preferred embodiments described should not be construed as limiting this disclosure to only the explicitly described embodiments. This description should be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of disclosed elements and / or preferred elements. Furthermore, unless the context otherwise indicates, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application.

[0105] Preferably, the terms used herein are defined as those set forth in "A multilingual glossary of biotechnological terms: (IUPAC recommendations)," H.G. W. Euenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0106] Unless otherwise noted, the implementation of this disclosure will involve conventional chemistry, biochemistry, cell biology, immunology, and recombinant DNA technologies as described in the literature in the relevant field. (cf., e.g. Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Rompp Chemie Lexikon, Falbe / Regitz(Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring (see Harbor 1989).

[0107] Throughout this specification and the claims, unless otherwise specified in the context, the word “comprise,” and variations such as “comprises” and “comprising,” shall be understood to mean including the described component, integer or step or group of component, integer or step, but not to exclude other component, integer or step or group of component, integer or step. The term “consisting essentially of” shall be understood to mean excluding other component, integer or step as being essentially important. The term “comprising” encompasses the term “consisting essentially of,” and further encompasses the term “consisting of.” Therefore, in each place in this application, the term “comprising” may be replaced with the term “consisting essentially of” or “consisting of.” Similarly, in each part of this application, the phrase "consisting essentially of" can be replaced with the phrase "consisting of".

[0108] The terms “a,” “an,” and “the,” as well as similar references, used in the context describing this disclosure (particularly in the context of the claims), shall be construed as including both singular and plural forms unless otherwise stated herein or the context clearly contradicts this.

[0109] All methods described herein may be performed in any suitable order, unless otherwise specified herein or unless the context clearly contradicts it.

[0110] Any use of any example or exemplary language provided herein (e.g., "etc.") is solely for the purpose of better describing the disclosure and does not limit the scope of the disclosure as otherwise requested. No language in this specification should be construed as indicating any unrequested element essential to the implementation of the disclosure.

[0111] As used herein, “and / or” shall be interpreted as a specific disclosure for each of two specified features or components, with or without the other. For example, “X and / or Y” shall be interpreted as a specific disclosure for each of (i) X, (ii) Y, and (iii) X and Y, as if they were individually described herein.

[0112] In the context of this disclosure, the term “approximately” indicates a range of precision that a person skilled in the art would understand to still guarantee the technical effect of the feature in question. Typically, this term refers to a deviation of ±10% from a given number, e.g., ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, e.g., a deviation of ±0.01%. In some embodiments, “approximately” refers to a deviation of ±10% from a given number. In some embodiments, “approximately” refers to a deviation of ±5% from a given number. In some embodiments, “approximately” refers to a deviation of ±4% from a given number. In some embodiments, “approximately” refers to a deviation of ±3% from a given number. In some embodiments, “approximately” refers to a deviation of ±2% from a given number. In some embodiments, “approximately” refers to a deviation of ±1% from a given number. In some embodiments, "approximately" indicates a deviation of ±0.9% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.8% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.7% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.6% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.5% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.4% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.3% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.2% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.1% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.05% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.01% from the indicated value. As will be understood by those skilled in the art, the specific deviation of the numerical value relating to a given technical effect depends on the nature of the technical effect. For example, natural or biotechnological effects may generally have larger deviations than artificial or engineering-technological effects.

[0113] The enumeration of value ranges in this specification is intended to serve simply as a convenient way to refer to each individual value that falls within the range individually. Unless otherwise specified herein, each individual value is incorporated into the specification as if it were individually enumerated herein.

[0114] This specification incorporates references to several documents. Each document referenced herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) is incorporated herein by reference in its entirety, whether as stated above or below. Nothing in this specification should be construed as admitting that the present invention has no prior rights to such disclosures based on prior art.

[0115] definition The following definitions are provided that apply to all aspects of this disclosure. Unless otherwise specified, the following terms have the meanings set forth below. Terms not defined have the meanings accepted in the art.

[0116] As used herein, terms such as “reduce” or “inhibit” mean the ability to cause an overall reduction of, for example, more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 40%, more than 50%, or more than 75%. The term “inhibit” or similar phrases include complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0117] As used herein, terms such as “enhance” and “increase” mean an overall increase in level, or the ability to cause an enhancement of, for example, at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 75%, or about 100% or more. In some embodiments, these terms relate to an enhancement or increase of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

[0118] As used herein, “physiological pH” refers to a pH of approximately 7.5 or approximately 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0119] In this specification, “physiological conditions” refers to conditions in a living organism, particularly in humans (especially pH and temperature). Preferably, physiological conditions mean physiological pH and / or a temperature of about 37°C.

[0120] As used in this disclosure, "%(w / v)" (or "%w / v") refers to a weight percentage, which is a unit of concentration used to measure the amount of solute in grams (g) and is expressed as a percentage of the total volume of the solution in milliliters (ml).

[0121] As used in this disclosure, “volume %” or “%(v / v)” (or “%v / v”) refers to volume percentage, which is a unit of concentration used to measure the amount of liquid substance in milliliters (ml), expressed as a percentage of the total volume of a solution in milliliters (ml).

[0122] As used in this disclosure, “weight percent” or “%(w / w)” (or “%w / w”) refers to weight percent, which is a unit of concentration used to measure the amount of a substance expressed in grams (g), and is expressed as a percentage of the total weight of the entire composition expressed in grams (g).

[0123] As used herein, "mol%" is defined as the ratio obtained by dividing the number of moles of one component by the total number of moles of all components, multiplied by 100.

[0124] In this specification, “moles of total lipids” is defined as the ratio obtained by dividing the number of moles of one lipid component by the total number of moles of all lipids, multiplied by 100. In this context, in some embodiments, the term “total lipids” includes lipids and lipid-like substances.

[0125] As used in this disclosure, the term "buffer system" means a system containing a buffering material. As used herein, "buffering material" means a mixture of a base and its protonated form (e.g., histidine and its protonated form [histidine-H] + This refers to a mixture of the base and the conjugate acid. Therefore, the amount of buffering material in the composition is the sum of the amounts of both the base and the conjugate acid in the composition. In some embodiments, the "buffering system" may consist essentially of "buffering material".

[0126] In this specification, “molar ratio” refers to the ratio between the molar amounts of any two substances. For example, if a composition contains 1 mmol (mmol) of substance A and 2 mmol (mmol) of substance B, the molar ratio of substance B to substance C is 1:2 or 0.5.

[0127] "Osmotic concentration" refers to the concentration of a particular solute, expressed as the number of osmoles of solute per kilogram of solvent.

[0128] The term "reconstitution" refers to the process of adding a solvent, such as water, to a dried product to return it to its original liquid state or other liquid state.

[0129] The term "freezing" typically refers to the process of solidifying a liquid by removing heat. In some embodiments, freezing is the reverse of thawing.

[0130] The term "thawing" usually refers to the process of liquefying a solid by applying heat. In some embodiments, thawing is the reverse of freezing.

[0131] In this specification, the term “aqueous phase” as used in relation to compositions / formulations containing particles, particularly LNPs, liposomes, and / or lipoplexes, means a mobile phase or liquid phase, i.e., a continuous aqueous phase containing all components dissolved therein but (formally) excluding particles. Therefore, if particles such as LNPs are dispersed in the aqueous phase and it is required that the aqueous phase is substantially free of compound X, the aqueous phase is free of X in a practically and realistically viable way. For example, the concentration of compound X in the aqueous composition is less than 1% by weight. However, at the same time, it is also possible that the particles dispersed in the aqueous phase contain more than 1% by weight of compound X.

[0132] In the context of this disclosure, the term “recombinant” means “genetically engineered.” In some embodiments, the “recombinant” in the context of this disclosure is not naturally occurring.

[0133] As used herein, the term “naturally occurring” means that a substance exists in nature, for example in living organisms (including viruses), can be isolated from a natural source, and is a peptide or nucleic acid that has not been intentionally modified by humans in a laboratory. The term “found in nature” means “naturally occurring” and includes not only known substances but also substances that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from nature in the future.

[0134] As used herein, the terms “room temperature” and “ambient temperature” are used interchangeably and refer to temperatures of at least about 15°C, preferably about 15°C to about 35°C, about 15°C to about 30°C, about 15°C to about 25°C, or about 17°C to about 22°C. Such temperatures include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0135] The term "alkyl" refers to a monoradical of a saturated linear or branched hydrocarbon. Preferably, an alkyl group has 1 to 12 (e.g., 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (C 1-12 Alkyl (abbreviated as alkyl), (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, C 1-10 Alkyl (abbreviated as alkyl), more preferably containing 1 to 8 carbon atoms, for example, 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl (2-propyl or 1-methylethyl), butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isoamyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, etc. "Substituted alkyl" means that one or more hydrogen atoms of an alkylene group (for example, from one to the maximum number of hydrogen atoms bonded to the alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are substituted with a substituent other than hydrogen (if multiple hydrogen atoms are substituted, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent as specified herein. Examples of substituted alkyls include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

[0136] The term "alkylene" refers to a diradical of a saturated straight-chain or branched hydrocarbon. Preferably, the alkylene contains 1 to 12 (e.g., 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1 to 8 carbon atoms, e.g., 1 to 6 or 1 to 4 carbon atoms. Examples of alkylene groups include methylene, ethylene (i.e., 1,1-ethylene, 1,2-ethylene), propylene (i.e., 1,1-propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2-propylene (-C(CH3)2-), and 1,3-propylene), butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans, or mixtures thereof), 1,4-butylene, 1,1-isobutylene, 1,2-isobutylene, and 1,3-isobutylene), pentylene isomers (e.g., 1,1-pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-iso-pentylene) (Lene, 1,1-sec-pentyl, 1,1-neo-pentyl), hexylene isomers (e.g., 1,1-hexylene, 1,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene, 1,1-isohexylene), heptylene isomers (e.g., 1,1-heptylene, 1,2-heptylene, 1,3-heptylene, 1,4- Examples include heptylene, 1,5-heptylene, 1,6-heptylene, 1,7-heptylene, and 1,1-isoheptylene, as well as octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3-octylene, 1,4-octylene, 1,5-octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene, and 1,1-isooctylene). Straight-chain alkylene groups, which have at least three carbon atoms and free valence at each end, can also be denoted by a multiple of the methylene group (for example, 1,4-butylene is also called the tetramethylene group).Generally, instead of using the suffix "-ylene" for alkylene groups as described above, the suffix "-diyl" can also be used (for example, 1,2-butylene is also called butane-1,2-diyl group). A "substituted alkylene" means that one or more hydrogen atoms of an alkylene group (for example, the maximum number of hydrogen atoms bonded to the alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, 1 to 3, or 1 or 2) are substituted with a substituent other than hydrogen (if multiple hydrogen atoms are substituted, the substituents may be the same or different). Preferably, the substituents other than hydrogen are first-level substituents as defined herein.

[0137] The term "alkenyl" refers to a monoradical of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximum number of carbon-carbon double bonds in an alkenyl group is equal to an integer obtained by dividing the number of carbon atoms in the alkenyl group by 2, and if the number of carbon atoms in the alkenyl group is odd, the result of the division is rounded down to the next integer. For example, in an alkenyl group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, an alkenyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. Preferably, the alkenyl group contains 2 to 12 (e.g., 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), and more preferably 2 to 8 carbon atoms, e.g., 2 to 6 carbon atoms or 2 to 4 carbon atoms. Therefore, in a preferred embodiment, the alkenyl group is C 2-12Alkenyls are abbreviated as such and contain 2 to 12 (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, for example 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bonds can be in a cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-hexenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl This includes 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, etc. When an alkenyl group is bonded to a nitrogen atom, the double bond cannot be located at the alpha position relative to the nitrogen atom."Substituted alkenyl" means that one or more hydrogen atoms of an alkenyl group (for example, from 1 to the maximum number of hydrogen atoms bonded to the alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are substituted with a non-hydrogen substituent (if multiple hydrogen atoms are substituted, the substituents may be the same or different). Preferably, the non-hydrogen substituent is a first-level substituent as specified herein.

[0138] The term "alkynyl" refers to a monovalent hydrocarbon segment, either linear or branched, having at least one carbon-carbon triple bond, with a total number of carbon atoms ranging from 6 to 30, typically 6 to 20, and often 6 to 18. An alkynyl group may have one or more carbon-carbon double bonds as needed. Generally, the maximum number of carbon-carbon triple bonds in an alkynyl group is equal to an integer obtained by dividing the number of carbon atoms in the alkynyl group by 2, where the result of the division is rounded down to the next integer if the number of carbon atoms is odd. For example, in an alkynyl group with 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, an alkynyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2 carbon-carbon triple bonds. Exemplary alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 5-heptynyl, 6-heptynyl, 1-octinyl, 2 Examples include -octinyl, 3-octinyl, 4-octinyl, 5-octinyl, 6-octinyl, 7-octinyl, 1-nonilyl, 2-noninyl, 3-noninyl, 4-noninyl, 5-noninyl, 6-noninyl, 7-noninyl, 8-noninyl, 1-decinyl, 2-decinyl, 3-decinyl, 4-decinyl, 5-decinyl, 6-decinyl, 7-decinyl, 8-decinyl, 9-decinyl, etc. When the alkynyl group is bonded to the nitrogen atom, the triple bond cannot be located at the alpha position relative to the nitrogen atom. "Substituting alkynyl" means that one or more hydrogen atoms of an alkynyl group (for example, from 1 to the maximum number of hydrogen atoms bonded to the alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are substituted with a substituent other than hydrogen (if multiple hydrogen atoms are substituted, the substituents may be the same or different). Preferably, substituents other than hydrogen are first-level substituents as specified herein.

[0139] The term "alkenylene" refers to a diradical of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximum number of carbon-carbon double bonds in an alkenylene group is equal to an integer obtained by dividing the number of carbon atoms in the alkenylene group by 2, and if the number of carbon atoms in the alkenylene group is odd, the result of the division is rounded down to the next integer. For example, for an alkenylene group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, an alkenylene group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. Preferably, the alkenylene group contains 2 to 12 (e.g., 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, for example, 2 to 6 carbon atoms or 2 to 4 carbon atoms. Therefore, in preferred embodiments, the alkenylene group contains 2 to 12 (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, for example, 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond can be in a cis (Z) or trans (E) configuration. Examples of alkenylene groups include ethene-1,2-diyl, vinylidene (also called etenylidene), 1-propene-1,2-diyl, 1-propene-1,3-diyl, 1-propene-2,3-diyl, alilidene, 1-butene-1,2-diyl, 1-butene-1,3-diyl, 1-butene-1,4-diyl, 1-butene-2,3-diyl, 1-butene-2,4-diyl, 1-butene-3,4-diyl, 2-butene-1,2-diyl, 2-butene-1,3-diyl, 2-butene-1,4-diyl, 2-butene-2,3-diyl, 2-butene-2,4-diyl, and 2-butene-3,4-diyl.When an alkenylene group is bonded to a nitrogen atom, it cannot be located at the alpha position relative to the double-bonded nitrogen atom. "Substituted alkenylene" means that one or more hydrogen atoms of the alkenylene group (for example, from 1 to the maximum number of hydrogen atoms bonded to the alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are substituted with a non-hydrogen substituent (if multiple hydrogen atoms are substituted, the substituents may be the same or different). Preferably, the non-hydrogen substituent is a first-level substituent as specified herein.

[0140] The term "alkynylene" refers to a diradical of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon triple bond (for example, the divalent version of the monoradical alkynyl as defined above). Generally, the maximum number of carbon-carbon triple bonds in an alkynylene group is equal to an integer obtained by dividing the number of carbon atoms in the alkynylene group by 2, and if the number of carbon atoms in the alkynylene group is odd, the result of the division is rounded down to the next integer. For example, for an alkynylene group with 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynylene group has 1 to 7 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, 6, or 7 (e.g., 1, 2, 3, or 4), more preferably 1 or 2 carbon-carbon triple bonds. Preferably, the alkynylene group contains 2 to 14 (e.g., 2 to 12 or 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10), more preferably 2 to 8 carbon atoms, for example, 2 to 6 or 2 to 4 carbon atoms. Therefore, in preferred embodiments, the alkynylene group comprises 2 to 14 (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5, 6, or 7 (e.g., 1, 2, 3, 4, or 5 (preferably 1, 2, or 3)) carbon-carbon triple bonds, more preferably comprising 2 to 8 carbon atoms and 1, 2, 3, or 4 (preferably 1 or 2) carbon-carbon triple bonds, for example, comprising 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon triple bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon triple bonds. Examples of alkynylene groups include ethyne-1,2-diyl, 1-propyne-1,3-diyl, 1-propyne-3,3-diyl, 1-butyne-1,3-diyl, 1-butyne-1,4-diyl, 1-butyne-3,4-diyl, and 2-butyne-1,4-diyl. When the alkynylene group is a nitrogen atom, the triple bond cannot be located at the alpha position relative to the nitrogen atom."Substituted alkynylene" means that one or more hydrogen atoms of an alkynylene group (for example, from 1 to the maximum number of hydrogen atoms bonded to the alkynylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are substituted with a non-hydrogen substituent (if multiple hydrogen atoms are substituted, the substituents may be the same or different). Preferably, the non-hydrogen substituent is a first-level substituent as specified herein.

[0141] The term "cycloalkyl" represents the cyclic non-aromatic version of "alkyl" and "alkenyl," preferably containing 3 to 14 carbon atoms, for example, 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclocodecyl, cyclocodecenyl, and adamantyl. Cycloalkyl groups may consist of one ring (monocyclic), two rings (bicyclic), or two or more rings (polycyclic).

[0142] The term “cycloalkylene” represents a cyclic non-aromatic version of “alkylene,” which is geminal, vicinal, or isolated diradical. In certain embodiments, a cycloalkylene is (i) monocyclic or polycyclic (e.g., bicyclic or tricyclic), and / or (ii) a 3- to 14-membered ring (i.e., a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered ring, e.g., a 3- to 12-membered ring or a 3- to 10-membered ring). In one embodiment, a cycloalkylene is monocyclic, bicyclic or tricyclic, and a 3- to 14-membered ring (i.e., a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered ring, e.g., a 3- to 12-membered ring or a 3- to 10-membered ring). Generally, instead of using "ylene" at the end of the cycloalkylene group specified above, you can also use "diyl" (for example, 1,2-cyclopropylene is also called cyclopropane-1,2-diyl). Examples of cycloalkylene groups include cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene, and adamantanylene (for example, tricyclo[3.3.1.1 3,7 Examples include decane-2,2-diyl. "Substituted cycloalkylene" means that one or more (for example, 1 to a maximum number, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of an alkylene group are replaced by substituents other than hydrogen (if one or more hydrogen atoms are replaced, those substituents may be the same or different). Preferably, the substituents other than hydrogen are first-level substituents as specified herein.

[0143] The term "cycloalkenylene" refers to the cyclic non-aromatic version of "alkenylene," which is geminal, vicinal, or isolated diradical. Generally, the maximum number of carbon-carbon double bonds in a cycloalkenylene group is equal to the integer obtained by dividing the number of carbon atoms in the cycloalkenylene group by 2, and if the number of carbon atoms in the cycloalkenylene group is odd, the result of the division is rounded down to the next integer. For example, in a cycloalkenylene group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, a cycloalkenylene group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. In certain embodiments, the cycloalkenylene is (i) monocyclic or polycyclic (e.g., bicyclic or tricyclic), and / or (ii) a 3- to 14-membered ring (i.e., a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered ring, e.g., a 3- to 12-membered ring or a 3- to 10-membered ring). In one embodiment, the cycloalkenylene is a monocyclic, bicyclic or tricyclic 3- to 14-membered ring (i.e., a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered ring, e.g., a 3- to 12-membered ring or a 3- to 10-membered ring) cycloalkenylene. Examples of cycloalkenylene groups include cyclohexenylene, cycloheptenylene, cyclopropenylene, cyclobutenylene, cyclopentenylene, and cyclooctenylene. "Substituted cycloalkenylene" means that one or more hydrogen atoms of the cycloalkenylene group (for example, from 1 to the maximum number of hydrogen atoms bonded to the cycloalkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are substituted with a non-hydrogen substituent (if multiple hydrogen atoms are substituted, the substituents may be the same or different). Preferably, the non-hydrogen substituent is a first-level substituent as specified herein.

[0144] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 carbon atoms (e.g., 5, 6, 7, 8, 9, or 10, e.g., 5, 6, or 10), which can be arranged in one ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenilium, cyclopentadienyl, phenyl, indenyl, naphthyl, azlenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Fullerenes are not included in the term aryl. "Substituting aryl" means that one or more hydrogen atoms of an aryl group (for example, from 1 to the maximum number of hydrogen atoms bonded to the aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are substituted with a non-hydrogen substituent (if multiple hydrogen atoms are substituted, the substituents may be the same or different). Preferably, the non-hydrogen substituent is a first-level substituent as specified herein.

[0145] The term "heteroaryl" means the aryl group as defined above, in which one or more carbon atoms (e.g., 1, 2, 3, or 4) in the aryl group are replaced by heteroatoms (e.g., O, S, or N). Preferably, a heteroaryl refers to a 3- to 8-membered (e.g., 5- to 6-membered) aromatic monocyclic ring, in which one or more carbon atoms (e.g., 1, 2, or 3) are replaced by the same or different O, N, or S heteroatoms. Alternatively, it means an aromatic bicyclic or tricyclic ring system in which 1, 2, 3, 4, or 5 carbon atoms are replaced by the same or different O, N, or S heteroatoms. Preferably, in each ring of the heteroaryl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. For example, heteroaryl groups with 3 to 14 member rings include monocyclic heteroaryls (e.g., 3, 5, or 6 member rings), bicyclic heteroaryls (e.g., 9 or 10 member rings), and tricyclic heteroaryls (e.g., 13 or 14 member rings). Exemplary heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazolyl, benzoisothiazolyl, benzotriazolyl Examples include quinolinil, isoinolinil, benzodiadinil, quinoxalinil, quinazolinil, benzotriazinil, pyridadinil, phenoxadinil, thiazolopyridinil, pyrrolothiazolyl, phenothiazinil, isobenzofuranil, clomenil, xanthenil, pyrrolidinil, indazolyl, purinil, quinolidinil, phthalazinil, naphthyridinil, sinnolinil, pteridinil, carbazolyl, phenanthridine, acridinil, perimidinil, phenanthrolinil, and phenadinil.Examples of 5- or 6-membered ring heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. "Substituted heteroaryl" means that one or more hydrogen atoms of the heteroaryl group (e.g., 1 to the maximum number of hydrogen atoms bonded to the heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are substituted with a non-hydrogen substituent (if multiple hydrogen atoms are substituted, the substituents may be the same or different). Preferably, the non-hydrogen substituent is a first-level substituent as specified herein.

[0146] The terms “heterocyclyl,” “heterocyclic,” and “heterocycloalkyl” (as used herein interchangeably) mean that one or more (e.g., 1, 2, 3, or 4) carbon atoms of the cycloalkyl group defined above are replaced by heteroatoms of O, N, Si, Se, P, or S, preferably O, S, or N. A heterocyclyl group has one or two rings containing preferably 3 to 10 (e.g., 3, 4, 5, 6, or 7) ring atoms. Preferably, in each ring of the heterocyclyl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term “heterocyclyl” also means that partially or completely hydrogenated forms (e.g., dihydro, tetrahydro, or perhydro forms) of the heteroaryl group described above are also included. Examples of heterocyclic groups include morpholinyl, pyrrolidinyl, imidazolidinyl, 2,3-dihydro-1H-imidazolyl, pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl, azetidinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydropyranyl, urotropinyl, lactones, lactams, cyclic imides, and cyclic anhydrides. A "substituted heterocyclyl" means that one or more hydrogen atoms of a heterocyclyl group (for example, from 1 to the maximum number of hydrogen atoms bonded to the heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) are substituted with a non-hydrogen substituent (if multiple hydrogen atoms are substituted, the substituents may be the same or different). Preferably, substituents other than hydrogen are first-level substituents as specified herein.

[0147] The term “heterocycloalkylene” means the diradical version of the monoradical heterocycloalkyl as defined above. Heterocycloalkylenes may be geminal, vicinal, or isolated diradicals. In certain embodiments, heterocycloalkylenes may be unsaturated (e.g., heterocycloalkenylene (i.e., heterocycloalkyl heterocycloalkylene groups as defined herein contain at least one carbon-carbon double bond) or heterocycloalkylynylene (i.e., heterocycloalkyl heterocycloalkylene groups as defined herein contain at least one carbon-carbon triple bond)), but must not be aromatic. In certain embodiments, the heterocycloalkylene is (i) monocyclic or polycyclic (e.g., bicyclic or tricyclic), and / or (ii) a 3- to 14-membered ring (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered ring, e.g., a 3- to 12-membered ring or a 3- to 10-membered ring). In one embodiment, the heterocycloalkylene is a monocyclic, bicyclic or tricyclic 3- to 14-membered ring (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered ring, e.g., a 3- to 12-membered ring or a 3- to 10-membered ring) heterocycloalkylene. In general, instead of using the suffix "-ylene" for the specified heterocycloalkylene portion, the suffix "-diyl" can also be used (for example, 1,3-imidazolidinylene is also called imidazolidin-1,3-diyl). Exemplary cycloalkylene groups include pyrrolidinylene, imidazolidinylene, 2,3-dihydro-1H-imidazoylene, pyrazolidinylene, piperidinylene, piperadinylene, and azetidinylene. "Substituted heterocycloalkylene" means that one or more hydrogen atoms of a heterocycloalkylene group (for example, 1 to the maximum number of hydrogen atoms bonded to the heterocycloalkylene group, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are substituted with a substituent other than hydrogen (if one or more hydrogen atoms are substituted, the substituents may be the same or different).Preferably, substituents other than hydrogen are first-level substituents as specified herein.

[0148] As used herein, the expression “partially hydrogenated form” of an unsaturated compound or group means that some of the unsaturation has been removed by formally adding hydrogen to the compound or group which was originally unsaturated, but not all of the unsaturation has been removed. The phrase “fully hydrogenated form” of an unsaturated compound or group is used herein interchangeably with the term “perhydro” and means that all of the unsaturation has been removed by formally adding hydrogen to the compound or group which was originally unsaturated. For example, a partially hydrogenated form of a five-membered heteroaryl group (containing two double bonds in the ring, e.g., furan) includes the dihydro form of the five-membered heteroaryl group (e.g., 2,3-dihydrofuran or 2,5-dihydrofuran), while the tetrahydro form of the five-membered heteroaryl group (e.g., tetrahydrofuran, i.e., THF) is the fully hydrogenated (or perhydro) form of the five-membered ring heteroaryl group. Similarly, in the case of a six-membered heteroaryl group having three double bonds in the ring (such as pyridyl), the partially hydrogenated forms include the dihydro and tetrahydro forms (such as dihydropyridyl and tetrahydropyridyl), while the hexahydro form (such as piperidinyl in the case of heteroarylpyridyl) is a fully hydrogenated (or perhydro) derivative of the said six-membered heteroaryl group. Thus, the hexahydro form of an aryl or heteroaryl can be considered a partially hydrogenated form in accordance with this disclosure only if the aryl or heteroaryl contains at least four unsaturated moieties consisting of double and triple bonds between ring atoms.

[0149] In the context of hydrocarbons, the term "aromatic" means that the entire molecule must be aromatic. For example, when a monocyclic aryl is hydrogenated (partially or completely), the resulting hydrogenated cyclic structure is classified as a cycloalkyl for the purposes of this disclosure. Similarly, when a bicyclic or polycyclic aryl (such as naphthyl) is hydrogenated, the resulting hydrogenated bicyclic or polycyclic structure (such as 1,2-dihydronaphthyl) is classified as a cycloalkyl for the purposes of this disclosure (even if one ring remains aromatic, as in 1,2-dihydronaphthyl). A similar distinction is made between heteroaryls and heterocyclyls in this disclosure. For example, indolinyl, i.e., a dihydro variant of indolyl, is classified as a heterocyclyl for the purposes of this disclosure because only one ring of the bicyclic structure is aromatic, and one of the ring atoms is a heteroatom.

[0150] As used herein, the term "hydrocarbyl" refers to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule. In some embodiments, the hydrocarbyl group is acyclic, for example, linear (linear) or branched. Typical examples of hydrocarbyl groups include alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, aryl groups, and combinations thereof (e.g., arylalkyl groups). A specific example of a hydrocarbyl group is C 1-6 Alkyl groups, aryl groups, and aryl ( C1-6 Examples include alkyl groups. In some embodiments, the hydrocarbyl group may be optionally substituted (for example, substituted with one or more first-level substituents as defined herein), as long as the polarity of the overall hydrocarbon remains relatively nonpolar.

[0151] The term "aliphatic" refers to any non-aromatic organic group, and in particular includes any non-aromatic hydrocarbyl group as defined herein, and diradical versions thereof. In some embodiments, where the aliphatic group is a monoradical, the aliphatic group includes alkyl, alkenyl, alkynyl, and cycloalkyl groups as defined herein (where each of alkyl, alkenyl, alkynyl, and cycloalkyl groups may be optionally substituted). In some embodiments, where the aliphatic group is a diradical, the aliphatic group includes alkylene, alkenylene, alkynylene, and cycloalkylene groups as defined herein (where each of alkylene, alkenylene, alkynylene, and cycloalkylene groups may be optionally substituted).

[0152] The term "alicyclic" refers to any organic group that is cyclic and non-aromatic, and in particular includes any non-aromatic cyclic hydrocarbyl group as defined herein, and diradical versions of such non-aromatic cyclic hydrocarbyl groups. In some embodiments, if the alicyclic group is monoradical, the alicyclic group includes a cycloalkyl group as defined herein (where the cycloalkyl group may be optionally substituted). In some embodiments, if the alicyclic group is diradical, the alicyclic group includes a cycloalkylene group as defined herein (where the cycloalkylene group may be optionally substituted).

[0153] The term "heteroaliphatic" means an aliphatic group as defined herein in which one or more (e.g., 1, 2, 3, or 4) carbon atoms in the aliphatic group are substituted with heteroatoms of O, N, Si, Se, P, or S, preferably O, S, or N. In some embodiments, when the heteroaliphatic group is a monoradical, the heteroaliphatic group encompasses alkyl, alkenyl, alkynyl, and heterocyclic groups as defined herein (each of which may be optionally substituted), where in each of the alkyl, alkenyl, and alkynyl groups one or more (e.g., 1, 2, 3, or 4) carbon atoms are substituted with heteroatoms of O, N, Si, Se, P, or S, preferably O, S, or N. In some embodiments, when the heteroaliphatic group is a diradical, the heteroaliphatic group is an alkylene group, alkenylene group, alkynylene group, and heterocycloalkylene group as defined herein (each of which may be optionally substituted), wherein in each of the alkylene group, alkenylene group, and alkynylene group, one or more (e.g., one, two, three, or four) carbon atoms are replaced by heteroatoms of O, N, Si, Se, P, or S, preferably O, S, or N.

[0154] Typical first-level substituents are C 1-3 Preferably selected from the group consisting of alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH2-z(CH3)z, -C(=O)OH, and -C(=O)OCH3, where z is 0, 1, or 2, and C 1-3 The alkyl group is methyl, ethyl, propyl, or isopropyl. Particularly preferred first-level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogens (such as F, Cl, or Br), and -CF3, e.g., halogens (e.g., F, Cl, or Br), and -CF3.

[0155] As used herein, the term “tertiary amine moiety” refers to a moiety comprising a nitrogen atom substituted with three organic substituents (where the substituents may be identical or different from one another). In some embodiments, the organic substituents are optionally substituted (e.g., with one or more first-level substituents as defined herein) with a hydrocarbyl group (e.g., alkyl groups, particularly C 1-6 Selected from (alkyl groups).

[0156] As used herein, the term “filtration” refers to any process that involves removing or separating at least one component of a liquid composition (such as a permeable molecule, like a salt, small protein, or solvent) based on the molecular size of the components contained in the composition. This separation may be performed using micromolecular permeable filters (e.g., for diafiltration or tangential flow filtration) or semipermeable membranes (e.g., for dialysis). Therefore, examples of filtration include dialysis, tangential flow filtration, and diafiltration.

[0157] A "monovalent" compound is a compound that has only one functional group of the type in question. For example, a monovalent acid is a compound that has only one acidic group (for example, only one carboxyl (-COOH) group). A monovalent cation is, for example, an alkali cation (for example, Na + , K + Li + ), ammonium cation (NH4 + The present invention relates to compounds having only one cationic group, such as organic compounds having one primary, secondary, or tertiary amine group (a protonated form such as triethylamine or trimethylamine), or organic compounds having one quaternary amine group.

[0158] A "divalent" or "dibasic" compound refers to a compound that has two functional groups of the same nature. For example, a dibasic organic acid has two carboxyl groups.

[0159] The term "polyvalent" or "polybasic" refers to compounds that have three or more functional groups of the choice. For example, polybasic organic acids have three or more acidic carboxyl groups.

[0160] As used herein, the expression “substantially free of X” means that the mixture (such as the aqueous phase of a composition or formulation described herein) is free of X to the extent that it is practically and realistically practicable. For example, if a mixture is substantially free of X, the amount of X in the mixture may be less than 1% by weight (e.g., less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.005%, less than 0.001%) based on the total weight of the mixture.

[0161] For example, as used herein, “substantially free of cryoprotectants” means that the mixture (such as the aqueous phase of a composition or formulation described herein) is free of cryoprotectants to the extent that it is practically and realistically feasible. For example, if a mixture is substantially free of cryoprotectants, the amount of cryoprotectant in the mixture may be less than 1% by weight (e.g., less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.005%, less than 0.001%) based on the total weight of the mixture. The same considerations apply to other expressions that include the phrase "substantially does not contain," such as "substantially does not contain chelating agents," "substantially does not contain EDTA," "substantially does not contain cationized lipids," "substantially does not contain additional lipids," "does not contain lipids," "substantially does not contain nucleic acids," and "does not contain one or more unwanted substances."

[0162] The expression "nucleic acid integrity" refers to the ratio of full-length nucleic acids (i.e., unfragmented nucleic acids) to the total amount of nucleic acids in a sample (i.e., the sum of unfragmented and fragmented nucleic acids). Nucleic acid integrity can be determined by separating nucleic acids by chromatography (e.g., using capillary electrophoresis), determining the peak area of ​​the major nucleic acid peak (i.e., the peak area of ​​full-length (i.e., unfragmented) nucleic acids), determining the peak area of ​​the total nucleic acids, and dividing the peak area of ​​the major nucleic acid peak by the peak area of ​​the total nucleic acids. Similarly, the expression "RNA integrity" refers to the ratio of full-length RNA (i.e., unfragmented RNA) to the total amount of RNA in a sample (i.e., the sum of unfragmented and fragmented RNA). RNA integrity can be determined by separating the RNA by chromatography (e.g., using capillary electrophoresis), determining the peak area of ​​the major RNA peak (i.e., the peak area of ​​full-length RNA (i.e., unfragmented) RNA), determining the peak area of ​​total RNA, and dividing the peak area of ​​the major RNA peak by the peak area of ​​total RNA.

[0163] As used herein, the term "isotonic agent" refers to a compound that is typically added to an injectable preparation to prevent osmotic shock at the injection site during administration, thereby reducing local irritation. Typical isotonic agents include salts (e.g., physiological saline), sugars (e.g., glucose, sucrose, dextrose, trehalose), and sugar alcohols (e.g., glycerin, mannitol). "Isotonicity" is preferably a colligative property that depends primarily on the number of dissolved particles in the solution. Therefore, the amount of isotonic agent added depends on the particular preparation. Typically, an osmotic pressure of 0.280 to 0.320 osmoles is considered isotonic.

[0164] The term "freeze-protecting agent" refers to a substance added to a formulation (e.g., a formulation or composition) to protect the active ingredients of the formulation during the freezing stage.

[0165] In this invention, the term "peptide" includes oligos and polypeptides and refers to substances containing about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150 consecutive amino acids linked to one another via peptide bonds. The term "polypeptide" refers to a large peptide, in particular a peptide containing at least about 151 amino acids. Both "peptide" and "polypeptide" are protein molecules, but in this specification, the terms "protein" and "polypeptide" are generally used as synonyms.

[0166] A “therapeutic polypeptide” has a positive or beneficial effect on a subject’s condition or disease when administered to the subject in a therapeutically effective dose. In some embodiments, therapeutic polypeptides have curative or mitigating properties and can be administered to improve, mitigate, reduce, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. Therapeutic polypeptides may have preventive properties and can be used to delay the onset of a disease or reduce the severity of such a disease or disease. The term “therapeutic polypeptide” includes the entire polypeptide or peptide, and may also refer to their therapeutically active fragments. It also includes therapeutically active variants of polypeptides. Examples of therapeutically active polypeptides include, but are not limited to, immunostimulants such as antigens for vaccination and cytokines. The terms “therapeutic polypeptide” and “pharmaceutically active peptide or polypeptide” are used interchangeably herein.

[0167] According to various embodiments of this disclosure, nucleic acids such as RNA (e.g., mRNA) encoding a peptide, polypeptide, or protein are taken up or introduced into a cell that may be present in vitro or within a subject, i.e., transfected or transduced, resulting in the expression of the peptide, polypeptide, or protein. The cell may express the encoded peptide, polypeptide, or protein intracellularly (e.g., in the cytoplasm and / or nucleus), secrete the encoded peptide, polypeptide, or protein, and / or express it on its surface.

[0168] According to the present invention, terms such as “nucleic acid expression” and “nucleic acid coding” or similar terms are used interchangeably herein, and with respect to a particular peptide, polypeptide, or protein, it means that when a nucleic acid is present in a suitable environment, preferably within a cell, it can be expressed to produce the peptide, polypeptide, or protein.

[0169] The term "part" refers to a fragment. In relation to specific structures such as amino acid sequences or proteins, the term "part" may refer to a continuous or discontinuous portion of that structure.

[0170] In this specification, the terms “part” and “fragment” are used interchangeably and refer to a contiguous element. For example, a part of an amino acid sequence or a structure such as a protein refers to a contiguous element of said structure. When used in the context of a composition, the term “part” means a portion of a composition. For example, a part of a composition may be any portion from 0.1% to 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0171] A “fragment” refers to a sequence representing a portion of an amino acid sequence (peptide, polypeptide, or protein), i.e., a shortened amino acid sequence at the N-terminus and / or C-terminus. A C-terminal shortened fragment (N-terminal fragment) is obtained, for example, by translation of a truncated open reading frame lacking the 3' end of the open reading frame. A C-terminal shortened fragment (C-terminal fragment) is obtained, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame, provided that the truncated open reading frame contains a start codon to initiate translation. An amino acid sequence fragment contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. Preferably, an amino acid sequence fragment contains at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence. A fragment of an amino acid sequence may, for example, contain up to eight, and more particularly, up to ten, twelve, fifteen, twenty, thirty, or fifty consecutive amino acid sequences.

[0172] According to the present invention, a part or fragment of a peptide, polypeptide, or protein preferably has at least one functional property of the peptide, polypeptide, or protein from which it is derived. Such functional properties include pharmacological activity, interaction with other peptides, polypeptides, or proteins, enzymatic activity, interaction with antibodies, and selective binding of nucleic acids. For example, a pharmacologically active fragment of a peptide, polypeptide, or protein has at least one of the pharmacological activities of the peptide, polypeptide, or protein from which the fragment is derived.

[0173] In this specification, as used with respect to amino acid sequences (peptides, polypeptides, or proteins), “variant” means an amino acid sequence that differs from the parent amino acid sequence by at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence may be a naturally occurring amino acid sequence or a wild-type (WT) amino acid sequence, or a modified version of a wild-type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, for example, a difference of 1 to about 20 amino acids, preferably a difference of 1 to about 10 or 1 to about 5 amino acids compared to the parent.

[0174] In amino acid sequences, "wild-type," "WT," or "native" refers to the naturally occurring amino acid sequence, including allele variations. Wild-type amino acid sequences, peptides, polypeptides, or proteins have an unmodified amino acid sequence. Similarly, in nucleic acid sequences, "wild-type," "WT," or "native" refers to the naturally occurring nucleic acid sequence, including allele variations. For example, a wild-type coding sequence refers to a naturally occurring coding sequence that has not been intentionally modified.

[0175] As used herein, "coding sequence" refers to the portion of nucleic acid (e.g., DNA or RNA of a gene) that codes for a protein.

[0176] The expression "guanosine / cytosine (G / C) content" or "G / C content" refers to the percentage of bases that are either guanine (G) or cytosine (C) in a DNA or RNA molecule. G / C content may be given for a specific portion of DNA or RNA, or for the entire genome. When G / C content refers to a portion, it may refer to the G / C content of an individual gene or part of a gene (domain), a group or cluster of genes, a non-coding region, a coding sequence, or a synthetic oligonucleotide such as a primer.

[0177] In this specification, “variants” of an amino acid sequence (peptide, protein, or polypeptide) include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term “variant” includes all mutants, splice variants, post-translational modification variants, conformations, isoforms, allelic variants, species variants, and species homologs, especially those occurring in nature. The term “variant” specifically includes fragments of amino acid sequences.

[0178] In some embodiments, the similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, optionally at least 80%, preferably at least 90%, most preferably at least 95% (e.g., 99%). The similarity or identity is preferably shown for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the amino acid region of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the similarity or identity is preferably expressed for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, and in some embodiments, for consecutive amino acids. In some embodiments, the similarity or identity is expressed for the entire length of the reference amino acid sequence.

[0179] "Sequence similarity" refers to the proportion of amino acids that are identical or represent a conserved amino acid substitution. "Sequence identity" between two amino acid sequences refers to the proportion of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the proportion of nucleotides that are identical between the sequences.

[0180] The terms "% identical" and "% identity," or similar terms, are intended to refer specifically to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences being compared. This percentage is purely statistical, and the differences between the two sequences may, but may not, be randomly distributed across the entire length of the sequences being compared. The comparison of two sequences is typically performed by comparing the sequences with respect to a segment or "comparison window" after optimal alignment, and identifying the local regions of the corresponding sequences. Optimal alignment for comparison can be performed manually, or using local homology algorithms such as those described by Smith and Waterman, 1981, Ads App. Math. 2, 482; Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444; or computer programs that utilize these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the identity percentage of two sequences is determined using the BLASTN or BLASTP algorithm available on the National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) setting the expected fragment threshold to 10; (ii) setting the fragment word size to 28; (iii) setting the maximum number of matches within the fragment query range to 0; (iv) setting the fragment match / mismatch score to 1, -2; (v) setting the fragment gap cost to linear; and (vi) using a filter for low fragment complexity regions. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) setting the expected threshold to 10; (ii) setting the word size to 3; (iii) setting the maximum number of matches within the query range to 0; (iv) setting the matrix to BLOSUM62; (v) setting the gap cost to exist: 11 and expand: 1; and (vi) conditional configuration score matrix adjustment.

[0181] The identity percentage is obtained by determining the number of corresponding identical positions in the arrays being compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference array), and multiplying the result by 100.

[0182] In some embodiments, a fragment or variant of an amino acid sequence (peptide, polypeptide, or protein) is preferably a “functional fragment” or “functional variant.” The term “functional fragment” or “functional variant” of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to the amino acid sequence from which it is derived, i.e., functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the terms “functional fragment” or “functional variant” refer in particular to a mutant molecule or sequence containing an amino acid sequence in which one or more amino acids are changed compared to the amino acid sequence of the parent molecule or sequence, and which can still perform one or more functions of the parent molecule or sequence, e.g., the function of inducing an immune response (immunogenic fragment). In one embodiment, the modification of the amino acid sequence of the parent molecule or sequence does not significantly affect or alter the properties of the molecule or sequence. In different embodiments, the function of a functional fragment or functional variant may be reduced but still significant; for example, the immunogenicity of a functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence. However, in other embodiments, the immunogenicity of a functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0183] The amino acid sequence (peptide, protein, or polypeptide) “derived” from a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the initial amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to that particular sequence or fragment. An amino acid sequence derived from a particular amino acid sequence may also be a variant of that particular sequence or fragment. For example, it will be understood by those skilled in the art that antigens suitable for use herein may be modified so as to have a different sequence from the naturally occurring or native sequence from which they are derived, while retaining the desired activity of the naturally occurring or native sequence.

[0184] In some embodiments, “isolation” means being modified or removed (e.g., purified) from its natural state or from an artificial composition such as a composition from a manufacturing process. For example, nucleic acids or peptides that are naturally present in living animals are not “isolated,” but the same nucleic acids or peptides that have been partially or completely separated from their naturally occurring coexisting substances are “isolated.” Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment such as a host cell. In some embodiments, the RNA (e.g., mRNA) used in this disclosure is in a substantially purified form. In some embodiments, a solution (preferably an aqueous solution) of the substantially purified form of RNA (e.g., mRNA) includes a first buffer system.

[0185] The terms “genetic modification” or simply “modification” include the transfection of cells with nucleic acids. The term “transfection” relates to the introduction of nucleic acids, particularly RNA, into cells. For the purposes of this disclosure, the term “transfection” also includes the introduction of nucleic acids into cells, or the uptake of nucleic acids by such cells, in which case the cells may be present in a subject, e.g., a patient. Accordingly, according to the present invention, cells for nucleic acid transfection described herein can be present in vitro (e.g., in a cell culture) or in vivo, and for example, the cells may form organs, tissues and / or parts of an organism of a patient.

[0186] As used in this disclosure, “antigen” encompasses any substance that elicits an immune response, and / or any substance to which an immune response or immune mechanism, such as a cellular response, is directed. This includes situations in which an antigen is processed into an antigenic peptide and an immune response or immune mechanism is directed to one or more antigenic peptides, particularly when presented in the context of MHC molecules. In particular, “antigen” refers to any substance, preferably a peptide or protein, that specifically reacts with an antibody or T lymphocyte (T cell). According to the present invention, the term “antigen” includes any molecule containing at least one epitope, such as a T cell epitope. Preferably, in the context of the present invention, an antigen is a molecule that, after being optionally processed, preferably induces an immune response specific to the antigen (including cells expressing the antigen). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0187] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. Disease-associated antigens are molecules containing epitopes that stimulate the host's immune system to trigger a cellular antigen-specific immune response and / or humoral antibody response to the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with infection by microorganisms (typically microbial antigens such as bacterial and viral antigens), or antigens associated with cancer (typically tumor antigens, such as tumor antigens).

[0188] In some embodiments, the antigen is a tumor antigen, i.e., a part of a tumor cell, particularly one that is primarily present within the tumor cell or present as a surface antigen of the tumor cell. In other embodiments, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, such as a virus, bacterium, single-celled organism, or parasite, such as a viral antigen, e.g., a viral ribonucleoprotein or coat protein. In particular, the antigen needs to be presented by an MHC molecule, resulting in a regulation such as the activation of immune system cells, preferably CD4+ and CD8+ lymphocytes, through the modulation of T cell receptor activity.

[0189] The term “tumor antigen” or “tumor-associated antigen” refers to components of cancer cells derived from the cytoplasm, cell surface, or cell nucleus. In particular, it refers to antigens produced intracellularly or as surface antigens of tumor cells. For example, tumor antigens include carcinoembryonic antigens, α1-fetoprotein, isoferritin, fetal glycoprotein sulfate, α2-H-ferroprotein, γ-fetoprotein, and various viral tumor antigens. According to some embodiments of this disclosure, tumor antigens include tumors or cancers with respect to type and / or level of expression, as well as any antigens characteristic of tumors or cancer cells.

[0190] The term "viral antigen" refers to a viral component that possesses antigenic properties, i.e., the ability to trigger an immune response in an individual. Viral antigens can be either the ribonucleoprotein or envelope protein of the virus.

[0191] The term "bacterial antigen" refers to a bacterial component that possesses antigenic properties, i.e., the ability to elicit an immune response in an individual. Bacterial antigens may originate from the bacterial cell wall or cytoplasmic membrane.

[0192] The terms “epitope,” “antigen fragment,” “immunogenic peptide,” and “antigen peptide” are used interchangeably herein and may, for example, relate to an antigen or an incomplete representation of an antigen that can elicit an immune response against cells expressing or containing an antigen. In some embodiments, these terms relate to the immunogenic portion of an antigen. Preferably, it is a portion of an antigen that is recognized (i.e., specifically binds) by a T cell receptor, particularly when presented in the context of an MHC molecule. Certain preferred immunogenic portions bind to MHC class I or class II molecules. The term “epitope” refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope may be recognized by a T cell, a B cell, or an antibody. The antigen epitope may include a continuous or discontinuous portion of the antigen and may be about 5 to about 100, for example, about 5 to about 50, more preferably about 8 to about 30, and most preferably about 8 to about 25 amino acid lengths. For example, the epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid lengths. In some embodiments, the epitope is about 10 to about 25 amino acid lengths.

[0193] Peptide antigens and protein antigens may be 2 to 100 amino acids long, for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids. In some embodiments, the peptide may be longer than 50 amino acids. In some embodiments, the peptide may be longer than 100 amino acids.

[0194] A peptide or protein antigen is any peptide or protein that can induce or enhance the immune system's ability to produce antibodies and T-cell responses against the peptide or protein.

[0195] In some embodiments, the vaccine antigen, i.e., an antigen that, when inoculated into a target, induces an immune response, is recognized by immune effector cells. In some embodiments, when the vaccine antigen is recognized by immune effector cells, in the presence of appropriate co-stimulatory signals, it can induce stimulation, priming, and / or proliferation of immune effector cells having antigen receptors that recognize the vaccine antigen. In the context of embodiments of this disclosure, the vaccine antigen is preferably presented or present on the surface of cells, preferably antigen-presenting cells. In some embodiments, the antigen is presented by lesion cells (e.g., tumor cells or infected cells).

[0196] The terms “immune response” and “immune reaction” are used herein interchangeably in their conventional sense and refer to an integrated bodily response to an antigen, which may refer to a cellular immune response, a humoral immune response, or both. According to this disclosure, the terms “immune response to” or “immune response against” a factor such as an antigen, cell, or tissue relate to an immune response such as a cellular response to a factor. An immune response is the production of antibodies against one or more antigens, and CD4 + and CD8 + T lymphocytes, for example, CD8 + The response may include one or more reactions selected from the group consisting of proliferation of antigen-specific T lymphocytes, such as T lymphocytes, which can be detected by various proliferation or cytokine production tests in vitro.

[0197] In the context of this disclosure, the terms “induce an immune response” and “trigger an immune response,” as well as similar terms, refer to the induction of an immune response, such as the induction of a cellular immune response, a humoral immune response, or both. An immune response may be protective / prophylactic and / or therapeutic. An immune response may occur in response to any immunogen, antigen, or antigenic peptide, preferably in response to tumor-associated antigens or pathogen-associated antigens (e.g., viral antigens (influenza virus (A, B, or C), CMV, or RSV, etc.)). “Induction” here may mean that there was no immune response to a particular antigen or pathogen prior to induction, or that there was a certain level of immune response to a particular antigen or pathogen prior to induction, and that the immune response is enhanced after induction. Thus, “induce an immune response” in this context also includes “enhance an immune response.” In some embodiments, after inducing an immune response in an individual, the individual is protected from developing diseases such as infectious diseases or cancer, or the disease state is improved by inducing an immune response.

[0198] The terms “vaccination” and “immunization” refer to a process of treating an individual for therapeutic or preventive reasons, and relate to a procedure that stimulates an immune response to the immunogen or antigen, cells, characterized by administering one or more immunogens or antigens, or derivatives thereof (in particular form of encoding RNA (in particular mRNA)) to an individual, or by presenting the said immunogen or antigen.

[0199] In the context of this disclosure, the term “transcription” refers to the process by which the genetic code in a DNA sequence is transcribed into RNA (particularly mRNA). RNA (particularly mRNA) can then be translated into peptides, polypeptides, or proteins.

[0200] In relation to RNA, the terms "expression" or "translation" refer to the process within a cell's ribosomes in which an mRNA chain directs the assembly of amino acid sequences to create peptides or proteins.

[0201] The medical preparations, in particular kits, described herein may include explanatory materials or instructions. “Explanatory materials” or “Instructions” as used herein may include publications, records, charts, or other expressive media that can be used to convey the usefulness of the compositions and methods of this disclosure. The explanatory materials for the kits of this disclosure may, for example, be affixed to the container containing the compositions of this disclosure, or they may be shipped together with the container containing the compositions. Alternatively, the explanatory materials may be shipped separately from the container for the purpose of joint use by the recipient of the explanatory materials and the compositions.

[0202] As used herein, the terms “optional” or “optionally” mean that the events, situations, or conditions described thereafter may or may not occur, and such descriptions include both cases in which such events, situations, or conditions occur and cases in which they do not occur.

[0203] In this specification, the structural formula of a compound may represent a specific isomer of that compound. However, it should be understood that the present invention encompasses all isomers and mixtures of isomers, including structurally arising geometric isomers, optical isomers based on chiral carbons, stereoisomers, tautomers, etc., and is not limited to the description of a specific formula. Furthermore, in this specification, the structural formula of a compound may represent a specific salt and / or solvate of that compound. However, it should be understood that the present invention encompasses all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g., hydrates), and is not limited to the description of a specific salt and / or solvate.

[0204] An "isomer" is a compound that has the same molecular formula but differs in structure ("structural isomer") or the geometric (spatial) arrangement of functional groups or atoms ("stereoisomer"). An "enantiomer" is a pair of stereoisomers that are mirror images of each other and cannot be superimposed. A "racemic mixture" or "racemic compound" contains equal amounts of enantiomers and is represented by the prefix (±). A "diastereomer" is a stereoisomer that cannot be superimposed on each other and is not a mirror image of each other. A "tautomer" is a structural isomer of the same chemical substance that, even in its pure form, can spontaneously and reversibly interconvert through the movement of individual atoms or groups of atoms; that is, tautomers are in a state of dynamic chemical equilibrium with each other. An example of a tautomer is the keto-enol tautomer. "Contour isomers" are stereoisomers that, while formally single-bonded, can be interconverted by rotation. In particular, they include those that form (hetero)cyclic rings with different three-dimensional structures, such as chair, semi-chair, boat, and twisted-boat cyclohexane.

[0205] In this specification, "solvate" refers to an adduct complex of a substance dissolved in a solvent (for example, an organic solvent (e.g., aliphatic alcohols (methanol, ethanol, n-propanol, isopropanol, etc.), acetone, acetonitrile, ether, etc.), water, or a mixture of two or more of these liquids), and the adduct complex exists in the form of crystals or mixed crystals. The amount of solvent contained in the adduct complex may be stoichiometric or non-stoichiometric. "Hydrate" refers to a solvate in which the solvent is water.

[0206] The term "average diameter" refers to the average hydrodynamic diameter of a particle measured by dynamic light scattering (DLS) using data analysis with the so-called cumulant algorithm, which has the dimension of length, known as Z average This results in a dimensionless multidispersion index (PDI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the particle's "average diameter", "diameter", or "size" is given by this Z. average It is used synonymously with the value of [this value].

[0207] In some embodiments, the "polydispersion index" is calculated based on dynamic light scattering measurements by so-called cumulant analysis, as described in the definition of "average diameter." Under certain preconditions, this can be adopted as a measure of the size distribution of the nanoparticle aggregate.

[0208] The "radius of rotation" of a particle around its axis of rotation (hereinafter referred to as R g (Abbreviated as R) is the radial distance from the axis of rotation to the point where, assuming the total mass of the particle is concentrated, the moment of inertia of the particle around a given axis is the same as that of the actual mass distribution. Mathematically, R g m is the root mean square distance from the center of mass or a given axis of the particle's components. For example, mass m i It consists of n mass elements (i=1, 2, 3, ..., n), with a fixed distance s from the center of mass. i In the case of polymers located at R g is the s of all mass elements i 2 It is the square root of the mass average of and can be calculated as follows:

number

[0209] The radius of rotation can be experimentally determined or calculated, for example, using light scattering. In particular, small scattering vectors

number

number

[0210] The "hydrodynamic radius" of a particle (sometimes called the "Stokes radius" or "Stokes-Einstein radius") is the radius of a hypothetical rigid sphere diffusing at the same velocity as the particle. The hydrodynamic radius is related to the particle's mobility, taking into account not only the particle's size but also solvent effects. For example, a larger hydrodynamic radius may be greater for smaller charged particles with stronger hydration than for larger charged particles with weaker hydration. This is because smaller particles drag more water molecules as they move through the solution. Since the actual dimensions of particles in a solvent cannot be directly measured, the hydrodynamic radius can be defined using the Stokes-Einstein equation.

number

[0211] As used herein, the term “aggregate” refers to a cluster of particles, where the particles are identical or very similar and are attached to each other non-covalently (e.g., via ionic interactions, H-bridge interactions, dipole interactions, and / or van der Waals interactions).

[0212] The term "light scattering" used here refers to a physical process in which light is forced to deviate from a straight trajectory in one or more paths due to local non-uniformity in the medium through which it passes.

[0213] The term "UV" refers to ultraviolet light, a band of the electromagnetic spectrum with wavelengths ranging from 10 nm to 400 nm, that is, wavelengths shorter than visible light but longer than X-rays.

[0214] As used herein, the terms “multi-angle light scattering” or “MALS” relate to a technique for measuring light scattered by a sample at multiple angles. “Multi-angle” means that scattered light can be detected at different discrete angles, for example, by a single detector moving over a range including a specific selected angle, or by an array of detectors fixed at a specific angular position. In a preferred embodiment, the light source used in MALS is a laser light source (MALLS: multi-angle laser light scattering). Based on the MALS signal of a particle-containing composition, the radius of gyration (R) can be determined by using an appropriate format (e.g., Zimm plot, Berry plot, or Debye plot). g ) can be determined, and thereby the size of the particles can be determined. Preferably, the Zimm plot is represented in graph form using the following equation,

number

number

number

[0215] As used herein, the terms “dynamic light scattering” or “DLS” refer to a technique for determining particle size and size distribution profiles, particularly the hydrodynamic radius of particles. A monochromatic light source (usually a laser) is passed through a polarizer and irradiated onto the sample. The scattered light then passes through a second polarizer, where it is detected, and the resulting image is projected onto a screen. When light strikes particles in a solution, the light diffracts in all directions. The diffracted light from the particles may interfere constructively (bright regions) or cancel each other out (dark regions). This process is repeated at short time intervals, and the resulting set of speckle patterns is analyzed by an autocorrelator that compares the intensity of light at each spot over time.

[0216] As used herein, the terms “static light scattering” or “SLS” refer to a technique for determining particle size and size distribution profiles, particularly the radius of gyration and / or molar mass of particles. A high-intensity monochromatic light (usually a laser) is shone onto a solution containing particles. One or more detectors are used to measure the scattering intensity at one or more angles. Angle dependence is necessary to obtain accurate measurements of the molar mass and size of all polymers at the radius. Therefore, simultaneous measurements at multiple angles with respect to the direction of incident light are known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS) and are generally considered a standard implementation of static light scattering.

[0217] nucleic acid The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. This term includes genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids can exist as single-stranded or double-stranded molecules, linear or covalently bound in a circular manner. Nucleic acids can be isolated. According to the present invention, the term "isolated nucleic acid" means that the nucleic acid is (i) amplified in vitro, for example, by polymerase chain reaction (PCR) in the case of DNA, or by in vitro transcription (for example, using RNA polymerase) in the case of RNA, (ii) recombinantly produced by cloning, (iii) purified, for example, by cleavage and separation by gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis. Nucleic acids may be mRNA, auto-amplified RNA (saRNA), or trans-amplified RNA (taRNA), and are preferably mRNA. In some embodiments, the nucleic acid may be RNA comprising a coding region (e.g., mRNA) and an inhibitory region (e.g., miRNA).

[0218] The term "nucleoside" (hereinafter abbreviated as "N") refers to compounds that are considered to be nucleotides without a phosphate group. Nucleosides are nucleic acid bases bonded to a sugar (e.g., ribose or deoxyribose), while nucleotides consist of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0219] The five standard nucleosides that typically make up natural nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. These five nucleosides are usually abbreviated with single-letter codes U, A, T, C, and G, respectively. However, thymidine is often written as "dT" (where "d" stands for "deoxy") because it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is present in deoxyribonucleic acid (DNA) but not in ribonucleic acid (RNA). Conversely, uridine is present in RNA but not in DNA. The remaining three nucleosides can be present in both RNA and DNA. In RNA, they are represented as A, C, and G, and in DNA, as dA, dC, and dG.

[0220] The modified purine (A or G) or pyrimidine (C, T, or U) base moiety is preferably one or more alkyl groups, more preferably one or more C 1-4 Modified with an alkyl group, more preferably one or more methyl groups. Specific examples of modified purine or pyrimidine base moieties include N 7 -alkylguanine, N 6 -Alkyl adenine, 5-alkylcytosine, 5-alkyluracil, and N(1)-alkyluracil, for example, N 7 -C 1-4 Alkylguanine, N 6 -C 1-4 Alkyl adenine, 5-C 1-4 Alkylcytosine, 5-C 1-4 Examples include alkyluracil and N(1)-C1-4 alkyluracil, preferably N7 -methylguanine, N 6 -These are methyladenine, 5-methylcytosine, 5-methyluracil, and N(1)-methyluracil.

[0221] In some embodiments of all aspects of this disclosure, the nucleic acid is DNA.

[0222] In this specification, the term “DNA” refers to nucleic acid molecules containing deoxyribonucleotide residues. In preferred embodiments, DNA contains all or most of the deoxyribonucleotide residues. In this specification, “deoxyribonucleotide” refers to a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA includes, but is not limited to, isolated DNA such as double-stranded DNA, single-stranded DNA, partially purified DNA, essentially pure DNA, synthetic DNA, recombinant DNA, and modified DNA that differs from native DNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may refer to the addition of nucleotides within the DNA or non-nucleotide substances to the DNA ends. Also in this specification, nucleotides in DNA may be chemically synthesized nucleotides or non-standard nucleotides such as ribonucleotides. In this disclosure, these modified DNAs are considered analogues of naturally occurring DNA. If the content of deoxyribonucleotide residues in a molecule exceeds 50% based on the total number of nucleotide residues in the molecule (for example, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), then the molecule contains “the majority of deoxyribonucleotide residues.” The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogues thereof).

[0223] The DNA may be recombinant DNA, and may be obtained by cloning nucleic acids, particularly cDNA. The cDNA may be obtained by reverse transcription of RNA.

[0224] RNA In some embodiments of all aspects of this disclosure, the nucleic acid is RNA.

[0225] According to the present invention, the term “RNA” means a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, RNA contains all or most of the ribonucleotide residues. As used herein, “ribonucleotide” refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA), essentially pure RNA, synthetic RNA, RNA produced by recombination, and modified RNA that differs from native RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to the addition of nucleotides within the RNA or non-nucleotide substances to the RNA terminus. It is intended herein that the nucleotides in RNA may be chemically synthesized nucleotides or non-standard nucleotides such as deoxynucleotides. In this disclosure, these modified nucleotides (or modified nucleosides) are referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNA containing such modified nucleotides or nucleosides (i.e., modified RNA) is referred to as an analog of naturally occurring RNA. A molecule contains “a majority of ribonucleotide residues” if the ribonucleotide residue content in the molecule exceeds 50% based on the total number of nucleotide residues in the molecule (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%). The total number of nucleotide residues in the molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0226] RNA includes mRNA, tRNA, ribosomal RNA (rRNA), nuclear small RNA (snRNA), self-amplified RNA (saRNA), trans-amplified RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (antisense ssRNA, small interfering RNA (siRNA), microRNA (miRNA), etc.), activating RNA (small activating RNA, etc.), and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.

[0227] In preferred embodiments, the RNA includes an open reading frame (ORF) encoding a peptide, polypeptide, or protein.

[0228] As used herein, the terms “in vitro transcription” or “IVT” mean that transcription (i.e., RNA production) is carried out in a manner that does not use cells. That is, IVT does not use living cells / cultured cells, but uses transcription mechanisms extracted from cells (e.g., including cell lysates or their isolated components, RNA polymerase (preferably T7, T3, or SP6 polymerase)).

[0229] mRNA In some embodiments of all aspects of this disclosure, the nucleic acid is mRNA.

[0230] In this invention, the term "mRNA" means "messenger RNA" and includes "transcripts" that can be produced using a DNA template. Generally, mRNA encodes a peptide, polypeptide, or protein. Typically, mRNA includes a 5'-UTR, a peptide / protein coding region, and a 3'-UTR. In the context of this disclosure, mRNA is preferably produced by in vitro transcription (IVT) from a DNA template. As previously stated, in vitro transcription methods are known to those skilled in the art, and various in vitro transcription kits are commercially available.

[0231] Although mRNA is single-stranded, it may contain self-complementary sequences, which allow parts of the mRNA to fold and pair, forming a double helix.

[0232] In this invention, "dsRNA" means double-stranded RNA, which is RNA having two partially or completely complementary strands.

[0233] In preferred embodiments of the present invention, mRNA refers to RNA transcripts that encode peptides, polypeptides, or proteins.

[0234] In some embodiments, preferably the RNA encoding a peptide, polypeptide, or protein has a length of at least 45 nucleotides (e.g., at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, for example, up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides, or up to 10,000 nucleotides.

[0235] As is established in the art, RNA (such as mRNA) generally comprises a 5' untranslated region (5'-UTR), a peptide / polypeptide / protein coding region, and a 3' untranslated region (3'-UTR). In some embodiments, RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis. In one embodiment, RNA (such as mRNA) is produced by in vitro transcription using a DNA template. In vitro transcription methods are known to those skilled in the art; for example, Molecular Cloning: A Laboratory Manual, 2 nd See Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. In addition, various in vitro transcription kits are commercially available from companies such as Thermo Fisher Scientific (TranscriptAid® T7 kit, MEGAscript® T7 kit, MAXIscript®, etc.), New England BioLabs Inc. (HiScribe® T7 kit, HiScribe® T7 ARCA mRNA kit, etc.), Promega (RiboMAX®, HeLaScribe®, Riboprobe® systems, etc.), Jena Bioscience (SP6 or T7 transcription kit, etc.), and Epicentre (AmpliScribe®, etc.). To provide modified RNA (e.g., mRNA), accordingly modified nucleotides, such as modified native nucleotides, non-native nucleotides, and / or modified non-native nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or the mRNA can be modified after transcription, and / or modifications can be added to the mRNA.

[0236] In some embodiments, the RNA (e.g., mRNA) is in vitro transcription RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for regulating transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases include T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is regulated by the T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0237] In some embodiments of this disclosure, RNA (e.g., mRNA) is “replicon RNA” (e.g., “replicon mRNA”) or simply “replicon,” and in particular is “self-replicating RNA” (e.g., “self-replicating mRNA”) or “self-amplified RNA” (or “self-amplified mRNA”). In one particularly preferred embodiment, the replicon or self-replicating RNA (e.g., self-replicating mRNA) is derived from or contains elements derived from ssRNA viruses, particularly positive-strand ssRNA viruses such as alphaviruses. Alphaviruses are typical representative examples of positive-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837–856 for the life cycle of alphaviruses). The total genome length of many alphaviruses is typically in the range of 11,000–12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The alphavirus genome encodes non-structural proteins (involved in the transcription, modification, replication, and protein modification of viral RNA) and structural proteins (formation of the viral particle). The genome typically contains two open reading frames (ORFs). The four non-structural proteins (nsP1-nsP4) are usually encoded together by a first ORF that begins near the 5' end of the genome, while the alphavirus structural proteins are encoded together by a second ORF located downstream of the first ORF and extending to near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences encoding non-structural proteins are translated from the genomic RNA, while the genetic information encoding structural proteins is translated from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol.87 pp.111-124).After infection, i.e., in the early stages of the viral life cycle, the (+) strand genomic RNA functions directly like messenger RNA in the translation of the open reading frame encoding the non-structural polyprotein (nsP1234). Alphavirus-derived vectors have been proposed for delivering foreign genetic information to target cells or target organisms. A simple method involves replacing the open reading frame encoding the structural protein of the alphavirus with an open reading frame encoding the protein of interest. Alphavirus-based trans replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule can be trans-replicated by that replicase (hence the name trans-replication system). Trans replication requires the presence of both of these nucleic acid molecules within a particular host cell. The nucleic acid molecule that can be trans-replicated by the replicase must contain specific alphavirus sequence elements to enable recognition and RNA synthesis by the alphavirus replicase.

[0238] In some embodiments of the present disclosure, RNA (such as mRNA) described herein (e.g., included in compositions of the present disclosure and / or used in methods of the present disclosure) includes one or more modifications, for example, to enhance its stability and / or to enhance its translation efficiency and / or to reduce its immunogenicity and / or to reduce its cytotoxicity. For example, to increase the expression of RNA (such as mRNA), it may be modified within the coding region, i.e., the sequence encoding the peptide or protein to be expressed, preferably without altering the sequence of the peptide or protein to be expressed. Such modifications are described, for example, in WO 2007 / 036366 and PCT / EP2019 / 056502 and include 5' cap structures, extension or cleavage of naturally occurring poly(A) tails, modification of the 5' and / or 3' untranslated region (UTR), e.g., introduction of UTRs unrelated to the coding region of the RNA, substitution of one or more naturally occurring nucleotides with synthetic nucleotides, and codon optimization (e.g., altering, preferably increasing, the G / C content of the RNA). In the context of modified mRNA as described herein, the term “modification” preferably refers to any modification of the RNA (e.g., mRNA) that is not naturally present.

[0239] In some embodiments, the RNA described herein (e.g., mRNA) includes a 5' cap structure. In one embodiment, the mRNA does not have an uncapped 5' triphosphate. In one embodiment, the RNA described herein (e.g., mRNA) may include a conventional 5' cap and / or a 5' cap analogue. The term “conventional 5' cap” refers to the cap structure found at the 5' end of an mRNA molecule, and generally consists of a guanosine 5' triphosphate (Gppp) attached to the 5' end of the next nucleotide of the mRNA via its triphosphate moiety (i.e., guanosine is attached to the rest of the mRNA via a 5'-5' triphosphate bond). 7 It may also be methylated at the position (as a result, the cap structure m 7(This results in Gppp). The term "5'-cap analogue" is based on the conventional 5'-cap, but to avoid the 5'-cap analogues being integrated in the reverse direction, m 7 This refers to a 5'-cap in which either the 2' or 3' position of the guanosine structure is modified (such 5'-cap analogs are also called anti-reverse cap analogs (ARCA)). Particularly preferred 5'-cap analogs have one or more substituents on the crosslinked and uncrosslinked oxygen atoms of the phosphate crosslink, for example, a 5'-cap analog with phosphorothioate modification of β-phosphate (e.g., m2 7,2'O Examples include G(5')ppSp(5')G (referred to as beta-S-ARCA or β-S-ARCA). Particularly preferred 5'-cap analogs are described in WO 2008 / 157688, WO 2011 / 015347, and WO 2019 / 175356. Providing RNA (e.g., mRNA) having the 5'-cap structure described herein can be achieved by in vitro transcription of a DNA template in the presence of the corresponding 5'-cap compound, in which case the 5'-cap structure is co-transcribed into the generated RNA (e.g., mRNA) strand, or the RNA (e.g., mRNA) may be generated, for example, by in vitro transcription, and the 5'-cap structure may be conjugated to the mRNA post-transcriptionally using a capping enzyme, for example, the capping enzyme of vaccinia virus.

[0240] In some embodiments, RNA (e.g., mRNA) includes cap0, cap1, or cap2, preferably cap1 or cap2. According to this disclosure, the term "cap0" refers to the structure "m 7 "GpppN" means "GpppN", where N is any nucleoside having an OH group at the 2' position. According to this disclosure, the term "cap1" refers to the structure "m 7 "GpppNm" means any nucleoside having an OCH3 group at the 2' position. According to this disclosure, the term "cap2" refers to the structure "m 7This means "GpppNmNm", where each Nm is an arbitrary nucleoside that independently has an OCH3 group at the 2' position.

[0241] In this specification, the terms “polyA tail” or “polyA sequence” refer to a contiguous or interrupted sequence of adenylate residues typically located at the 3' end of an RNA (e.g., mRNA) molecule. PolyA tails or polyA sequences are known to those skilled in the art and may follow the 3'-UTR in RNA (e.g., mRNA) described herein. An uninterrupted polyA tail is characterized by a contiguous sequence of adenylate residues.

[0242] The poly-A tail may be of any length. In some embodiments, the poly-A tail may contain, essentially consist of, or consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, in particular about 120 A nucleotides.

[0243] In some embodiments, the poly(A) tail is added during RNA transcription, for example, during the preparation of in vitro transcribed RNA, based on a DNA template containing repeating dT nucleotides (deoxythymidylate) in a strand complementary to the coding strand. The DNA sequence encoding the poly(A) tail (coding strand) is called the poly(A) cassette.

[0244] In some embodiments, the poly(A) cassette present in the coding strand of DNA is essentially composed of dA nucleotides but interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). Such a random sequence may be 5–50, 10–30, or 10–20 nucleotides in length. Such cassettes are disclosed in International Publication No. 2016 / 005324, which is incorporated herein by reference. Any poly(A) cassette disclosed in International Publication No. 2016 / 005324 may be used in this disclosure. Accordingly, in some embodiments, the poly(A) tail contained in the RNA (in particular, mRNA) molecule described herein is essentially composed of A nucleotides but interrupted by a random sequence of four nucleotides (A, C, G, U). Such a random sequence may be 5–50, 10–30, or 10–20 nucleotides in length.

[0245] In some embodiments, nucleotides other than A nucleotides are not present on either side of the poly-A tail at its 3' end; that is, the poly-A tail is not masked by or followed by nucleotides other than A at its 3' end.

[0246] In some embodiments, the poly-A tail may contain at least 20, at least 30, at least 40, at least 80, or at least 100-500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100-500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100-500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail contains at least 100 nucleotides. In some embodiments, the poly-A tail contains about 150 nucleotides. In some embodiments, the poly-A tail contains about 120 nucleotides. In some embodiments, the polyA tail contains or consists of the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the polyA sequence has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 3.

[0247] In some embodiments, the RNA (such as mRNA) used in this disclosure includes a 5'-UTR and / or a 3'-UTR. The terms “untranslated region” or “UTR” refer to a region within a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region within an RNA molecule such as an mRNA molecule. Untranslated regions (UTRs) can be located at the 5' (upstream) end of an open reading frame (5'-UTR) and / or at the 3' (downstream) end of an open reading frame (3'-UTR). The 5'-UTR, if present, is located at the 5' end upstream of the start codon of the protein-coding region. The 5'-UTR is downstream of the 5'-cap (if present), for example, directly adjacent to the 5'-cap. The 3'-UTR, if present, is located at the 3' end downstream of the stop codon of the protein-coding region, although the term “3'-UTR” preferably does not include a polyA sequence. Thus, the 3'-UTR is upstream of the polyA sequence (if present), for example, directly adjacent to the polyA sequence. Incorporating a 3'-UTR into the 3'-untranslated region of an RNA (preferably mRNA) molecule improves translation efficiency.

[0248] In some embodiments, the RNA (such as mRNA) used in this disclosure includes a 5'-UTR containing the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the RNA (such as mRNA) used in this disclosure includes a 3'-UTR containing the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO: 2.

[0249] The RNA (e.g., mRNA) described herein may have modified ribonucleotides to enhance its stability and / or reduce its immunogenicity and / or reduce its cytotoxicity. For example, in some embodiments, the uridine in the RNA (e.g., mRNA) described herein is (partially or completely, preferably completely) replaced by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0250] In some embodiments, the modified uridines that substitute for uridine are selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudridine (m1ψ), 5-methyl-uridine (m5U), and combinations thereof.

[0251] In this specification, RNA (preferably mRNA) modified with pseudouridine (with partial or complete, preferably complete, substitution of uridine) is referred to as “Ψ-modified,” and the term “m1Ψ-modified” means that the RNA (preferably mRNA) contains N(1)-methylpseudridine (with partial or complete, preferably complete, substitution of uridine). Furthermore, the term “m5U-modified” means that the RNA (preferably mRNA) contains 5-methyluridine (with partial or complete, preferably complete, substitution of uridine). Such Ψ-, m1Ψ-, or m5U-modified RNAs are generally less immunogenic than their unmodified forms and are therefore preferred for applications where it is necessary to avoid or minimize the induction of an immune response. In some embodiments, the RNA (preferably mRNA) contains N(1)-methylpseudridine with complete uridine substitution.

[0252] The codons of the RNA (preferably mRNA) described in the present invention may be further optimized, for example, to increase the G / C content of the RNA and / or to replace rare codons in the cells (or subjects) expressing the peptide or protein of interest with synonymous frequent codons in the cells (or subjects). In some embodiments, the amino acid sequence encoded by the RNA described in the present invention is encoded by a codon-optimized coding sequence and / or a coding sequence with increased G / C content compared to the wild-type coding sequence. This includes embodiments in which one or more sequence regions of the coding sequence are codon-optimized and / or have increased G / C content compared to the corresponding sequence regions of the wild-type coding sequence. In one embodiment, the codon optimization and / or increase in G / C content preferably do not alter the sequence of the encoded amino acid sequence.

[0253] The term "codon optimization" refers to modifying codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism, preferably without altering the amino acid sequence encoded by the nucleic acid molecule. In the context of this disclosure, the coding region is preferably codon-optimized for optimal expression in a target treated with the RNA (preferably mRNA) described herein. Codon optimization is based on the finding that translation efficiency is also determined by differences in the occurrence frequency of tRNAs within a cell. Therefore, the sequence of RNA (preferably mRNA) can be modified to insert codons where frequently occurring tRNAs are available, instead of "rare codons."

[0254] In some embodiments, the guanosine / cytosine (G / C) content of the coding region of the RNA (preferably mRNA) described herein is increased compared to the G / C content of the corresponding coding sequence of wild-type RNA, and the amino acid sequence encoded by the RNA (preferably mRNA) is preferably unmodified compared to the amino acid sequence encoded by wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region being translated is important for the efficient translation of that RNA (preferably mRNA). Sequences with increased G (guanosine) / C (cytosine) content are more stable than sequences with increased A (adenosine) / U (uracil) content. With respect to the fact that several codons encode one amino acid (so-called degeneracy of the genetic code), it is possible to determine the most preferred codon for stability (so-called alternative codon use). Depending on the amino acids encoded by the RNA (preferably mRNA), there are various possibilities for modifying the RNA sequence compared to the wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by substituting these codons with other codons that encode the same amino acids but do not contain A and / or U, or contain a small amount of A and / or U nucleotides.

[0255] In various embodiments, the G / C content of the coding region of the RNA (in particular mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or more compared to the G / C content of the coding region of wild-type RNA.

[0256] The combination of the above modifications, namely the incorporation of a 5'-cap structure, the incorporation of a poly(A) sequence, the unmasking of a poly(A) sequence, the modification of 5'- and / or 3'-UTRs (such as the incorporation of one or more 3'-UTRs), the substitution of one or more native nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (Ψ), or N(1)-methylpseudridine (m1Ψ) or 5-methyluridine (m5U) for uridine), and codon optimization, have a synergistic effect on increasing the stability and translation efficiency of RNA (preferably mRNA). Accordingly, in some embodiments, the RNA (preferably mRNA) described herein, in particular the RNA (preferably mRNA) encoding an antigen or epitope for inducing an immune response disclosed herein, includes at least two, at least three, at least four, or all five of the above modifications, namely, (i) incorporation of a 5'-cap structure, (ii) incorporation of a polyA sequence, unmasking of a polyA sequence, (iii) modification of a 5'- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), (iv) substitution of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine instead of cytidine, and / or pseudouridine (Ψ) or N(1)-methylpseudridine (m1Ψ) or 5-methyluridine (m5U) instead of uridine), and (v) codon optimization. In some embodiments, the RNA (preferably mRNA) described herein includes a cap1 or cap2 structure, preferably a cap1 structure. In some embodiments, the polyA sequence includes at least 100 nucleotides. In some embodiments, the polyA sequence includes or consists of the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the 5'-UTR includes the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO: 1.In some embodiments, the 3'-UTR includes the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO: 2.

[0257] In some embodiments, after administration of the RNA (particularly mRNA) composition described herein, at least a portion of the RNA is delivered to a target cell or target organ. In some embodiments, at least a portion of the RNA is delivered to the cytoplasm of the target cell. In some embodiments, the RNA is RNA (preferably mRNA) encoding a peptide or protein, and the RNA is translated by the target cell to produce a peptide or protein. In some embodiments, the target cell is a cell in the liver. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is an endothelial cell. In some embodiments, the target cell is a tumor cell or a cell in the tumor microenvironment. In some embodiments, the target cell is a blood cell. In some embodiments, the target cell is a cell in a lymph node. In some embodiments, the target cell is a cell in the lung. In some embodiments, the target cell is a cell in the skin. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen-presenting cell, such as a pro-antigen-presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is a B cell. In some embodiments, the target cell is an NK cell. In some embodiments, the target cells are monocytes. Therefore, the nucleic acid (e.g., RNA) particle (e.g., RNA LNP) compositions described herein can be used to deliver nucleic acids (e.g., RNA, preferably mRNA) to such target cells. Accordingly, this disclosure also relates to a method for delivering nucleic acids (e.g., RNA, preferably mRNA) to target cells in a subject, comprising administering the nucleic acid (e.g., RNA, preferably mRNA) compositions described herein to the subject. In some embodiments, the RNA is delivered to the cytoplasm of the target cells. In some embodiments, the RNA is RNA (preferably mRNA) encoding a peptide or protein, and the RNA is translated by the target cells to produce a peptide or protein.

[0258] Inhibitory RNA In some embodiments of all aspects of this disclosure, the nucleic acid is an inhibitory RNA such as siRNA or miRNA.

[0259] As used herein, the term “inhibitory RNA” means RNA that selectively hybridizes to a target mRNA and / or is specific to the target mRNA, thereby inhibiting (e.g., reducing) its transcription and / or translation. Inhibitory RNAs include RNA molecules having an antisense sequence relative to the target mRNA. The length of a suitable inhibitory oligonucleotide is typically 5 to several hundred nucleotides, more typically 20 to 70 nucleotides or less, and even more typically 10 to 30 nucleotides. Examples of inhibitory RNAs include antisense RNAs, ribozymes, iRNAs, siRNAs, and miRNAs. In some embodiments of all aspects of this disclosure, the inhibitory RNA is an siRNA.

[0260] As used herein, the term “antisense RNA” refers to RNA that, under physiological conditions, hybridizes to DNA containing a specific gene or to the mRNA of said gene, thereby inhibiting the transcription of said gene and / or the translation of said mRNA. Antisense transcripts of nucleic acids or parts thereof can form double helixes with naturally occurring mRNA, thereby inhibiting mRNA accumulation or translation. Another possibility is the use of ribozymes to inactivate nucleic acids. Antisense RNA can hybridize to the N-terminus or 5' upstream region, such as a translation initiation site, transcription initiation site, or promoter site. In some embodiments, antisense RNA can hybridize to a 3' untranslated region or mRNA splicing site.

[0261] The size of antisense RNA can vary from 15 to 15,000 nucleotides, preferably 20 to 12,000, particularly 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000, 300 to 5,000 nucleotides, for example, 15 to 2,000, 20 to 1,000, 25 to 800, 30 to 600, 35 to 500, 40 to 400, 45 to 300, 50 to 250, 55 to 200, 60 to 150, or 65 to 100 nucleotides.

[0262] Antisense RNA can target any range of approximately 19 to 25 consecutive nucleotides within a target mRNA sequence (hereinafter referred to as the "target sequence"). Generally, the target sequence on the target mRNA can be selected from a predetermined cDNA sequence corresponding to the target mRNA, and it is preferable to start 50 to 100 nt downstream from the start codon (i.e., in the 3' direction). However, the target sequence can also be located in the 5' or 3' untranslated region, or in a region near the start codon.

[0263] As used herein, “small interfering RNA” or “siRNA” means an RNA molecule, preferably 10 nucleotides or longer, more preferably 15 nucleotides or longer, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long, that can specifically bind to a portion of a target mRNA. This binding induces a process in which the portion of the target mRNA is cleaved or degraded, thereby inhibiting gene expression of the target mRNA. The size of the siRNA is most preferably in the range of 19 to 25 nucleotides. In principle, the sense and antisense strands of an siRNA consist of two complementary single-stranded RNA molecules, but the siRNA according to this disclosure consists of a single molecule in which two complementary parts form base pairs and are covalently linked by a single-stranded “hairpin” region. That is, the sense and antisense regions can be covalently linked via a linker molecule. The linker molecule may be a polynucleotide or a non-nucleotide linker, but is preferably a polynucleotide linker. While we do not wish to be bound by any theory, it is thought that the hairpin region of the siRNA molecule is cleaved within the cell by a "dicer" protein (or equivalent) to form siRNAs of two separate base-pair RNA molecules.

[0264] As used herein, “target mRNA” refers to an RNA molecule that is targeted for downregulation. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide as specified herein. In some embodiments, the pharmaceutically active peptide or polypeptide is associated with disease when its expression (in particular, increased expression compared to expression in healthy subjects) is associated with cancer. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide is associated with cancer when its expression (in particular, increased expression compared to expression in healthy subjects) is associated with cancer.

[0265] According to this disclosure, siRNA can target any sequence of approximately 19 to 25 consecutive nucleotides in any target mRNA sequence (hereinafter, “target sequence”). Techniques for selecting target sequences for siRNA are described, for example, in Tuschl T. et al., “The siRNA User Guide” (revised October 11, 2002), the full disclosure of which is incorporated herein by reference. “The siRNA User Guide” is published on the World Wide Web on a website run by Dr. Thomas Tuschl of the Laboratory of RNA Molecular Biology, Rockefeller University, New York, USA, and can be found by visiting the Rockefeller University website and searching for the keyword “siRNA”. Further guidance on target sequence selection and / or siRNA design can be found on the Protocol Online (www.protocol-online.com) webpage using the keyword “siRNA”. Thus, in some embodiments, the sense strand of the siRNA used in this disclosure contains a nucleotide sequence substantially identical to any sequence of approximately 19 to 25 consecutive nucleotides in the target mRNA.

[0266] As used herein, the term “miRNA” (microRNA) refers to non-coding RNA having a length of 21–25 (e.g., 21–23, preferably 22) nucleotides that induces degradation and / or inhibits translation of target mRNA. miRNAs are commonly found in plants, animals, and some viruses, and are encoded by nuclear DNA in eukaryotes of plants and animals, and viral DNA (viruses whose genomes are DNA-based), respectively. miRNAs are post-transcriptional regulators that bind to a complementary sequence on a target messenger RNA transcript (mRNA), typically resulting in translational repression or degradation of the target and gene silencing.

[0267] Antisense RNA, siRNA, or miRNA can be obtained using various techniques known to those skilled in the art. Preferably, antisense RNA, siRNA, or miRNA is transcribed from a recombinant circular or linear DNA plasmid using any suitable promoter.

[0268] Pharmacologically active peptides or polypeptides "Encoding" refers to the inherent property of a specific nucleotide sequence within a polynucleotide, such as a gene, cDNA, or RNA (preferably mRNA), to function as a template for the synthesis of other polymers and macromolecules in biological processes having a defined nucleotide sequence (rRNA, tRNA, mRNA, etc.) or a defined amino acid sequence, and the resulting biological properties. Therefore, if a protein is produced in a cell or other biological system by the transcription and translation of RNA (preferably mRNA) corresponding to a gene, then that gene codes for a protein. Both the coding strand, whose nucleotide sequence is identical to the RNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for the transcription of a gene or cDNA, can be said to code for the protein or other product of that gene or cDNA.

[0269] In some embodiments, the RNA (preferably mRNA) described herein comprises one or more polypeptides, e.g., peptides or proteins, preferably nucleic acid sequences (e.g., ORFs) encoding pharmaceutically active peptides or polypeptides.

[0270] In some embodiments, the RNA (preferably mRNA) described in this disclosure comprises a nucleic acid sequence (e.g., ORF) encoding a peptide or polypeptide, preferably a pharmaceutically active peptide or polypeptide, which can express the peptide or polypeptide, particularly when introduced into a cell or subject. Therefore, in some embodiments, the RNA (preferably mRNA) described in this disclosure comprises a coding region (ORF) encoding a peptide or polypeptide, preferably a pharmaceutically active peptide or polypeptide. In this regard, “open reading frame” or “ORF” is a sequence of codons beginning with a start codon and ending with a stop codon. Such RNA (preferably mRNA) encoding a pharmaceutically active peptide or polypeptide is also referred to herein as “pharmaceutically active RNA” (or “pharmaceutically active mRNA”). In some embodiments, the RNA (preferably mRNA) described in this disclosure comprises a nucleic acid sequence encoding one or more peptides or polypeptides, such as two, three, four, or more peptides or polypeptides.

[0271] According to the present invention, the term “pharmaceutically active peptide or polypeptide” means a peptide or polypeptide that can be used to treat an individual whose expression is beneficial, for example, in improving the symptoms of a disease or disorder. Preferably, the pharmaceutically active peptide or polypeptide has therapeutic or mitigating properties and can be administered to improve, mitigate, reduce, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. In some embodiments, the pharmaceutically active peptide or polypeptide, when administered to an individual in a therapeutically effective amount, has a positive or beneficial effect on the individual's condition or disease state. The pharmaceutically active peptide or polypeptide may have preventive properties and can be used to delay the onset of a disease or disorder and to reduce the severity of such a disease or disorder. The term “pharmacologically active peptide or polypeptide” includes the whole peptide or polypeptide and may also refer to their pharmacologically active fragments. It also includes pharmacologically active analogs of peptides or polypeptides. The terms “pharmacologically active peptide or polypeptide” and “therapeutic polypeptide” are used interchangeably herein.

[0272] Specific examples of pharmaceutically active peptides and polypeptides include, but are not limited to, immunostimulants such as cytokines, hormones, adhesion molecules, immunoglobulins, immunoactive compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genome-engineered proteins, and blood proteins. In some embodiments, pharmaceutically active peptides and polypeptides include substitution proteins.

[0273] An "immunostimulant" is a substance that stimulates the immune system by inducing or enhancing the activation of its components, particularly immune effector cells. Immunostimulants can be pro-inflammatory (e.g., in the treatment of infections or cancer) or anti-inflammatory (e.g., in the treatment of autoimmune diseases).

[0274] In some embodiments, the pharmaceutically active peptide or polypeptide is a cytokine or a variant thereof. Examples of cytokines include interferons (e.g., interferon-α (IFN-α) or interferon-γ (IFN-γ)), interleukins (e.g., IL2, IL7, IL12, IL15, and IL23), colony-stimulating factors (e.g., M-CSF and GM-CSF), and tumor necrosis factors.

[0275] The term "cytokine" refers to proteins with a molecular weight of approximately 5–60 kDa (e.g., approximately 5–20 kDa) that are involved in cellular signaling (e.g., paracrine signaling, endocrine signaling, and / or autocrine signaling). In particular, when cytokines are released, they affect the behavior of cells in the vicinity of the release site. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factor (TNF). According to this disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they usually act at a much wider range of concentrations than hormones, and (ii) they are generally produced by a wide range of cells (almost all nuclear cells can produce cytokines). Interferons are typically characterized by antiviral, antiproliferative, and immunomodulatory activity. Interferons are proteins that bind to regulated interferon receptors on the cell surface to alter and regulate the transcription of genes within the cell, thereby preventing viral replication within the cell. Interferons can be classified into two types. IFN-γ is the only type II interferon; all others are type I interferons. Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factor (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (INFγ), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), and interleukin-21 (IL-21).

[0276] According to the present invention, cytokines may be natural cytokines or their functional fragments or variants. Cytokines may be human cytokines, or may be derived from any vertebrate, particularly any mammal. One particularly preferred cytokine is interferon-alpha.

[0277] In some embodiments, the pharmaceutically active peptide or polypeptide includes a substitution protein. In these embodiments, the disclosure provides a method for treating a subject having a disorder requiring protein supplementation (e.g., a protein deficiency disorder), the method comprising administering RNA encoding the substitution protein described herein to the subject. The term “protein supplementation” means introducing a protein (including its functional variants) into a subject that is deficient in such a protein. The term also means introducing a protein into a subject that needs or would benefit from the provision of a protein, e.g., a subject suffering from protein deficiency. The term “disorder characterized by protein deficiency” means any disorder that presents with a pathology caused by a lack or deficiency of a protein. The term includes protein folding disorders, i.e., structural abnormalities that result in biologically inactive protein products. Protein deficiency may be related to disorders caused by infection, immunosuppression, organ failure, glandular problems, radiation sickness, malnutrition, poisoning, or other environmental or external factors.

[0278] The term “hormone” refers to a class of signaling molecules produced by glands, and signaling typically involves the following steps: (i) synthesis of hormones in a particular tissue, (ii) storage and secretion, (iii) transport of hormones to their targets, (iv) binding of hormones by receptors, (v) relaying and amplification of signals, and (vi) degradation of hormones. Hormones differ from cytokines in that (1) hormones usually have little variation in concentration and (2) are generally produced by specific types of cells. In some embodiments, “hormones” are peptide or protein hormones such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormones, growth hormones (such as human growth hormone or bovine somatotropin), oxytocin, atrial natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptin.

[0279] The term "adhesion molecule" refers to proteins located on the surface of cells that are involved in the binding of cells to other cells or the extracellular matrix (ECM). Adhesion molecules are typically transmembrane receptors and can be classified into calcium-independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Specific examples of adhesion molecules include integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins.

[0280] Integrins are also involved in signal transduction. Specifically, when a ligand binds, integrins regulate cell signal transduction pathways, such as those of transmembrane protein kinases like receptor tyrosine kinases (RTK). Such regulation can lead to cell growth, division, survival, or differentiation, or apoptosis. Specific examples of integrins include α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α L β2, α M β2, α IIb β3, α V β1, α V β3, α V β5, α V β6, α V β8, and α6β4.

[0281] The term "immunoglobulin" or "immunoglobulin superfamily" refers to molecules involved in the processes of cell recognition, binding, and / or adhesion. Molecules belonging to this superfamily share the common feature of containing a region known as the immunoglobulin domain or fold. Members of the immunoglobulin superfamily include antibodies (e.g., IgG), T cell receptors (TCR), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor accessory molecules (e.g., CD-3γ, CD3-δ, CD-3ε, CD79a, CD79b), co-stimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), etc.

[0282] In some embodiments, the RNA (in particular mRNA) described herein comprises a nucleic acid sequence encoding a peptide or polypeptide containing an epitope for inducing an immune response to an antigen in a subject. The “peptide or polypeptide containing an epitope for inducing an immune response to an antigen in a subject” is also referred herein to as “vaccine antigen,” “peptide and protein antigen,” or simply “antigen.”

[0283] In some embodiments, the RNA encoding the vaccine antigen is expressed within the target cells to provide the vaccine antigen. In some embodiments, the vaccine antigen is expressed on the cell surface. In some embodiments, the vaccine antigen is presented in relation to the MHC. In some embodiments, the RNA encoding the vaccine antigen is transiently expressed within the target cells. In some embodiments, the RNA encoding the vaccine antigen is administered systemically. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen occurs in the spleen. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen occurs in antigen-presenting cells, preferably pro-antigen-presenting cells. In some embodiments, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, and B cells. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen is absent or substantially absent in the lungs and / or liver. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen in the spleen is at least five times higher than expression in the lungs.

[0284] A vaccine antigen contains an epitope for inducing an immune response to the antigen in a subject. Therefore, a vaccine antigen contains an antigen sequence for inducing an immune response to the antigen in a subject. Such an antigen sequence may correspond to a target antigen or disease-related antigen, such as a protein or tumor antigen of an infectious agent (e.g., a viral or bacterial antigen), or an immunogenic variant thereof, or an immunogenic fragment of a target antigen or disease-related antigen or its immunogenic variant. Therefore, an antigen sequence may contain at least one epitope of a target antigen or disease-related antigen or their immunogenic variants.

[0285] In some embodiments, a secretory sequence, for example, a sequence containing the amino acid sequence of SEQ ID NO: 4, may be fused to the N-terminus of an antigenic peptide or polypeptide.

[0286] In some embodiments, the amino acid sequence that enhances antigen processing and / or presentation includes the amino acid sequence of SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence of SEQ ID NO: 5, or a functional fragment of the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the amino acid sequence that enhances antigen processing and / or presentation includes the amino acid sequence of SEQ ID NO: 5.

[0287] Accordingly, in some embodiments, the RNA described herein comprises at least one coding region encoding an antigen peptide or polypeptide and an amino acid sequence that enhances the processing and / or presentation of the antigen, wherein the amino acid sequence that enhances the processing and / or presentation of the antigen is preferably fused to the antigen peptide or polypeptide, and more preferably fused to the C-terminus of the antigen peptide or polypeptide described herein.

[0288] The following describes the embodiments of vaccine RNA, and the specific terms used to describe its elements have the following meanings: Cap: m2 7,2'O G(5')ppSp(5')G (especially its D1 diastereomer), m2 7,3'O G(5')ppp(5')G, and m2 7,3'-O Gppp(m1 2'-O A 5'-cap structure selected from the group consisting of ApG. hAg-Koza: A 5'-UTR sequence of human alpha-globin mRNA containing a Kozak sequence optimized to enhance translation efficiency. sec / MITD is a fusion protein tag derived from a sequence encoding the human MHC class I complex (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3), and has been shown to improve antigen processing and presentation. sec corresponds to a 78 bp fragment encoding a secretory signaling peptide that translocates nascent polypeptide chains to the endoplasmic reticulum. MITD corresponds to the transmembrane and cytoplasmic domains of the MHC class I molecule and is also called the MHC class I transport domain. Antigen: The sequence that codes for each vaccine antigen / epitope. Glycine-serine linker (GS): A sequence that codes for a short peptide linker, mainly consisting of the amino acids glycine (G) and serine (S), commonly used in fusion proteins. P2P16: A sequence encoding a tetanus toxoid-derived helper epitope that breaks immune tolerance. The FI element:3'-UTR is a combination of two sequence elements derived from "amino-terminal split enhancer" (AES) mRNA (referred to as F) and mitochondrial-encoded 12S ribosomal RNA (referred to as I). These sequences, which confer RNA stability and enhance total protein expression, were identified by an ex vivo selection process. A30L70: A 110-nucleotide poly(A) tail consisting of a sequence of 30 adenosine residues, followed by a 10-nucleotide linker sequence and 70 adenosine residues designed to enhance RNA stability and translation efficiency in dendritic cells.

[0289] In some embodiments, the vaccine RNA described herein has one of the following structures: cap-hAg-Kozak-sec-GS(1)-Antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70 beta-S-ARCA(D1)-hAg-Kozak-sec-GS(1)-Antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70

[0290] In some embodiments, the vaccine antigen described herein has the following structure: sec-GS(1)-Antigen-GS(2)-P2P16-GS(3)-MITD

[0291] In some embodiments, hAg-Kozak includes the nucleotide sequence of SEQ ID NO: 1. In some embodiments, sec includes the amino acid sequence of SEQ ID NO: 4. In some embodiments, P2P16 includes the amino acid sequence of SEQ ID NO: 6. In some embodiments, MITD includes the amino acid sequence of SEQ ID NO: 5. In some embodiments, GS(1) includes the amino acid sequence of SEQ ID NO: 7. In some embodiments, GS(2) includes the amino acid sequence of SEQ ID NO: 8. In some embodiments, GS(3) includes the amino acid sequence of SEQ ID NO: 8. In some embodiments, FI includes the nucleotide sequence of SEQ ID NO: 2. In some embodiments, A30L70 includes the nucleotide sequence of SEQ ID NO: 3.

[0292] In some embodiments, the sequence encoding the vaccine antigen / epitope comprises a modified nucleoside that (partially or completely, preferably completely) substitutes uridine, the modified nucleoside being selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudridine (m1ψ), and 5-methyl-uridine.

[0293] In some embodiments, the sequence encoding the vaccine antigen / epitope is codon-optimized.

[0294] In some embodiments, the G / C content of the vaccine antigen / epitope encoding sequence is increased compared to the wild-type encoding sequence.

[0295] Therefore, in some embodiments, the pharmaceutically active peptide or polypeptide is (i) a cytokine, preferably selected from the group consisting of erythropoietin (EPO), interleukin 4 (IL-2), and interleukin 10 (IL-11), more preferably EPO; (ii) an adhesion molecule, particularly an integrin; (iii) an immunoglobulin, particularly an antibody; (iv) an immunoactive compound, particularly an antigen, e.g., a viral antigen or a bacterial antigen, e.g., the antigen of SARS-CoV-2; (v) a hormone, particularly vasopressin, insulin, or growth hormone; (vi) a growth factor, particularly VEGFA; (vii) a protease inhibitor, particularly α1-antitrypsin; (viii) an enzyme, preferably herpes simplex virus 1 Selected from the group consisting of type thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes, and lactase; (ix) receptors, especially growth factor receptors; (x) apoptosis regulators, especially BAX; (xi) transcription factors, especially FOXP3; (xii) tumor suppressor proteins, especially p53; (xiii) structural proteins, especially surfactant protein B; (xiv) reprogramming factors, e.g., OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG; (xv) genome engineering proteins, especially clustered regular-spacing palindromic repeat sequence CRISPR-related protein 9 (CRISPR-Cas9); (xvi) blood proteins, especially fibrinogen.

[0296] In some embodiments, the pharmaceutically active peptide or polypeptide comprises one or more antigens or one or more epitopes, i.e., administration of the peptide or polypeptide to a subject induces an immune response in the subject to one or more antigens or one or more epitopes, which may be therapeutic, partial, or complete protective.

[0297] In certain embodiments, the RNA (preferably mRNA) encodes at least one epitope, for example, at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes.

[0298] In certain embodiments, the target antigen is a tumor antigen, and the antigen sequence (e.g., an epitope) is derived from the tumor antigen. The tumor antigen may be a “standard” antigen that is commonly known to be expressed in various cancers. The tumor antigen may be a “novel antigen” that is specific to the tumor of an individual and has not been previously recognized by the immune system. A neoantigen or neoepitope may arise from one or more cancer-specific mutations in the genome of a cancer cell, which may result in an amino acid change. If the tumor antigen is a neoantigen, the vaccine antigen preferably includes an epitope or fragment of the neoantigen containing one or more amino acid changes.

[0299] Examples of tumor antigens include p53, ART-4, BAGE, beta-catenin / m, and Bcr-abL. CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family, e.g., CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, C-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE- Examples include, but are not limited to, A10, MAGE-A11 or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, Myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Pml / RARa, PRAME, Proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, and WT-1.

[0300] Cancer mutations vary from person to person. Therefore, cancer mutations encoding novel epitopes (neoepitopes) are attractive targets in the development of vaccine compositions and immunotherapies. The effectiveness of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes that can induce a potent immune response in the host. RNA can be used to deliver patient-specific tumor epitopes to patients. Dendritic cells (DCs) present in the spleen are particularly interesting antigen-presenting cells for RNA expression of antigens such as immunogenic epitopes or tumor epitopes. The use of multiple epitopes has been shown to enhance the therapeutic effect of tumor vaccine compositions. Rapid sequencing of tumor mutanomes may provide multiple epitopes for personalized vaccines, for example, by the RNAs (particularly mRNA) described herein, where the epitopes may be encoded as single polypeptides, for example, with the epitopes selectively isolated by a linker. In certain embodiments of this disclosure, RNA (particularly mRNA) encodes at least one epitope, at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes. Exemplary embodiments include RNA (particularly mRNA) encoding at least five epitopes (referred to as "pentatopes") and RNA (particularly mRNA) encoding at least ten epitopes (referred to as "decatopes").

[0301] In certain embodiments, the epitope is derived from a pathogen-associated antigen. In some embodiments, the pharmaceutically active polypeptide and / or antigen or epitope is derived from, or is derived from, a pathogen protein, an immunogenic variant of that protein, or an immunogenic fragment of the protein or its immunogenic variant.

[0302] In some embodiments, the pathogen is selected from viruses, bacteria, fungi, parasites, and other microorganisms.

[0303] Exemplary viruses include severe acute respiratory syndrome coronaviruses (SARS-CoV), such as SARS-CoV-2; human immunodeficiency virus (HIV); Epstein-Barr virus (EBV); cytomegalovirus (CMV) (e.g., CMV5); human herpesvirus (HHV) (e.g., HHV6, 7, or 8); herpes simplex virus (HSV); bovine herpesvirus (BHV) (e.g., BHV4); equine herpesvirus (EHV) (e.g., EHV2); human T-cell leukemia virus (HTLV) 5; and varicella-zoster virus. This list includes, but is not limited to, (VZV), measles virus, papovavirus (JC and BK), hepatitis virus (e.g., HBV or HCV), myxoma virus, adenovirus, rhinovirus, enterovirus, parvovirus, polyomavirus, influenza virus, papillomavirus (such as human papillomavirus (HPV)), poxviruses such as vaccinia virus, molluscum contagiosum virus (MCV), lyssavirus, rotavirus, norovirus, rubella virus, and mumps virus. Exemplary diseases caused by viral infections include, but are not limited to, SARS, acquired immunodeficiency syndrome (AIDS), measles, chickenpox, cytomegalovirus infection, genital herpes, hepatitis (such as hepatitis B or C), influenza (influenza, e.g., human influenza, swine influenza, canine influenza, equine influenza, and avian influenza), HPV infection, shingles, rabies, the common cold, gastroenteritis, rubella, and mumps.

[0304] Exemplary bacteria include, but are not limited to, Campylobacter (e.g., Campylobacter jejuni), Enterobacter species, Enterococcus faecium, Enterococcus faecalis, Escherichia coli (e.g., F. coli O157:H7), Group A Streptococcus, Haemophilus influenzae, Helicobacter pylori, Listeria monocystis, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Salmonella, Shigella, Staphylococcus aureus, Staphylococcus epidermidis, Borrelia and Rickettsia, Chlamydiaceae, Neisseria gonorrhoeae, Bordetella pertussis, Tetanus, Neisseria meningitidis, Streptococcus (e.g., Streptococcus pneumoniae or Streptococcus pyogenes), and Treponema pallidum. Exemplary diseases caused by bacterial infections include, but are not limited to, anthrax, cholera, diphtheria, food poisoning, leprosy, meningitis, peptic ulcerative disease, pneumonia, sepsis, septic shock, tetanus, tuberculosis, typhoid fever, urinary tract infections, Lyme disease, Rocky Mountain spotted fever, chlamydia, gonorrhea, pertussis, tetanus, meningitis, scarlet fever, and syphilis.

[0305] Exemplary parasites include, but are not limited to, malaria, trypanosoma, leishmaniasis, trichomoniasis, bivalve amoeba, giardiasis, dysentery amoeba, Naegleria, Isospora, Toxoplasma, sarcoma mycosis, rhinosporidiosis, and balantidium. Exemplary diseases caused by parasitic infections include, but are not limited to, malaria, trypanosomiasis, Chagas disease, leishmaniasis, trichomoniasis, bivalve amoeba disease, giardiasis, amoebic dysentery, coccidiosis, toxoplasmosis, sarcoma mycosis, rhinosporidiosis, and balantidium.

[0306] In some embodiments, the pathogen is an infectious pathogen, particularly one that causes infectious diseases such as viral diseases, bacterial diseases, or parasitic diseases. In some embodiments, the pathogen is a virus, a bacterium, or a parasite. Therefore, in these embodiments, the RNA (particularly mRNA) and / or compositions described herein can be used to prevent and / or treat infectious diseases caused by the pathogen.

[0307] In certain embodiments, the epitope is derived from a viral antigen.

[0308] In some embodiments, the antigen or epitope is derived from the coronavirus protein, its immunogenic variant, or an immunogenic fragment of the coronavirus protein or its immunogenic variant. Thus, in some embodiments, the mRNA used in this disclosure encodes an amino acid sequence comprising the coronavirus protein, its immunogenic variant, or an immunogenic fragment of the coronavirus protein or its immunogenic variant.

[0309] In some embodiments, the antigen or epitope is derived from the coronavirus S protein, its immunogenic variant, or an immunogenic fragment of the coronavirus S protein or its immunogenic variant. Therefore, in some embodiments, the RNA (in particular, mRNA) described herein encodes an amino acid sequence comprising the coronavirus S protein, its immunogenic variant, or an immunogenic fragment of the coronavirus S protein or its immunogenic variant. In some embodiments, the coronavirus is MERS-CoV. In some embodiments, the coronavirus is SARS-CoV. In some embodiments, the coronavirus is SARS-CoV-2.

[0310] particle The nucleic acids described herein, particularly mRNA, may be present in particles comprising (i) nucleic acids and (ii) at least one cationic or cationically ionizable compound. In some embodiments, the particles comprise (i) nucleic acids (RNA, particularly mRNA, etc.); (ii) at least one cationic or cationically ionizable compound disclosed herein; (iii) steroids disclosed herein; (iv) neutral lipids disclosed herein; and (v) optionally polymer-bound lipids disclosed herein.

[0311] Previously, various types of RNA-containing particles have been described as suitable for delivering particulate RNA (see, for example, Kaczmarek, JCet al., 2017, Genome Medicine 9, 60). In the case of nonviral RNA delivery media, encapsulating RNA with nanoparticles physically protects the RNA from degradation and, in response to specific chemical reactions, can facilitate uptake into cells and exit from endosomes.

[0312] Particle formation involves electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acids. As a result, nucleic acid particles form complexes and are formed spontaneously.

[0313] In the context of this disclosure, the term “particle” refers to a structural entity formed by a molecule or molecular complex, in particular by a particle-forming compound. In some embodiments, a particle comprises an envelope (e.g., one or more layers or lamellae) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids, amphiphilic polymers, and / or amphiphilic proteins / polypeptides). In this context, the expression “amphiphilic substance” means that the substance possesses both hydrophilic and lipophilic properties. The envelope may also contain additional substances that do not need to be amphiphilic (e.g., additional lipids and / or additional polymers). Thus, a particle may be a monolayer or multilayer structure, and the substances constituting one or more layers or lamellae may include a combination of one or more types of amphiphilic substances (in particular, selected from the group consisting of amphiphilic lipids, amphiphilic polymers, and / or amphiphilic proteins / polypeptides) and, optionally, additional substances that do not need to be amphiphilic (e.g., additional lipids and / or additional polymers). In some embodiments, the term “particle” refers to a micro-sized or nano-sized structure, e.g., a micro-sized or nano-sized compact structure. In this regard, the term “micro-size” means that all three external dimensions of the particle are on the microscale, i.e., 1–5 μm. According to this disclosure, the term “particle” includes lipoplex particles (LPX), lipid nanoparticles (LNP), and liposomes.

[0314] "Nucleic acid particles" can be used to deliver nucleic acids (e.g., RNA, especially mRNA) to target sites of interest (e.g., cells, tissues, organs, etc.). Nucleic acid particles can be formed from nucleic acids with at least one cationic or cationic ionized lipid or lipid-like substance, at least one cationic polymer (e.g., protamine), or a mixture thereof. Nucleic acid particles include lipid nanoparticle (LNP)-based, lipoplex (LPX)-based, and / or liposome-based formulations.

[0315] While not bound by any theory, it is believed that cationic lipids or cationic ionized lipids bind to nucleic acids to form aggregates, and that this aggregation produces colloidally stable particles. In some embodiments, the particles comprise cationic lipids or cationic ionized amphiphilic lipids described herein, and nucleic acids (e.g., RNA, particularly mRNA). In some embodiments, the particles comprise or consist of cationic / cationic ionized lipids (specifically, cationic ionized lipids of formula (X) disclosed herein, cationic ionized lipids having one of structures A-G disclosed herein, or cationic ionized lipids of formula (XI) disclosed herein); and additional lipids such as neutral lipids (e.g., phospholipids), steroids (e.g., cholesterol), and combinations thereof; and optionally polymer-bound lipids.

[0316] In some embodiments, the nucleic acid (e.g., RNA) particles disclosed herein include (i) nucleic acids described herein (e.g., RNA, particularly mRNA); (ii) amphiphilic lipids, particularly cationic or cationically ionized amphiphilic lipids (e.g., cationic ionized lipids of formula (X) disclosed herein; cationic ionized lipids having any of structures A to G disclosed herein; cationic ionized lipids of formula (XI) disclosed herein; or cationic lipids disclosed herein); (iii) steroids (e.g., cholesterol), neutral lipids (e.g., phospholipids); and (v) optionally polymer-bound lipids. In some embodiments, the steroid is cholesterol, and the neutral lipid is selected from the group consisting of DSPC, DOPC, and DOPE. In some embodiments, the particles are dispersed in an aqueous phase, where the aqueous phase includes a buffer system containing histidine or HEPES as a buffering agent. In some embodiments, the steroid is cholesterol, the neutral lipid is selected from the group consisting of DSPC, DOPC, and DOPE, and the particles are dispersed in an aqueous phase, where the aqueous phase includes a buffer system containing histidine or HEPES as a buffering agent.

[0317] In some embodiments, in nucleic acid particles (e.g., RNA particles) described herein, the nucleic acid (e.g., RNA, in particular mRNA) is bound to a cationic ionized lipid (in particular, a cationic ionized lipid of formula (X) disclosed herein, a cationic ionized lipid having any of structures A to G disclosed herein, or a cationic ionized lipid of formula (XI) disclosed herein) that occupies the central nucleus of the LNP in the case of LNPs. In some embodiments, phospholipids, together with polymer-bound lipids, for example, form the surface of the particle (e.g., LNP). In some embodiments, the particle is substantially free of PEG lipids having at least 30 consecutive ethylene glycol repeating units. In some embodiments, the surface includes a bilayer. In some embodiments, cholesterol and cationic ionized lipids (in particular, a cationic ionized lipid of formula (X) disclosed herein, a cationic ionized lipid having any of structures A to G disclosed herein, or a cationic ionized lipid of formula (XI) disclosed herein) can be distributed throughout the particle, such as an LNP, in both charged and uncharged forms.

[0318] Generally, lipoplex (LPX) is obtained by mixing two aqueous phases, namely a phase containing nucleic acids (particularly RNA) and a phase containing a lipid dispersion. In some embodiments, the lipid phase contains liposomes.

[0319] In some embodiments, liposomes are self-closed monolayer or multilayer vesicular particles, where the lamellae comprise a lipid bilayer and the enclosed lumen comprises an aqueous phase. A prerequisite for using liposomes to form nanoparticles is that the lipids in the mixture can, if necessary, form a lamellar (bilayer) phase in the applied aqueous environment.

[0320] In some embodiments, liposomes comprise a monolayer or multilayer phospholipid bilayer surrounding an aqueous core (also referred herein as an aqueous lumen). These can be prepared from materials having polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, the cationic lipids used in the formulation of liposomes designed for nucleic acid delivery are inherently amphiphilic and consist of positively charged (cationic) amine head groups bonded to a hydrocarbon chain or cholesterol derivative via glycerol.

[0321] In some embodiments, lipoplexes are multilayer liposome-based formulations formed by electrostatic interactions between cationic liposomes and nucleic acids (e.g., RNA). In some embodiments, the formed lipoplexes have a distinct internal arrangement of molecules resulting from the conversion from liposome structures to compact nucleic acid-lipoplexes (e.g., RNA-lipoplexes).

[0322] In some embodiments, LPX particles comprise amphiphilic lipids described herein, particularly cationic or cationic ionized amphiphilic lipids, and nucleic acids (e.g., RNA, particularly mRNA). In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, particularly cationic or cationic ionized amphiphilic lipids) and negatively charged nucleic acids (e.g., RNA, particularly mRNA) result in complex formation and the spontaneous formation of nucleic acid lipoplex particles. Positively charged liposomes can generally be synthesized using cationic ionized lipids of formula (I), cationic or cationic ionized amphiphilic lipids such as DOTMA and / or DODMA, and additional lipids such as DSPC or DOPC. In some embodiments, the nucleic acid (e.g., RNA, particularly mRNA) lipoplex particles are nanoparticles.

[0323] Generally, lipid nanoparticles (LNPs) are obtained by directly mixing nucleic acids (such as RNA) in an aqueous phase with lipids in a phase containing an organic solvent such as ethanol. In the case of LNPs, the lipids or lipid mixtures used for particle formation usually do not form a lamellar (bilayer) phase in water. In some embodiments, the lipids include cationic ionized lipids (in particular, cationic ionized lipids of formula (X) disclosed herein, cationic ionized lipids having one of structures A to G disclosed herein, or cationic ionized lipids of formula (XI) disclosed herein), steroids disclosed herein (such as cholesterol), and neutral lipids disclosed herein (such as phospholipids).

[0324] In some embodiments, the particles described herein include cationic ionized lipids disclosed herein (in particular, cationic ionized lipids of formula (X) disclosed herein, cationic ionized lipids having one of structures A to G disclosed herein, or cationic ionized lipids of formula (XI) disclosed herein), steroids disclosed herein (such as cholesterol), neutral lipids disclosed herein (such as phospholipids), and optionally polymer-bound lipids.

[0325] In some embodiments, nucleic acid particles (in particular RNA particles such as RNA LNPs (e.g. mRNA particles such as mRNA LNPs)) comprise one or more types of nucleic acid molecules, where the molecular parameters of the nucleic acid molecules may be similar or different from one another with respect to basic structural elements such as molar mass or molecular structure, capping, coding region or other features.

[0326] In some embodiments, the nucleic acids described herein (e.g., RNA, e.g., mRNA) may be non-covalently bound to the particles described herein. In some embodiments, the nucleic acids (e.g., RNA, particularly mRNA) may be attached to the outer surface of the particles (surface nucleic acids (e.g., surface RNA, particularly surface mRNA)) and / or contained within the particles (inclusion nucleic acids (e.g., surface RNA, particularly inclusion mRNA)).

[0327] In this specification, “nanoparticles” means particles comprising nucleic acids (especially RNA such as mRNA) and at least one cationic lipid as described herein, wherein the three external dimensions of the particles are all nanoscale, i.e., at least about 1 nm and less than about 1000 nm (preferably 10 to 990 nm, e.g., 15 to 900 nm, 20 to 800 nm, 30 to 700 nm, 40 to 600 nm, or 50 to 500 nm). Preferably, the longest axis and the shortest axis do not differ significantly. Preferably, the size of the particle is its diameter.

[0328] Nucleic acid particles (in particular RNA particles, e.g., mRNA particles) described herein may exhibit a polydispersity index (PDI) of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. For example, nucleic acid particles may exhibit a polydispersity index in the range of about 0.01 to about 0.4 or about 0.1 to about 0.3.

[0329] In the context of this disclosure, the term “lipoplex particles” refers to particles comprising amphiphilic lipids, particularly cationic lipids, and nucleic acids (especially RNA such as mRNA) as described herein. A complex is formed by electrostatic interaction between a positively charged liposome (made from one or more amphiphilic lipids, particularly cationic amphiphilic lipids) and a negatively charged nucleic acid (especially RNA such as mRNA), thereby spontaneously forming nucleic acid lipoplex particles. Positively charged liposomes can generally be synthesized using cationic amphiphilic lipids such as DOTMA and additional lipids such as DSPC or DOPC. In one embodiment, the nucleic acid (especially RNA such as mRNA) lipoplex particles are nanoparticles.

[0330] The term "nucleic acid-containing particles" refers to particles to which nucleic acids (especially RNA such as mRNA) are bound, as described herein. In this regard, nucleic acids (especially RNA such as mRNA) may be attached to the outer surface of the particle (surface nucleic acids (especially surface RNA such as surface mRNA)) and / or contained within the particle (inclusion nucleic acids (especially inclusion RNA such as inclusion mRNA)).

[0331] In one embodiment, the particles contained in the composition of the Disclosure and / or utilized in the method and use of the Disclosure are in the range of about 10 to about 2000 nM, for example, at least about 15 nM (preferably at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, less (at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, at least about 95 nM, or at least about 100 nM) and / or up to 1900 nM (preferably up to about 1900 nM, up to about 1800 nM, up to about 1700 nM, up to about 1600 nM, up to about 1500 nM, up to about 1400 nM, up to about 1300 nM, up to about 1200 nM, up to about 1100 n) M particles, maximum approximately 1000 nM particles, maximum approximately 950 nM particles, maximum approximately 900 nM particles, maximum approximately 850 nM particles, maximum approximately 800 nM particles, maximum approximately 750 nM particles, maximum approximately 700 nM particles, maximum approximately 650 nM particles, maximum approximately 600 nM particles, maximum approximately 550 nM, or maximum approximately 500 nM), preferably in the range of approximately 20 to approximately 1500 nM, for example, approximately 30 to approximately 1200 nM, approximately 40 to approximately 1100 nM, approximately 50 to approximately 1000 nM, approximately 60 to approximately 900 nM, approximately 70 to 800 nM, The size ranges from approximately 80 to 700 nM, approximately 90 to 600 nM, or approximately 50 to 500 nM or approximately 100 to 500 nM, for example, 10 to 1000 nM, 15 to 500 nM, 20 to 450 nM, 25 to 400 nM, 30 to 350 nM, 40 to 300 nM, 50 to 250 nM, 60 to 200 nM, or 70 to 150 nM (preferably twice the diameter, i.e., twice the radius, for example, twice the radius of rotation (Rg) value, or twice the hydrodynamic radius).

[0332] In some embodiments, the particles described herein (e.g., LNP and LPX) are, in some embodiments, approximately 50 nM to approximately 1000 nM, approximately 50 nM to approximately 800 nM, approximately 50 nM to approximately 700 nM, approximately 50 nM to approximately 600 nM, approximately 50 nM to approximately 500 nM, approximately 50 nM to approximately 450 nM, approximately 50 nM to approximately 400 nM, approximately 50 nM to approximately 350 nM, and approximately 50 nM to approximately 300 nM. nM, about 50nM to about 250nM, about 50nM to about 200nM, about 100nM to about 1000nM, about 100nM to about 800nM, about 100nM to about 700nM, about 100nM to about 60 0nM, about 100nM to about 500nM, about 100nM to about 450nM, about 100nM to about 400nM, about 100nM to about 350nM, about 100nM to about 300nM, about 100nM to about 2 50nM, about 100nM to about 200nM, about 150nM to about 1000nM, about 150nM to about 800nM, about 150nM to about 700nM, about 150nM to about 600nM, about 150nM ~about 500nM, about 150nM to about 450nM, about 150nM to about 400nM, about 150nM to about 350nM, about 150nM to about 300nM, about 150nM to about 250nM, about 150nM It has an average diameter in the range of M ~ approximately 200 nM, approximately 200 nM ~ approximately 1000 nM, approximately 200 nM ~ approximately 800 nM, approximately 200 nM ~ approximately 700 nM, approximately 200 nM ~ approximately 600 nM, approximately 200 nM ~ approximately 500 nM, approximately 200 nM ~ approximately 450 nM, approximately 200 nM ~ approximately 400 nM, approximately 200 nM ~ approximately 350 nM, approximately 200 nM ~ approximately 300 nM, or approximately 200 nM ~ approximately 250 nM.

[0333] Regarding nucleic acid particles (RNA lipid particles, especially RNA LNPs such as mRNA LNPs), the N / P ratio or N / P value indicates the ratio of the number of nitrogen groups in the lipid (especially positively chargeable polymer amine (N = nitrogen) groups) to the number of negatively charged phosphate (P) groups in the nucleic acid. It correlates with the charge ratio, where nitrogen atoms (pH-dependent) are usually positively charged and phosphate groups are negatively charged. The N / P ratio at which charge equilibrium exists is pH-dependent. Since positively charged nanoparticles are considered favorable for transfection, lipid formulations are often formed with an N / P ratio greater than 4 and up to 12. In that case, RNA is considered to be completely bound to the nanoparticles.

[0334] The nucleic acid particles described herein (in particular RNA particles such as mRNA particles) can be prepared using a wide range of methods, including obtaining a colloid from at least one cationic lipid or cationic ionized lipid and / or at least one cationic polymer, and mixing this colloid with nucleic acid to obtain nucleic acid particles.

[0335] As used herein, the term “colloid” refers to a type of homogeneous mixture in which dispersed particles do not precipitate. The insoluble particles in the mixture are microscopic, with particle sizes ranging from 1 to 1000 nanometers. This mixture is sometimes called a colloid or colloidal suspension. The term “colloid” may also refer to the particles in the mixture only, rather than the entire suspension.

[0336] For the preparation of colloids containing at least one cationic lipid or a lipid that can be cationically ionized, the present invention can be applied by appropriately adapting methods conventionally used for the preparation of liposomal vesicles. The most common methods for preparing liposomal vesicles share the following basic steps: (i) dissolution of the lipid in an organic solvent, (ii) drying of the resulting solution, and (iii) hydration of the dried lipid (using various aqueous media).

[0337] In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried to form a thin film at the bottom of a flask. The resulting lipid film is then hydrated with a suitable aqueous medium to produce a liposome dispersion. Furthermore, an additional downsizing step may be included.

[0338] Reverse-phase evaporation is an alternative method for preparing liposome vesicles to membrane hydration, forming a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. This mixture needs to be homogenized by short-term sonication. Removing the organic phase under reduced pressure yields a milky gel, which then transforms into a liposome suspension.

[0339] The "ethanol injection method" refers to the process of rapidly injecting an ethanol solution containing lipids into an aqueous solution through a needle. This operation disperses the lipids throughout the solution and promotes the formation of lipid structures, such as lipid vesicle formation including liposomes. Generally, the nucleic acid (especially RNA such as mRNA) lipoplex particles described herein are obtained by adding nucleic acids (especially RNA such as mRNA) to a colloidal liposome dispersion. Using the ethanol injection method, in one embodiment, such colloidal liposome dispersions are formed as follows: an ethanol solution containing lipids, e.g., cationic lipids (cateionic lipids of formula (X) disclosed herein, cationic lipids having one of structures A to G disclosed herein, cationic lipids of formula (XI) disclosed herein, DOTMA and / or DODMA) and additional lipids (e.g., polymer-bound lipids (e.g., polyethylene glycol (PEG) lipids, or polysarcosine-lipid conjugates or conjugates of polysarcosine and lipid-like substances), neutral lipids (e.g., phospholipids), steroids (e.g., cholesterol), and combinations) is injected into an aqueous solution with stirring. In some embodiments, the nucleic acid (in particular RNA such as mRNA) lipoplex particles described herein are obtained without an extrusion step.

[0340] The terms "extrusion" or "stretching" refer to the creation of particles with a fixed cross-sectional profile. In particular, it refers to the downsizing of particles by passing them through a filter with predetermined pores.

[0341] According to this disclosure, other methods having the property of not including organic solvents can also be used for the production of colloids.

[0342] LNPs typically consist of four components: cationic ionized lipids or cationic lipids, neutral lipids such as phospholipids, steroids such as cholesterol, and polymer-bound lipids such as PEG lipids or polysarcosine-bound lipids (sometimes called "stealth lipids" or "stereostabilizing lipids"). Each component is responsible for protecting the payload and enabling effective intracellular delivery. However, due to the drawbacks of commonly used PEG lipids, i.e., PEG lipids having at least 30 consecutive ethylene glycol repeating units (the most common polymer-bound lipids), it is preferable that the LNPs of this disclosure are substantially free of PEG lipids having at least 30 consecutive ethylene glycol repeating units.

[0343] Various types of nucleic acid-containing particles have been described as suitable for the delivery of particulate nucleic acids (see, for example, Kaczmarek, JCet al., 2017, Genome Medicine 9, 60). In the case of nonviral nucleic acid delivery media, nanoparticle encapsulation of nucleic acids can physically protect them from degradation and, depending on specific chemical reactions, can aid in their uptake into cells and exit from endosomes.

[0344] In some embodiments, LNPs comprising nucleic acids (such as RNA) and at least one cationic ionized lipid as described herein are prepared by (a) providing (e.g., preparing) a nucleic acid solution comprising water and a first buffer system; (b) providing (e.g., preparing) an organic (e.g., ethanol) solution comprising cationic ionized lipids, steroids, and neutral lipids, and optionally one or more additional lipids (e.g., polymer-bound lipids); (c) mixing the nucleic acid solution provided (e.g., prepared) in (a) with the organic (e.g., ethanol) solution provided (e.g., prepared) in (b), thereby preparing a first intermediate formulation comprising LNP dispersed in a first aqueous phase comprising a first buffer system; and (d) filtering (e.g., dialysis, tangential flow filtration, or diafiltration) and / or diluting the first intermediate formulation prepared in (c) using a final aqueous buffer containing a final buffer system, thereby preparing a formulation comprising LNP dispersed in a final aqueous phase containing a final buffer system. Step (d) may be followed by one or more steps selected from dilution and filtration, such as dialysis, tangential flow filtration, or diafiltration. In some embodiments, the first buffer system is different from the final buffer system. In other embodiments, the first buffer system and the final buffer system are the same. In some embodiments, in particular, if the first aqueous phase is very similar to or substantially equivalent to the final aqueous phase, or if the first aqueous phase can be converted to the final aqueous phase simply by diluting it with a suitable diluent (e.g., the final aqueous buffer), the step of filtering the first intermediate formulation prepared in (c) with the final aqueous buffer containing the final buffer system may be replaced with the step of diluting the first intermediate formulation prepared in (cc) with a diluent (e.g., the final aqueous buffer containing the final buffer system).

[0345] In some embodiments, LNPs comprising nucleic acids (such as RNA) and at least one cationic ionized lipid as described herein are prepared by (a') providing (e.g., preparing) a liposome or colloidal formulation of the cationic ionized lipid and, if present, one or more additional lipids in an aqueous phase; (b') providing (e.g., preparing) a nucleic acid (such as RNA) solution comprising water and a buffer system; and (c') mixing the liposome or colloidal formulation provided (e.g., prepared) in (a') with the nucleic acid (such as RNA) solution provided (e.g., prepared) in (b'). Step (c') may be followed by one or more steps selected from dilution and filtration, e.g., dialysis, tangential flow filtration, or diafiltration.

[0346] The present invention describes a composition comprising a nucleic acid (e.g., RNA, particularly mRNA) and at least one cationic ionized lipid that binds to the nucleic acid (e.g., RNA) to form nucleic acid particles. The nucleic acid particles may include nucleic acids (e.g., RNA) complexed with the particles in various forms by non-covalent interactions. The particles described herein are not viral particles, and in particular are not infectious viral particles; that is, the particles described herein cannot virally infect cells.

[0347] Appropriate cationic ionized lipids are lipids that form nucleic acid particles and are included in the term "particle-forming components" or "particle-forming agents." The term "particle-forming components" or "particle-forming agents" refers to any component that binds to nucleic acids to form nucleic acid particles. Such components include any component that can become part of a nucleic acid particle.

[0348] In some embodiments, nucleic acid (e.g., RNA) particles (in particular, mRNA particles) comprise one or more types of nucleic acid (e.g., RNA) molecules, where the molecular parameters of the nucleic acid molecules may be similar or different from one another with respect to molar mass or basic structural elements such as molecular structure, capping, coding region, or other features.

[0349] In particulate formulations, each nucleic acid (such as RNA) species can be formulated separately as individual particulate formulations. In this case, each individual particulate formulation contains one nucleic acid (such as RNA) species. Individual particulate formulations can exist as separate entities, for example, in separate containers. Such formulations are obtained by providing each nucleic acid (such as RNA) species separately (typically in the form of nucleic acid-containing solutions) with a particle-forming agent, thereby enabling particle formation. Each particle contains only the specific nucleic acid (e.g., RNA) species provided when the particle is formed (individual particulate formulation). In some embodiments, a composition, such as a pharmaceutical composition, comprises multiple individual particulate formulations. Each pharmaceutical composition is called a mixed particulate formulation. A mixed particulate formulation according to this disclosure is obtained by forming individual particulate formulations separately and then mixing the individual particulate formulations. The mixing step yields a formulation containing a mixed population of nucleic acid-containing particles. The individual particle populations may exist together in a single container and contain a mixed population of individual particulate formulations. Alternatively, all nucleic acid (e.g., RNA) species in a pharmaceutical composition can be formulated together as a composite particulate formulation. Such formulations are obtained by providing a formulation (typically a mixed solution) in which all nucleic acid (such as RNA) species are mixed with a particle-forming agent, thereby enabling particle formation. In contrast to mixed particle formulations, mixed particle formulations typically contain particles containing one or more nucleic acid (such as RNA) species. In mixed particle compositions, different nucleic acid (such as RNA) species typically coexist within a single particle.

[0350] Lipids In this specification, the terms “lipid” and “lipid-like substance” are broadly defined as molecules containing one or more hydrophobic moieties or groups, and optionally one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are also called amphiphilic substances. Lipids are typically insoluble or sparingly soluble in water, but soluble in many organic solvents. In aqueous environments, due to their amphiphilic nature, molecules self-assemble to form phases distinct from the organized structures. One of these phases consists of a lipid bilayer, which exists in aqueous environments as vesicles, multilayer / monolayer liposomes, or membranes. Hydrophobicity can be conferred by the inclusion of nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups may include polar and / or charged groups, and include carbohydrates, phosphate groups, carboxylic acid groups, sulfate groups, amino groups (e.g., tertiary amino groups), sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups.

[0351] In this specification, the term "hydrophobic" refers to a molecule, part, or group that is substantially miscible or insoluble in aqueous solution. The term hydrophobic group includes hydrocarbons having at least six carbon atoms. Monovalent radicals of hydrocarbons are referred to herein as hydrocarbyls. Hydrophobic groups may have functional groups (e.g., ethers, esters, halides, etc.) and atoms other than carbon and hydrogen, insofar as they satisfy the condition of being substantially miscible or insoluble in aqueous solution.

[0352] The term "hydrocarbon" includes acyclic hydrocarbon groups, such as alkyl, alkenyl, or alkynyl groups, as defined herein, e.g., linear or branched hydrocarbon groups. It should be understood that one or more hydrogen atoms of an alkyl, alkenyl, or alkynyl group may be substituted with other atoms, such as halogens, oxygen, or sulfur. Unless otherwise specified, hydrocarbon groups also include cyclic (alkyl, alkenyl, or alkynyl) groups or aryl groups, as long as the polarity of the hydrocarbon as a whole remains relatively nonpolar.

[0353] As used herein, the term “amphiphilic” refers to a molecule having both a polar and a nonpolar moiety. Often, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar moiety is soluble in water, while the nonpolar moiety is insoluble in water. Furthermore, the polar moiety may formally have a positive charge or a formally negative charge. Alternatively, the polar moiety may formally have both positive and negative charges, or it may be an amphoteric ion or an intramolecular salt. For the purposes of this disclosure, amphiphilic compounds may be one or more natural or non-natural lipids and lipid-like compounds, but are not limited to these.

[0354] The terms “lipid-like substances,” “lipid-like compounds,” or “lipid-like molecules” refer to substances that are structurally and / or functionally related to lipids but are not considered lipids in the strict sense. For example, this term includes compounds that can form amphiphilic layers because they exist in vesicles, multilayer / monolayer liposomes, or membranes in an aqueous environment, as well as surfactants or synthetic compounds that have both hydrophilic and hydrophobic parts. Generally speaking, this term refers to molecules composed of hydrophilic and hydrophobic parts with different structural configurations, which may or may not be similar to lipids. Examples of lipid-like compounds that can spontaneously integrate into cell membranes include functional lipid components such as synthetic functional spacer lipid components (FSLs) and synthetic functional spacer sterol components (FSSs), and artificial amphiphilic molecules. Lipids containing two long alkyl chains and a polar head group are generally cylindrical. The area occupied by the two alkyl chains is approximately the same as the area occupied by the polar head group. Such lipids have low monomer solubility and tend to aggregate into a water-insoluble planar bilayer. Conventional surfactant monomers, which consist of only one linear alkyl chain and a hydrophilic head group, are generally conical in shape. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain. In some embodiments, surfactants tend to aggregate into water-soluble spherical or elliptical micelles. Lipids also have the same general structure as surfactants (polar hydrophilic head group and nonpolar hydrophobic tail), but differ from surfactants in monomer shape, the type of aggregates formed in solution, and the concentration range required for aggregation. As used herein, the term “lipid” is to be interpreted as covering both lipids and lipid-like substances unless otherwise stated herein or the context clearly indicates otherwise.

[0355] Specific examples of amphiphilic compounds included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0356] In certain embodiments, amphiphilic compounds are lipids. The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids can be classified into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (obtained from the condensation of ketoacyl subunits), sterol lipids, and prenolipids (obtained from the condensation of isoprene subunits). The term "lipid" is sometimes used as a synonym for fat, but fat is a subgroup of lipids called triglycerides. Lipids include fatty acids and their derivatives (triglycerides, diglycerides, monoglycerides, phospholipids, etc.), as well as molecules such as steroids, i.e., sterol-containing metabolites such as cholesterol or its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

[0357] Fatty acids, or fatty acid residues, are a diverse group of molecules made up of hydrocarbon chains ending in a carboxylic acid group; this arrangement gives the molecule a polar, hydrophilic end and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain typically consists of 4 to 24 carbon atoms, can be saturated or unsaturated, and can be bonded to functional groups including oxygen, halogens, nitrogen, and sulfur. When fatty acids contain double bonds, either cis or trans geometric isomers can occur, significantly affecting the molecular structure. Cis double bonds cause the fatty acid chain to bend, and this effect increases with the number of double bonds in the chain. Other major lipid classes in the fatty acid category are fatty acid esters and fatty acid amides.

[0358] Glycerolipids are composed of mono, di, and trisubstituted glycerols, the most well-known being fatty acid triesters of glycerol called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, each of the three hydroxyl groups of glycerol is esterified, usually by a different fatty acid. An additional subclass of glycerolipids is represented by glycosylglycerols, characterized by the presence of one or more sugar residues bonded to glycerol via glycosidic bonds.

[0359] Glycerophospholipids are amphiphilic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core, which is bonded to two fatty acid-derived "tails" by ester bonds and to a single "head" group by a phosphate ester bond. Examples of glycerophospholipids (usually called phospholipids, although sphingomyelin is also classified as a phospholipid) include phosphatidylcholine (also called PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0360] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base skeleton. The main sphingoid base in mammals is commonly called sphingosine. Ceramides (N-acyl sphingoid bases) are a major subclass of sphingoid base derivatives that contain amide-linked fatty acids. These fatty acids are typically saturated or monounsaturated, with chain lengths of 16–26 carbon atoms. The main sphingophospholipid in mammals is sphingomyelin (ceramide phosphocholine), while insects primarily contain ceramide phosphoethanolamine, and fungi have phytoceramide phosphoinositol and mannose-containing head groups. Sphingoglycolipids are a diverse family of molecules composed of one or more sugar residues linked to a sphingoid base via glycosidic bonds. Examples include simple and complex sphingoglycolipids such as cerebrosides and gangliosides.

[0361] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelin.

[0362] Glycolipids are compounds in which fatty acids are directly bound to a sugar backbone, forming a structure that is compatible with the membrane bilayer. In glycolipids, monosaccharides replace the glycerol backbone present in glycerolipids and glycerophospholipids. The best-known glycolipid is the acylated glucosamine precursor, the lipid A component of lipopolysaccharides in Gram-negative bacteria. A typical lipid A molecule is a glucosamine disaccharide derivatized by as many as seven fatty acid acyl chains. The minimum amount of lipopolysaccharide required for E. coli growth is Kdo2-lipid A, a hexaacylated disaccharide of glucosamine glycosylated by two 3-deoxy-D-mann-octurosonic acid (Kdo) residues.

[0363] Polyketides are synthesized by polymerization of acetyl and propionyl subunits using classical enzymes, as well as repeating and multimodular enzymes that share mechanistic features with fatty acid synthases. Polyketides are structurally diverse, comprising numerous secondary metabolites and natural products from animals, plants, bacteria, fungi, and marine resources. Many polyketides are cyclic molecules, and their backbone is often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0364] Cationic lipids and cationic ionized lipids The nucleic acid (e.g., RNA) compositions and nucleic acid particles (in particular RNA LNPs) described herein contain at least one cationic lipid or cationic ionizable lipid as a particle-forming agent. The cationic lipids and cationic ionizable lipids intended for use in the present invention include any cationic / cationic ionizable lipid or lipid-like substance that can electrostatically bind to nucleic acids. In one embodiment, the cationic / cationic ionizable lipids intended for use in the present invention can bind to nucleic acids, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is encapsulated or enclosed.

[0365] In this specification, "cationic lipid" or "cationic lipid-like substance" refers to a lipid or lipid-like substance that has a net positive charge. Cationic lipids or lipid-like substances bind to negatively charged nucleic acids through electrostatic interactions. Generally, cationic lipids have lipophilic moieties such as sterols, acyl chains, diacyl chains or more, and the lipid head group is usually positively charged.

[0366] In certain embodiments, cationic lipids or lipid-like substances have a net positive charge only at a specific pH, particularly an acidic pH, but preferably have no net positive charge at a different pH, preferably a higher pH, such as a physiological pH, i.e., they are neutral. This ionization behavior is thought to enhance efficacy by facilitating escape from endosomes and reducing toxicity compared to particles that remain cationic at physiological pH.

[0367] As used herein, "cationic ionic lipids" refers to lipids or lipid-like substances that have a net positive charge or are neutral, i.e., lipids that are not permanently cationic. Therefore, depending on the pH of the composition in which the cationic ionic lipids are dissolved, the cationic ionic lipids will be either positively charged or neutral.

[0368] In one embodiment, the cationic ionized lipid comprises a head group containing at least one nitrogen atom (N) that is positively charged or protonable, preferably under physiological conditions. In some embodiments, the cationic ionized lipid comprises a head group containing at least one tertiary amine moiety.

[0369] Examples of cationic lipids or cationic ionized lipids include N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP); 1, 2-Diacyloxy-3-dimethylammoniumpropane; 1,2-Dialkyloxy-3-dimethylammoniumpropane; Dioctadecyldimethylammonium chloride (DODAC), 1,2-Distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-Di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-Dimiristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-Dimiristoyl-3-trimethylammoniumpropane (DMTAP) , 1,2-dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5- En-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3] -Dioxolane (DLin-KC2-DMA), Heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propaneaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N ,N-dimethyl-2,3-bis(dodecyloxy)-1-propaneaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-aminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammoniumpropane (DMDAP), 1,2-dipalmitoyl-3-dimethylammoniumpropane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamide)ethyl]-3,4-Di[oleyloxy]benzamide (MVL5), 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-Bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-aminium bromide (DMORIE), Di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azandiyl)dioctanoate (ATX), N,N-dimethyl -2,3-bis(dodecyloxy)propane-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoylethyl)-{2-[(2-dodecylcarbamoylethyl)-2-{(2-dodecylcarbamoylethyl)-[2-(2-dodecylcarbamoylethylamino)-ethyl]-amino}ethylamino)propionamide (Lipidoid 98N, 12 -5) 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecylamino]ethyl]piperazine-1-yl]ethyl]amino]dodecane-2-ol (lipidoid C12-200) and the following structures (XV-1)~(XV-6): [ka] [ka] [ka] [ka] [ka] [ka] These include, but are not limited to, the following:

[0370] Preferred are DODMA, DOTMA, DOTAP, DODAC, and DOSPA. In certain embodiments, the cationic lipid or cationic ionized lipid is DODMA.

[0371] DOTMA is a cationic lipid containing a quaternary amine group. The structure of DOTMA is represented as follows: [ka]

[0372] DODMA is a cationic lipid with a tertiary amine group. The structure of DODMA is represented as follows: [ka]

[0373] In certain embodiments, the composition comprises cationic ionized lipids (particularly when the composition comprises lipid nanoparticles (LNPs)).

[0374] In certain embodiments, the composition includes cationic lipids (particularly when the composition includes lipoplex (LPX)).

[0375] Examples of cationic ionized lipids are disclosed, for example, in WO 2016 / 176330 and WO 2018 / 078053. In some embodiments, the cationic ionized lipid is expressed by formula (X): [ka] (X) [In the formula, L 10 and L 20 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NRa C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- and L 10 and L 20 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or direct bond; G1 and G 2 Each of them is independent of the unsubstituted C1-C 12 Alkylene or C 2-12 It is an alkenylene; G 3 C 1-24 Alkylene, C 2-24 Alkenylene, C 3-8 Cycloalkylene, or C 3-8 It is a cycloalkenylene; R a is H or C 1-12 It is alkyl; R 35 and R 36 Each is independent of C 6-24 Alkyl or C 6-24 It is an alkenil; R 37 H, OR 50 , CN, -C(=O)OR 40 -OC(=O)R 40 or -NR 50 C(=O)R 40 and; R 40 C 1-12 It is alkyl; R 50 is H or C 1-6 It is alkyl; and x is 0, 1, or 2. A compound having the structure shown, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0376] In some of the aforementioned embodiments of formula (X), the lipid has the following structure (XA) or (XB): [ka] (XA) (XB) [In the formula, A is a cycloalkyl or cycloalkylene group of 3 to 8 members; R 60 In each occurrence, independently, H, OH, or C1-C 24 It is alkyl; n1 is an integer between 1 and 15. It has one of the following.

[0377] In some of the aforementioned embodiments of formula (X), the lipid has structure (XA), and in other embodiments, the lipid has structure (XB).

[0378] In other embodiments of formula (X), lipids have the following structure (XC) or (XD): [ka] (XC) (XD) [In the formula, y and z are each independent integers between 1 and 12.] It has one of the following.

[0379] In any of the above embodiments of formula (X), L 10 and L 20 One of them is -O(C=O)-. For example, in some embodiments, L 10 and L 20 Each of them is -O(C=O)-. In any of the several different embodiments described above, L 10 and L 20Each of these is independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 10 and L 20 Each of these is -(C=O)O-.

[0380] In several different embodiments of formula (X), the lipid has the following structure (XE) or (XF): [ka] (XE) or [ka] (XF) It has one of the following.

[0381] In some of the aforementioned embodiments of formula (X), the lipid has the following structure: (XG), (XH), (XJ), or (XK): [ka] (XG) [ka] (XH) [ka] (XJ) or [ka] (XK) It has one of the following.

[0382] In some of the aforementioned embodiments of formula (X), n1 is an integer in the range of 2 to 12, for example, 2 to 8 or 2 to 4. For example, in some embodiments, n1 is 3, 4, 5 or 6. In some embodiments, n1 is 3. In some embodiments, n1 is 4. In some embodiments, n1 is 5. In some embodiments, n1 is 6.

[0383] In some of the other embodiments of equation (X) described above, y and z are each independently integers in the range of 2 to 10. For example, in some embodiments, y and z are each independently integers in the range of 4 to 9 or 4 to 6.

[0384] In some of the aforementioned embodiments of formula (X), R 60 is H. In other embodiments described above, R 60 C1-C 24 It is alkyl. In other embodiments, R 60 It is OH.

[0385] In some embodiments of equation (X), G 3 In other embodiments, G 3 is replaced. In various different embodiments, G 3 is a linear C1-C 24 Alkylene or linear C2-C 24 It is alkenylene. C2-C 24 Alkenylene.

[0386] In some other embodiments of equation (X), R 35 or R 36 , or both, C6-C 24 It is an alkenyl. For example, in some embodiments, R 35 and R 36 Each of them is independent and has the following structure: [ka] It has, During the ceremony: R 7a and R 7b These are, independently, H or C1-C 12 It is alkyl; and a is an integer between 2 and 12. Here, R 7a , R 7b and a are R 35 and R36 Each of these is independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer in the range of 5 to 9 or 8 to 12.

[0387] In some of the aforementioned embodiments of formula (X), R 7a At least one occurrence of is H. For example, in some embodiments, R 7a In each occurrence, is H. In the other different embodiments described above, R 7b At least one of the occurrences is a C1-C8 alkyl group. For example, in some embodiments, the C1-C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0388] In a different embodiment of equation (X), R 35 or R 36 , or both, have the following structure: [ka] It has one of the following.

[0389] In some of the aforementioned embodiments of equation (X), R 37 OH, CN, -C(=O)OR 40 -OC(=O)R 4 Or -NHC(=O)R 40 In some embodiments, R 40 It is either methyl or ethyl.

[0390] In various different embodiments, the cationic ionized lipid of formula (X) has one of the following structures. [Table 1] TIFF2026511086000034.tif234164TIFF2026511086000035.tif249164TIFF2026511086000036.tif21116 4TIFF2026511086000037.tif222164TIFF2026511086000038.tif217164TIFF2026511086000039.tif53164

[0391] In various different embodiments, the cationic ionized lipid has one of the structures shown in the table below. [Table 1]

[0392] In some embodiments, cationic ionized lipids are expressed by formula (XI): [ka] [In the formula, R1 and R2 are each independently R5 or -G1-L1-R6, where at least one R1 and R2 is -G1-L1-R6; R3 and R4 are independent of each other, C 1-6 Alkyl, C 2-6 Alkenyl, aryl, and C 3-10 Selected from the group consisting of cycloalkyl groups; R5 and R6 are each independently acyclic hydrocarbyl groups having at least 10 carbon atoms; G1 and G2 are independent of each other, unsubstituted C 1-12 Alkylene or C 2-12 It is an alkenylene; L1 and L2 are independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x Selected from the group consisting of -, -SS-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa-, and -NRaC(=O)O-; Ra is H or C 1-12 It is alkyl; m is 0, 1, 2, 3, or 4; and x is 0, 1, or 2. It has the structure shown in [image / diagram].

[0393] In some of the aforementioned embodiments of formula (XI), each G1 independently represents an unsubstituted C1-C 12 Alkylene or unsubstituted carbon dioxide 2-12 Alkenylenes, for example, unsubstituted, linear carbon chains. 1-12 Alkylene or unsubstituted, linear carbon chain 2-12 It is an alkenylene. In some embodiments, each G1 is independently an unsubstituted C 6-12 Alkylene or unsubstituted carbon dioxide 6-12 Alkenylenes, for example, unsubstituted, linear carbon chains. 6-12 Alkylene or unsubstituted, linear carbon chain 6-12 It is an alkenylene. In some embodiments, each G1 is independently an unsubstituted C 8-12 Alkylene or unsubstituted carbon dioxide 8-12 Alkenylenes, for example, unsubstituted, linear carbon chains. 8-12 Alkylene or unsubstituted, linear carbon chain 8-12 It is an alkenylene. In some embodiments, each G1 is independently an unsubstituted C 6-10 Alkylene or unsubstituted carbon dioxide 6-10 Alkenylenes, for example, unsubstituted, linear carbon chains. 6-10 Alkylene or unsubstituted, linear carbon chain 6-10 It is an alkenylene. In some embodiments, each G1 is independently an unsubstituted alkylene having 8, 9, or 10 carbon atoms, for example, an unsubstituted, linear alkylene having 8, 9, or 10 carbon atoms. In some embodiments, if both R1 and R2 are independently -G1-L1-R6, the G1 of R1 may be different from the G1 of R2. In some of these embodiments, for example, the G1 of R1 is an unsubstituted, linear C 1-12 It is an alkylene, and G1 of R2 is unsubstituted, linear C 2-12 Is it an alkenylene; or is G1 of R1 an unsubstituted, linear C? 1-12 It is an alkylene group, and G1 of R2 is a different unsubstituted, linear C1-12 It is an alkylene group. In some embodiments, if both R1 and R2 are independently -G1-L1-R6, the G1 of R1 may be the same as the G1 of R2. In some of these embodiments, for example, each G1 is the same unsubstituted, linear C 8-12 Alkylenes, for example, unsubstituted, linear carbon chains. 8-10 Either alkylene or each G1 is the same unsubstituted, linear C 6-12 It is alkenylene.

[0394] In some of the aforementioned embodiments of formula (XI), each L1 is independently selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, and -C(=O)NRa-. In some embodiments, Ra of L1 is H or C 1-12 It is alkyl. In some embodiments, Ra of L1 is H or C 1-6 Alkyl, for example, H or C 1-3It is alkyl. In some embodiments, Ra of L1 is H, methyl, or ethyl. In some embodiments, each L1 is independently selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, and -SC(=O)-. In some embodiments, each L1 is independently -O(C=O)- or -(C=O)O-. In some embodiments, if both R1 and R2 are independently -G1-L1-R6, L1 of R1 may be different from L1 of R2. In some of these embodiments, for example, L1 of R1 is one part selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, and -C(=O)NRa- (for example, L1 of R1 is -O(C=O)-), and L1 of R2 is a different part selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, and -C(=O)NRa- (for example, L1 of R2 is -(C=O)O-). In some embodiments, if both R1 and R2 are independently G1-L1-R6, then L1 of R1 may be identical to L1 of R2. In some of these embodiments, for example, each L1 is the same part selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, and -C(=O)NRa-, for example, each L1 is -O(C=O)-, or each of L1 is -(C=O)O-.

[0395] In some of the aforementioned embodiments of formula (XI), each R6 is independently an acyclic hydrocarbyl group having at least 10 carbon atoms, for example, a linear hydrocarbyl group having at least 10 carbon atoms. In some embodiments, each R6 is independently having up to 30 carbon atoms, for example, up to 28, up to 26, up to 24, up to 22, or up to 20. In some embodiments, each R6 is independently an acyclic hydrocarbyl group having at least 10 to 30 carbon atoms (for example, 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), for example, a linear hydrocarbyl group having at least 10 to 30 carbon atoms (for example, 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms). In some embodiments, each R6 is bonded to L1 via the internal carbon atoms of R6. In some embodiments, each R6 independently has up to 30 carbon atoms (e.g., up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms), and each R6 is bonded to L1 via its internal carbon atoms. In some embodiments, each R6 independently is an acyclic hydrocarbyl group having at least 10 carbon atoms, for example, a linear hydrocarbyl group having at least 10 carbon atoms, and each R6 is bonded to L1 via its internal carbon atoms. In some embodiments, each R6 is independently an acyclic hydrocarbyl group having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), for example a linear hydrocarbyl group having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), and each R6 is bonded to L1 via the internal carbon atoms of R6. In some embodiments, the hydrocarbyl group of R6 is an alkyl or alkenyl group, for example C 10-30It is an alkyl or alkenyl group. Therefore, in some embodiments, each R6 is independently an acyclic alkyl group having at least 10 carbon atoms or an acyclic alkenyl group having at least 10 carbon atoms, for example, a linear alkyl group having at least 10 carbon atoms or a linear alkenyl group having at least 10 carbon atoms. In some embodiments, each R6 is independently an acyclic alkyl group having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms) or an acyclic alkenyl group having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), for example, a linear alkyl group having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms) or a linear alkenyl group having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms). In some embodiments, each R6 is independently an acyclic alkyl group having 11 to 19 carbon atoms (e.g., 11, 13, 15, 17, or 17 carbon atoms), for example, a linear alkyl group having 11 to 19 carbon atoms (e.g., 11, 13, 15, 17, or 17 carbon atoms). In some embodiments, each R6 is independently an acyclic alkyl group having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), or an acyclic alkenyl group having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), for example, a linear alkyl group having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), and each R6 is bonded to L1 via the internal carbon atoms of R6.In some embodiments, each R6 is independently an acyclic alkyl group having 11 to 19 carbon atoms (e.g., 11, 13, 15, 17, or 17 carbon atoms), for example, a linear alkyl group having 11 to 19 carbon atoms (e.g., 11, 13, 15, 17, or 17 carbon atoms), and each R6 is bonded to L1 via an internal carbon atom of R6. The expression “internal carbon atom” means that the carbon atom of R6 to which R6 is bonded to L1 is directly bonded to at least two other carbon atoms of R6. For example, C below. 11 In the case of alkyl groups, any carbon atom at positions 2, 3, 4, 5, and 7 is eligible as an "internal carbon atom" according to the present invention, but carbon atoms at positions 1, 6, 8, 9, 10, and 11 are not eligible. [ka]

[0396] As a result, R6 is C 11 And, if it is bonded to L1 via the internal carbon of R6, then the following groups: [ka] This includes, and here, [ka] This represents a bond where R6 is attached to L1. Furthermore, in the case of a linear alkyl group, for example, a linear C 11 In alkyl groups, the first and last carbon atoms of a linear alkyl group are excluded (i.e., linear C 11Each carbon atom (except for the carbon atoms at the 1st and 11th positions of the alkyl group) is eligible as an "internal carbon atom". Thus, in some embodiments, a linear alkyl group having p carbon atoms, where R6 is bonded to L1 via its internal carbon atoms, means that R6 is bonded to L1 via its carbon atoms at any one position from 2 to (p-1) (this excludes the terminal C atoms at positions 1 and p). In some embodiments, R6 is a linear alkyl group having p' carbon atoms (p' is even), where R6 is bonded to L1 via its internal carbon atoms, where R6 is bonded to L1 via its carbon atoms at any of the (p' / 2-1), (p' / 2), and (p' / 2+1) positions (for example, if p' is 10, R6 is bonded to L1 via its carbon atoms at any of the 4th, 5th, and 6th positions). In some embodiments, R6 is a linear alkyl having p'' carbon atoms (where p'' is odd), and is bonded to L1 via the internal carbon atoms of R6, and R6 is bonded to L1 via the carbon atoms at either position (p''-1) / 2 and (p''+1) / 2 of R6 (for example, if p'' is 11, R6 is bonded to L1 via the carbon atoms at either position 5 and 6 of R6). Generally, if both R1 and R2 are -G1-L1-R6, and each R6 is bonded to L1 via the internal carbon atoms of R6, then it is understood that R6 of R1 is bonded to L1 of R1 (but not to L1 of R2) via the internal carbon atoms of R6, and R6 of R2 is bonded to L1 of R2 (but not to L1 of R1) via the internal carbon atoms of R6. In some embodiments, each R6 independently belongs to the group consisting of: [ka] Selected from, here, [ka] This represents the bond in which R6 is attached to L1. In some embodiments, when both R1 and R2 are independently -G1-L1-R6, R6 of R1 is different from R6 of R2. In some of these embodiments, for example, R6 of R1 may be an acyclic, preferably linear, hydrocarbyl group having at least 10 carbon atoms (for example, R6 of R1 is [ka] (where R2's R6 is,) R2's R6 may be a different acyclic, preferably linear, hydrocarbyl group having at least 10 carbon atoms (for example, R2's R6 is, [ka] (This is the case.) In some embodiments, if both R1 and R2 are independently -G1-L1-R6, then R6 of R1 is identical to R6 of R2. In some of these embodiments, for example, each R6 is the same acyclic, preferably linear, hydrocarbyl group having at least 10 carbon atoms (for example, each R6 is [ka] (That is.)

[0397] In some of the aforementioned embodiments of formula (XI), R5 is an acyclic hydrocarbyl group having at least 10 carbon atoms, for example, a linear hydrocarbyl group having at least 10 carbon atoms. In some embodiments, R5 is an acyclic hydrocarbyl group having at least 12 carbon atoms, for example, at least 14, at least 16, or at least 18 carbon atoms, for example, a linear hydrocarbyl group having at least 12, at least 14, at least 16, or at least 18 carbon atoms. In some embodiments, R5 has up to 30 carbon atoms, for example, up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms in some embodiments. R5 is an acyclic hydrocarbyl group, for example, a linear hydrocarbyl group, where each hydrocarbyl group has 10 to 30 carbon atoms (for example, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20 carbon atoms, or 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20 carbon atoms, etc.) (or 14-30, 14-28, 14-26, 14-24, 14-22, 14-20 carbon atoms, or 16-30, 16-28, 16-26, 16-24, 16-22, 16-20 carbon atoms, or 18-30, 18-28, 18-26, 18-24, 18-22, or 18-20 carbon atoms). In some embodiments, the hydrocarbyl group of R5 is an alkyl or alkenyl group, for example, C 10-30It is an alkyl or alkenyl group. Therefore, in some embodiments, R5 is an acyclic alkyl group having at least 10 carbon atoms (e.g., at least 12, at least 14, at least 16, or at least 18 carbon atoms) or an acyclic alkenyl group having at least 10 carbon atoms (e.g., at least 12, at least 14, at least 16, or at least 18 carbon atoms), for example, a linear alkyl group having at least 10 carbon atoms (e.g., at least 12, at least 14, at least 16, or at least 18 carbon atoms), or a linear alkenyl group having at least 10 carbon atoms (e.g., at least 12, at least 14, at least 16, or at least 18 carbon atoms). In some embodiments, R5 is an acyclic alkyl group or acyclic alkenyl group, for example, a linear alkyl group or a linear alkenyl group, where each alkyl and alkenyl group independently comprises 10 to 30 carbon atoms (for example, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20 carbon atoms, or 12 to 30, 12 to 28, 12 to 26, 12 to 24, The group has 12-22 carbon atoms, 12-20 carbon atoms, or 14-30, 14-28, 14-26, 14-24, 14-22, 14-20 carbon atoms, or 16-30, 16-28, 16-26, 16-24, 16-22, 16-20 carbon atoms, or 18-30, 18-28, 18-26, 18-24, 18-22, or 18-20 carbon atoms. In some embodiments, the alkenyl group has at least two carbon-carbon double bonds, e.g., two or three carbon-carbon double bonds, e.g., two carbon-carbon double bonds. In some embodiments, the alkenyl group has at least one carbon-carbon double bond in the cis configuration, e.g., one, two or three carbon-carbon double bonds in the cis configuration, e.g., two carbon-carbon double bonds.Therefore, in some embodiments, R5 is an acyclic alkyl group or an acyclic alkenyl group, for example, a linear alkyl group or a linear alkenyl group, where each alkyl and alkenyl group independently has 10 to 30 carbon atoms (for example, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20 carbon atoms, or 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20 carbon atoms, or The group has 14-30, 14-28, 14-26, 14-24, 14-22, or 14-20 carbon atoms, or 16-30, 16-28, 16-26, 16-24, 16-22, or 18-30, 18-28, 18-26, 18-24, 18-22, or 18-20 carbon atoms, and the alkenyl group has at least two carbon-carbon double bonds, for example, two or three carbon-carbon double bonds. In some embodiments, R5 is an acyclic alkyl group or acyclic alkenyl group, for example, a linear alkyl group or a linear alkenyl group, where each alkyl and alkenyl group independently comprises 10 to 30 carbon atoms (for example, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20 carbon atoms, or 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20 carbon atoms, or 14 to 30 carbon atoms). The alkenyl group has 14-28, 14-26, 14-24, 14-22, or 14-20 carbon atoms, or 16-30, 16-28, 16-26, 16-24, 16-22, or 18-30, 18-28, 18-26, 18-24, 18-22, or 18-20 carbon atoms, and the alkenyl group has at least one carbon-carbon double bond in cis configuration, e.g., 1, 2, or 3 carbon-carbon double bonds. In some embodiments, R5 has the following structure: [ka] It has, and here, [ka] This represents a bond in which R5 attaches to the rest of the compound.

[0398] In some of the aforementioned embodiments of formula (XI), L2 is selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -SS-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa-, and -NRaC(=O)O-. In some embodiments, L2 is selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, and -C(=O)NRa-. In some embodiments, Ra of L2 is H or C 1-12 It is alkyl. In some embodiments, Ra of L2 is H or C 1-6 Alkyl, for example, H or C 1-3 It is alkyl. In some embodiments, Ra of L2 is H, methyl, or ethyl. In some embodiments, L2 is selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, and -SC(=O)-. In some embodiments, L2 is -O(C=O)- or -(C=O)O-.

[0399] In some of the aforementioned embodiments of formula (XI), G2 is an unsubstituted C 1-12 Alkylene or unsubstituted carbon dioxide 2-12 Alkenylenes, for example, unsubstituted, linear carbon chains. 1-12 Alkylene or unsubstituted, linear carbon chain 2-12 It is an alkenylene. In some embodiments, G2 is an unsubstituted C 2-10 Alkylene or unsubstituted carbon dioxide 2-10 Alkenylenes, for example, unsubstituted, linear carbon chains. 2-10 Alkylene or unsubstituted, linear carbon chain 2-10 It is an alkenylene. In some embodiments, G2 is an unsubstituted C 2-6 Alkylene or unsubstituted carbon dioxide 2-6 Alkenylenes, for example, unsubstituted, linear carbon chains. 2-6 Alkylene or unsubstituted, linear carbon chain 2-6It is an alkenylene. In some embodiments, G2 is an unsubstituted C 2-4 Alkylene or unsubstituted carbon dioxide 2-4 Alkenylenes, for example, unsubstituted, linear carbon chains. 2-4 Alkylene or unsubstituted, linear carbon chain 2-4 It is an alkenylene. In some embodiments, G2 is ethylene or trimethylene.

[0400] In some of the embodiments of formula (XI) described above, R3 and R4 are independently C 1-6 Alkyl or C 2-6 It is an alkenyl. In some embodiments, R3 and R4 are each independently C 1-4 Alkyl or C 2-4 It is an alkenyl. In some embodiments, R3 and R4 are each independently C 1-3 It is alkyl. In some embodiments, R3 and R4 are independently methyl or ethyl. In some embodiments, R3 and R4 are each methyl.

[0401] In some of the aforementioned embodiments of formula (XI), m is 0, 1, 2, or 3. In some embodiments, m is 0 or 2. In some embodiments, m is 0. In some embodiments, m is 2.

[0402] In some of the aforementioned embodiments of formula (XI), the cationic ionized lipid is formula (XIIa) or (XIIb): [ka] (XIIa) [ka] (XIIb) [In the formula, R3 and R4 are independently C1-C6 alkyl or C 2-6 It is an alkenil; R5 is a linear hydrocarbyl group having at least 14 carbon atoms (e.g., at least 16 carbon atoms), where the hydrocarbyl group preferably has at least 2 carbon-carbon double bonds; Each R6 is independently a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 carbon atoms, and / or each R6 is bonded to L1 via the internal carbon atoms of R6, preferably each R6 is independently a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 carbon atoms, and each R6 is bonded to L1 via the internal carbon atoms of R6; Each G1 is independently unsubstituted, linear C 4-12 Alkylene or C 4-12 Alkenylenes, for example, unsubstituted, linear carbon chains. 6-12 Alkylene or C 6-12 Alkenylenes, for example, unsubstituted, linear carbon chains. 8-12 Alkylene or unsubstituted, linear carbon chain 8-12 It is an alkenylene; G2 is unsubstituted C2-C 10 Alkylene or C 2-10 Alkenylenes, preferably unsubstituted C2-C6 alkylenes or C 2-6 It is an alkenylene; L1 and L2 are independently -O(C=O)- or -(C=O)O-; and m is 0, 1, 2, or 3, preferably 0 or 2. It has the structure shown in [image / diagram].

[0403] In some of the aforementioned embodiments of formula (XIIa), R5 has up to 30 carbon atoms, e.g., up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms. In some embodiments of formula (XIIa), R5 is a linear hydrocarbyl group having 14 to 30 carbon atoms (e.g., 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms). In some embodiments of formula (XIIa), R5 is a linear alkyl or alkenyl group having 14 to 30 carbon atoms (e.g., 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms). In some embodiments of formula (XIIa), the alkenyl group has at least two carbon-carbon double bonds, e.g., two or three carbon-carbon double bonds, e.g., two carbon-carbon double bonds. In some embodiments, the alkenyl group has at least one carbon-carbon double bond in the cis configuration, for example, 1, 2, or 3 carbon-carbon double bonds in the cis configuration, for example, 2 carbon-carbon double bonds. Therefore, in some embodiments of formula (XIIa), R5 is a linear alkyl group or a linear alkenyl group, where the alkyl and alkenyl groups each independently have 14 to 30 carbon atoms (for example, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms), and the alkenyl group has at least two carbon-carbon double bonds, for example, two or three carbon-carbon double bonds.In some embodiments of formula (XIIa), R5 is a linear alkyl group or a linear alkenyl group, where the alkyl and alkenyl groups each independently have 14 to 30 carbon atoms (for example, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms), and the alkenyl group has at least one carbon-carbon double bond in cis configuration, for example, 1, 2, or 3 carbon-carbon double bonds. In some embodiments of formula (XIIa), R5 is a linear alkyl group or a linear alkenyl group, where the alkyl and alkenyl groups each independently have 14 to 30 carbon atoms (e.g., 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms), and the alkenyl group has 2 or 3 carbon-carbon double bonds, where at least 1 carbon-carbon double bond, e.g., 1, 2, or 3 carbon-carbon double bonds, is in the cis configuration. In some embodiments of equation (XIIa), R5 has the following structure: [ka] It has, and here, [ka] R5 represents a bond to the rest of the compound. In some embodiments of formula (XIIa), R6 has up to 30 carbon atoms, e.g., up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms. In some embodiments of formula (XIIa), R6 is an acyclic hydrocarbyl group (e.g., an acyclic alkyl group) having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), e.g., a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms). In some embodiments of formula (XIIa), R6 is a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 carbon atoms, and R6 is bonded to L1 via the internal carbon atoms of R6. In some embodiments of formula (XIIa), R6 is an acyclic hydrocarbyl group (e.g., an acyclic alkyl group) having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), for example, a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), and R6 is bonded to L1 via the internal carbon atoms of R6. In some embodiments of formula (XIIa), G1 is independently an unsubstituted, linear C 4-12 Alkylene or C 4-12 Alkenylenes, for example, unsubstituted, linear carbon chains. 6-12 Alkylene or C 6-12It is an alkenylene. In some embodiments of formula (XIIa), R5 is a linear hydrocarbyl group, e.g., a linear alkenyl group, having at least 14 carbon atoms (e.g., 14 to 30 carbon atoms) and 2 or 3 carbon-carbon double bonds; R6 is a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 carbon atoms (e.g., 10 to 30 carbon atoms), and R6 is bonded to L1 via the internal carbon atoms of R6; and G1 is independently an unsubstituted, linear C 4-12 Alkylene or C 4-12 Alkenylenes, for example, unsubstituted, linear carbon chains. 6-12 Alkylene or C 6-12 It is alkenylene.

[0404] In some of the aforementioned embodiments of formula (XIIb), each R6 independently has up to 30 carbon atoms, for example, up to 28, up to 26, up to 24, up to 22, or up to 20. In some embodiments of formula (XIIb), each R6 independently is a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms, or 11 to 19 carbon atoms, for example, 11, 13, 15, 17, or 17 carbon atoms). In some embodiments of formula (XIIb), each R6 is independently a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms, or 11 to 19 carbon atoms, e.g., 11, 13, 15, 17, or 17 carbon atoms), and each R6 is bonded to L1 via the internal carbon atoms of R6. In some embodiments of formula (XIIb), each R6 is independently a group consisting of: [ka] Selected from, here, [ka] This represents a bond in which R6 attaches to L1. In some embodiments of formula (XIIb), each G1 is independently an unsubstituted, linear C 6-12 Alkylene or C 6-12 It is an alkenylene. In some embodiments of formula (XIIb), each G1 is independently an unsubstituted, linear C 8-12 Alkylene or C 8-12 It is an alkenylene. In some embodiments of formula (XIIb), each R6 is independently a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 carbon atoms (e.g., 10-28, 10-26, 10-24, 10-22, or 10-20 carbon atoms, or 11-19 carbon atoms, e.g., 11, 13, 15, 17, or 17 carbon atoms), and each R6 is bonded to L1 via the internal carbon atoms of R6; each G1 is independently an unsubstituted, linear C 8-12 Alkylene or C 8-12 It is alkenylene.

[0405] In some of the aforementioned embodiments of formula (XI), the cationic ionized lipid is formula: (XIIIa) or (XIIIb): [ka] (XIIIa) [ka] (XIIIb) [In the formula, R3 and R4 are independent of each other, C 1-4 Alkyl or C 2-4 Alkenil, more preferably C 1-3 Alkyl, for example, methyl or ethyl; R5 is a linear alkyl or alkenyl group having at least 16 carbon atoms, where the alkenyl group preferably has at least two carbon-carbon double bonds; Each R6 is independently a linear hydrocarbyl group having at least 10 carbon atoms, where R6 is bonded to L1 via the internal carbon atoms of R6; Each G1 is independently unsubstituted, linear C 6-12 Alkylene or unsubstituted, linear carbon chain 6-12 Alkenylenes, for example, unsubstituted, linear carbon chains. 8-12 Alkylene or unsubstituted, linear carbon chain 8-12 Alkenylenes, for example, unsubstituted, linear carbon chains. 8-10 Alkylene or unsubstituted, linear carbon chain 8-10 Alkenylenes, for example, unsubstituted, linear C8 alkylenes; G2 is unsubstituted C 2-6 Alkylene or C 2-6 Alkenylene, preferably unsubstituted C 2-4 Alkylene or C 2-4 Alkenylenes, such as ethylene or trimethylene; L1 and L2 are independently -O(C=O)- or -(C=O)O-; and m is 0, 1, 2, or 3, preferably 0 or 2. It has the structure shown in [image / diagram].

[0406] In some of the aforementioned embodiments of formula (XIIIa), R5 has up to 30 carbon atoms, for example, up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms. In some embodiments of formula (XIIIa), R5 is a linear alkyl or alkenyl group having 16 to 30 carbon atoms (for example, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20). In some embodiments of formula (XIIIa), the alkenyl group has at least two carbon-carbon double bonds, for example, two or three carbon-carbon double bonds, for example, two carbon-carbon double bonds. In some embodiments, the alkenyl group has at least one carbon-carbon double bond in the cis configuration, e.g., 1, 2, or 3 in the cis configuration, e.g., 2 carbon-carbon double bonds. Thus, in some embodiments of formula (XIIIa), R5 is a linear alkyl group or a linear alkenyl group, where the alkyl group and the alkenyl group each independently have 16 to 30 carbon atoms (e.g., 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20), and the alkenyl group has at least two carbon-carbon double bonds, e.g., 2 or 3 carbon-carbon double bonds. In some embodiments of formula (XIIIa), R5 is a linear alkyl group or a linear alkenyl group, where each alkyl group and alkenyl group independently comprises 16 to 30 carbon atoms (e.g., 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms), and the alkenyl group has at least one carbon-carbon double bond, e.g., one, two, or three carbon-carbon double bonds in a cis configuration.In some embodiments of formula (XIIIa), R5 is a linear alkyl group or a linear alkenyl group, where each alkyl group and alkenyl group independently has 16 to 30 carbon atoms (e.g., 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms), and the alkenyl group has two or three carbon-carbon double bonds, where at least one carbon-carbon double bond, e.g., one, two, or three carbon-carbon double bonds, is in the cis configuration. In some embodiments of formula (XIIIa), R5 has the following structure: [ka] It has, and here, ...

Claims

1. A composition comprising particles dispersed in an aqueous phase, wherein the aqueous phase includes a buffer system and has a pH of about 4.0 to 5.5, and the particles comprise (i) nucleic acids and (ii) cationic or cationically ionizable lipids.

2. The composition according to claim 1, wherein the pH of the aqueous phase is less than 5.5 and / or greater than 4.0, for example, at least 4.1 to less than 5.5, at least 4.2 to less than 5.5, at least 4.3 to less than 5.5, at least 4.4 to less than 5.5, or at least 4.5 to less than 5.

5.

3. The composition according to claim 1 or 2, wherein the concentration of the buffer system in the aqueous phase is about 1 mM to about 50 mM, preferably about 2 mM to about 40 mM, more preferably about 3 mM to about 30 mM, more preferably about 4 mM to about 25 mM, for example, about 5 mM to about 20 mM.

4. The composition according to any one of claims 1 to 3, wherein the buffer system contains histidine.

5. The composition according to any one of claims 1 to 3, wherein the buffer system comprises HEPES.

6. The composition according to any one of claims 1 to 5, wherein the aqueous phase further comprises a chelating agent such as EDTA.

7. The composition according to claim 6, wherein the concentration of the chelating agent in the aqueous phase is about 0.1 mM to about 20 mM, for example, about 0.2 mM to about 15 mM, about 0.3 mM to about 12 mM, about 0.4 mM to about 11 mM, or about 0.5 mM to about 10 mM.

8. The composition according to any one of claims 1 to 7, wherein the aqueous phase further comprises one or more isotonic agents, for example, a salt such as sodium chloride, and / or a sugar such as sucrose or glucose.

9. The composition according to claim 8, wherein the concentration of one or more isotonic agents is such that the composition is most isotonic, particularly when compared to human blood.

10. The composition according to any one of claims 1 to 9, wherein the particles have a size of about 30 nm to about 500 nm.

11. The composition according to any one of claims 1 to 10, wherein the particles are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and two or more combinations thereof.

12. The composition according to any one of claims 1 to 11, wherein water is the main component of the composition, and / or the total amount of solvents other than water contained in the composition is less than about 0.5% (v / v).

13. The composition according to any one of claims 1 to 12, wherein the concentration of nucleic acid in the composition is about 0.1 mg / l to about 500 mg / l, for example, about 0.5 mg / l to about 400 mg / l, about 1 mg / l to about 300 mg / l, about 2 mg / l to about 200 mg / l, about 3 mg / l to about 150 mg / l, or about 5 mg / l to about 100 mg / l.

14. The composition according to any one of claims 1 to 13, wherein the N / P value is at least about 2, for example, in the range of about 2 to about 12, about 4 to about 10, about 4 to about 8, or about 5 to about 7, for example, about 6.

15. The composition according to any one of claims 1 to 14, wherein the cationic ionized lipid comprises a head group having at least one nitrogen atom that can be protonated under physiological conditions.

16. Cationic ionized lipids, formula (X): 【Chemistry 1】 (X) [In the formula, L a , a , a , a , a , a , a , a , x , a , a , a , x , a , 10 , 20 and L 20 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- and L 10 and L 20 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond; G 1 and G 2 Each of these is independently of the non-substituted C 1 -C 12 Alkylene or C 2-12 It is alkenylene; G 3 C 1-24 Alkylene, C 2-24 Alkenylene, C 3-8 Cycloalkylene, or C 3-8 It is a cycloalkenylene; R a is H or C 1-12 It is alkyl; R 35 and R 36 Each is independent of C 6-24 Alkyl or C 6-24 It is an alkenil; R 37 H, OR 50 , CN, -C(=O)OR 40 -OC(=O)R 40 or -NR 50 C(=O)R 40 And; R 40 C 1-12 It is alkyl; R 50 is H or C 1-6 It is alkyl; and x is 0, 1, or 2. The composition according to any one of claims 1 to 15, which is a compound having the structure shown, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

17. Cationic ionized lipids, formula (XI): 【Chemistry 2】 [In the formula, R 1 and R 2 Each is independent of R 5 or -G 1 -L 1 -R 6 And here, at least one R 1 and R 2 is, -G 1 -L 1 -R 6 And; R 3 and R 4 Each is independent of C 1-6 Alkyl, C 2-6 Alkenyl, aryl, and C 3-10 Selected from the group consisting of cycloalkyl groups; R 5 and R 6 Each of these is independently an acyclic hydrocarbyl group having at least 10 carbon atoms; G 1 and G 2 Each of these is independently of the non-substituted C 1-12 Alkylene or C 2-12 It is alkenylene; L 1 and L 2 These are independent of each other: -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x Selected from the group consisting of -, -SS-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa-, and -NRaC(=O)O-; Ra is H or C 1-12 It is alkyl; m is 0, 1, 2, 3, or 4; and x is 0, 1, or 2. The composition according to any one of claims 1 to 15, wherein the compound is a compound having the structure shown.

18. Cationic or cationic ionized lipids [(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldodecanoate) (ALC-315); 1,2-Dioleoyloxy-3-dimethylaminopropane (DODMA); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-Dioxolane (DLin-KC2-DMA); Heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA); Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate(L319); Bis-(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)nonanamide)-nonadecanedioate(A9); (Heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate)(L5); Heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate)(SM-102); O-[N-{(9Z,12Z)-octadeca-9,12-dien-1-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (HY501); 2-(di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl 4-(dimethylamino)butanoate (EA-2); 4-((di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutan-4-amine (HYAM-2); ((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azandiylbis(octane 8,1-diyl)bis(2-hexyldodecanoate) (EA-405); (2-(4-(dimethylamino)butanoyl)oxy)azandiylbis(octane 8,1-diyl)bis(2-hexyldodecanoate)(HY-405); Palmitoyl oleoyl-nor-arginine (PONA); Guanidino-di[(heptadecyl)methyl]carboxylic acid (GUADACA); 4-Methylpyridinium-di(heptadecyl)methylcarboxylic acid (MPDACA); 1,2-Dioleoyl-3 Trimethylammonium Propane (DOTAP); 1,2-Dioleoyl-3-dimethylammonium propane (DODAP); or 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); 7,7'-((4-hydroxybutyl)azandiyl)bis(N-hexyl-N-octylheptan-1-sulfonamide)(BNT-51); 7,7'-((4-(3,3-dimethylthioureido)butyl)azandiyl)bis(N-hexyl-N-octylheptane-1-sulfonamide)(BNT-52); Di(heptadecan-9-yl) 3,3'-((2-(4-methylpiperazine-1-yl)ethyl)azandiyl)dipropionate (BHD-C2C2-PipZ); Bis(2-octyldodecyl) 3,3'-((2-(1-methylpyrrolidine-2-yl)ethyl)azandiyl)dipropionate (BODD-C2C2-1Me-Pyr); Bis(2-octyldodecyl) 3,3'-((2-(pyrrolidine-1-yl)ethyl)azandiyl)dipropionate (BODD-C2C2-Pyr); Bis(2-octyldodecyl) 3,3'-(((1-methylpiperidine-3-yl)methyl)azandiyl)dipropionate (BODD-C2C1-1Me-3PipD); Bis(2-octyldodecyl) 3,3'-(((1-methylpiperidine-4-yl)methyl)azandiyl)dipropionate (BODD-C2C1-1Me-PipD); Bis(2-octyldodecyl) 3,3'-((2-(dimethylamino)ethyl)azandiyl)dipropionate (BODD-C2C2-DMA); Bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazine-1-yl)butyl)azandiyl)dipropionate (BODD-C2C4-PipZ); Bis(2-octyldodecyl) 3,3'-((4-(pyrroridine-1-yl)butyl)azandiyl)dipropionate (BODD-C2C4-Pyr); A composition according to any one of claims 1 to 15, comprising bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azandiyl)dipropionate (BHD-C2C4-PipZ); or a mixture thereof.

19. The composition according to any one of claims 1 to 18, wherein cationic or cationic ionized lipids constitute about 20 mol% to about 80 mol%, preferably about 25 mol% to about 65 mol%, more preferably about 30 mol% to about 50 mol%, for example, about 40 mol% to about 50 mol%, of the total lipids present in the composition.

20. The composition according to any one of claims 1 to 19, wherein the particles preferably further comprise one or more additional lipids selected from the group consisting of polymer-bound lipids, neutral lipids, steroids, and combinations thereof.

21. The polymer-bound lipid includes a pegylated lipid, where the pegylated lipid is preferably selected from the group consisting of (i) DSPE-PEG, DOPE-PEG, DPPE-PEG, and DMPE-PEG, and (ii) has the following structure: 【Transformation 3】 [In the formula, R 12 and R 13 Each of these is independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain being optionally interrupted by one or more ester bonds; and the average value of w is in the range of 30 to 60. The composition according to claim 20, having the structure shown by, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

22. The composition according to claim 20, wherein the polymer-bound lipid comprises a polysarcosine-lipid conjugate or a conjugate of polysarcosine and a lipid-like substance, preferably the polysarcosine-lipid conjugate or the conjugate of polysarcosine and a lipid-like substance is a member selected from the group consisting of a polysarcosine-diacylglycerol conjugate, a polysarcosine-dialkyloxypropyl conjugate, a polysarcosine-phospholipid conjugate, a polysarcosine-ceramide conjugate, and mixtures thereof.

23. The composition according to any one of claims 20 to 22, wherein the polymer-bound lipids constitute about 0.5 mol% to about 5 mol%, preferably about 1 mol% to about 5 mol%, and more preferably about 1 mol% to about 4.5 mol%, of the total lipids present in the composition.

24. The neutral lipid is a phospholipid, preferably selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin, more preferably distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine A composition according to any one of claims 20 to 23, selected from the group consisting of zilcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 lysoPC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), and diphytanoylphosphatidylethanolamine (DPyPE).

25. The composition according to any one of claims 20 to 24, wherein neutral lipids constitute about 5 mol% to about 40 mol%, preferably about 5 mol% to about 20 mol%, and more preferably about 5 mol% to about 15 mol%, of the total lipids present in the composition.

26. The composition according to any one of claims 20 to 25, wherein the steroid is a sterol such as cholesterol.

27. The composition according to any one of the cationic or cationic ionized lipid claims 20 to 26, wherein the steroid constitutes about 10 mol% to about 65 mol%, preferably about 20 mol% to about 60 mol%, more preferably about 30 mol% to about 50 mol%, of the total lipids present in the composition.

28. The composition according to any one of claims 20 to 27, wherein the particles comprise cationic or cationic ionized lipids, neutral lipids (e.g., phospholipids), and steroids, and optionally polymer-bound lipids.

29. The composition according to claim 28, wherein cationic or cationic ionized lipids constitute about 30 mol% to about 50 mol%, for example, about 40 mol% to about 50 mol%, of the total lipids present in the composition; neutral lipids (for example, phospholipids) constitute about 5 mol% to about 15 mol%, of the total lipids present in the composition; steroids constitute about 30 mol% to about 50 mol%, of the total lipids present in the composition; and polymer-bound lipids, if present, constitute about 1 mol% to about 4.5 mol%, of the total lipids present in the composition.

30. The composition according to any one of claims 1 to 29, wherein the nucleic acid is RNA such as mRNA or self-replicating RNA.

31. The composition according to claim 30, wherein the RNA (i) comprises a modified nucleoside instead of uridine, the modified nucleoside being selected from pseudouridine (ψ), N1-methyl-pseudridine (m1ψ), and 5-methyl-uridine, (ii) having a codon-optimized coding sequence; and / or (iii) having a coding sequence with increased G / C content compared to a wild-type coding sequence.

32. The composition according to claim 30 or 31, wherein the RNA comprises at least one of a 5' cap, 5' UTR, 3' UTR, and polyA sequence, or preferably all of the 5' cap, 5' UTR, 3' UTR, and polyA sequence.

33. The composition according to claim 32, wherein the polyA sequence comprises at least 100 A nucleotides, and the polyA sequence is preferably a suspended sequence of A nucleotides.

34. The composition according to claim 32 or 33, wherein the 5' cap is a cap 1 or cap 2 structure.

35. The composition according to any one of claims 30 to 34, wherein the RNA encodes one or more polypeptides, preferably one or more polypeptides are pharmaceutically active peptides or polypeptides and / or comprise an epitope for inducing an immune response to an antigen in a subject.

36. The composition according to claim 35, wherein the pharmaceutically active polypeptide and / or antigen or epitope is derived from, or is derived from, a pathogen protein, an immunogenic variant of the protein, or an immunogenic fragment of the protein or its immunogenic variant.

37. The composition according to any one of claims 1 to 36, wherein the composition is liquid at a temperature of about 2°C to about 10°C, for example, about 2°C to about 8°C.

38. A method for delivering nucleic acids to target cells, comprising administering the compositions described in claims 1 to 37 to the target cells.

39. A method for delivering a therapeutic peptide or polypeptide to a subject, comprising administering the composition described in claims 1 to 37 to the subject, wherein the nucleic acid encodes the therapeutic peptide or polypeptide.

40. A method for treating or preventing a disease or disorder in a subject, comprising administering the compositions described in claims 1 to 37 to the subject, wherein the delivery of nucleic acids to the cells of the subject is beneficial for treating or preventing the disease or disorder.

41. A method for treating or preventing a disease or disorder in a subject, comprising administering the compositions described in claims 1 to 37 to the subject, wherein the nucleic acid encodes a therapeutic peptide or polypeptide, and the delivery of the therapeutic peptide or polypeptide to the subject is beneficial for treating or preventing the disease or disorder.

42. The method according to any one of claims 38 to 41, wherein the subject is a mammal.

43. The method according to claim 42, wherein the mammal is a human.

44. A composition according to any one of claims 1 to 37 for use in treatment.

45. A method for preparing the composition according to any one of claims 1 to 37, (a) To provide a nucleic acid solution containing water and a first buffer system; (b) To provide an organic solution comprising cationic or cationic ionized lipids, and, if present, one or more additional lipids; (c) Mixing the nucleic acid solution provided in (a) and the organic solution provided in (b) to prepare an intermediate formulation containing particles dispersed in an aqueous phase containing a first buffer system; and (d) The intermediate formulation prepared in (c) is filtered and / or diluted with a final aqueous buffer containing the final buffer system to prepare the composition; Methods that include...

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