Lipid composition targeting antigen-presenting cell, and use of the same

Abstract

To provide a lipid composition targeting an antigen-presenting cell, and use of the composition.SOLUTION: A lipid composition consists of (1) permanent anionic lipid, (2) permanent cationic lipid, and (3) neutral lipid, and the ratio of permanent anionic lipid, permanent cationic lipid and neutral lipid in the lipid composition by a molar ratio is 14 to 33:40 to 57:22 to 40. Because a composition provided by the present invention has satisfactory organ and cell targeting effect, a protein expression level of an antigen in the spleen can be significantly improved, and percentage of cells expressing antigen in antigen-presenting cells (e.g., B cell, pDC cell, cDC cell, macrophage) in the spleen is significantly increased, the composition can be used for immunotherapy of a disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention is in the field of molecular biology and relates to lipid compositions that target antigen-presenting cells and uses thereof. [Background technology]

[0002] Inducing anti-tumor T cell responses by expressing tumor antigens in antigen-presenting cells (APCs) using mRNA encoding them holds great promise for tumor immunotherapy. Negatively charged mRNA molecules cannot directly enter antigen-presenting cells, and most conventional delivery techniques generally require the delivery of large amounts of mRNA to the liver or lungs. For immunotherapy, immune responses to mRNA in the lungs or liver are generally harmful. Therefore, the key to successful mRNA immunotherapy is the highly selective delivery of mRNA encoding a specific antigen to antigen-presenting cells (APCs).

[0003] The lipid-RNA composition, which is produced by encapsulating mRNA in a lipid composition, is one of the methods of mRNA cellular delivery commonly used in mRNA immunotherapy. Currently, researchers have developed the following lipid systems for lipid-RNA compositions: Lipid nanoparticles (LNPs) are spherical vesicles composed of one (unilamellar) or multiple (multilamellar) phospholipid bilayers. LNP delivery technology involves encapsulating mRNA in nanoliposome particles composed of four components: cationic lipids (e.g., DLin-MC3-DMA, C12-200), neutral lipids (e.g., DSPC, DOPE), structural lipids (e.g., cholesterol), and polymer-bound lipids (e.g., DMG-PEG2000). Among these, cationic lipids include permanent cationic lipids (containing quaternary ammonium groups) and ionizable cationic lipids (containing primary, secondary, or tertiary amine groups). Since the use of permanent cationic lipids to prepare LNPs increases cytotoxicity, ionizable cationic lipids are used instead.

[0004] Adding a fifth lipid (a selective organ targeting (SORT) lipid) to conventional four-component liver-targeting lipid nanoparticles can achieve organ targeting of mRNA drugs. For example, CN112996519A describes a composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein the lipid nanoparticle composition comprises [1] a cationic ionizable lipid, a phospholipid (i.e., a neutral lipid), a steroid (i.e., a structured lipid such as cholesterol), a PEGylated lipid (i.e., a polymer-bound lipid such as dimyristoyl-sn-glycerol), and a permanent cationic lipid (i.e., a liver-targeting SORT lipid), or [2] a cationic ionizable lipid, a phospholipid (i.e., a neutral lipid), a steroid (i.e., a structured lipid such as cholesterol), a PEGylated lipid (i.e., a polymer-bound lipid such as dimyristoyl-sn-glycerol), and a permanent anionic lipid (i.e., a spleen-targeting SORT lipid), and the apparent pK of the lipid nanoparticle composition comprising the selective spleen-targeting compound is 0.05. a is varied to adjust the targeting effect of the prepared composition formulation on different organs.

[0005] 2. Lipopolyplexes (LPPs) are bilayer structures consisting of a polymer-encapsulated mRNA core and a phospholipid capsule shell. Negatively charged mRNA aggregates within the positively charged polymer, forming a dense polyplex "core" structure with a diameter of several to several hundred nanometers. The bilayer nanostructure of LPPs better encapsulates and protects mRNA than conventional LNPs, allowing the mRNA molecules to be gradually released as the polymer degrades. Furthermore, their excellent dendritic cell targeting effect can enhance antigen presentation and activate T cell immune responses, resulting in ideal immunotherapy effects.

[0006] For example, CN115845040A describes a lipopolyplex mRNA vaccine consisting of a poly-(β-amino ester) polymer mRNA core encapsulated in a 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine / 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine / 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (permanent cationic lipid EDOPC / neutral lipid DOPE / helper lipid DSPE-PEG) lipid shell. In this shell / core-mRNA vaccine, the polyplex "core" is encapsulated by a hydrophilic phospholipid bilayer "shell," which enhances dendritic cell uptake and protects the mRNA molecules within the inner core from degradation by intracellular nucleases. Also described as an effective approach to enhance dendritic cell uptake of vaccine particles is "functionalization" by conjugating affinity moieties (e.g., sugar moieties such as mannose, binding proteins, or antibodies specific for one or more DC-expressed epitopes) to the surface of the lipid shell of mRNA vaccines to strengthen the interaction and / or increase binding between the vaccine particles and the antigen-presenting cells (dendritic cells, macrophages, B cells, etc.) targeted by the vaccine core / shell complex.

[0007] 3. Lipoplexes (LPX), developed by BioNTech, are complex delivery carriers composed of cationic lipids and neutral helper lipids, in which mRNA molecules are embedded between the bilayer lipids. For example, CN109331176A discloses that lipoplexes may be formed from permanently cationic (positively charged) liposomes and anionic (negatively charged) nucleic acids, with the addition of neutral lipids as helper lipids. By optimizing the lipid-to-RNA ratio in RNA-lipoplexes (RNA-LPX) and adjusting the net charge of the formulation to neutral or negative, intravenous injection can be precisely targeted to dendritic cells (DCs), eliminating the need for targeting nanoparticles.

[0008] In the simplest case, lipoplexes are formed spontaneously by mixing nucleic acids and liposomes using a specific mixing protocol, but various other protocols can also be applied. Electrostatic interactions between positively charged liposomes and negatively charged nucleic acids are the driving force for lipoplex formation. In addition to the lipid composition, the charge ratio between cationic and anionic moieties plays an important role for efficient condensation and transfection. It is generally believed that excess positive charge in lipoplexes is necessary for efficient transfection (Templeton, NS et al., (1997) Nature Biotechnology 15(7):647-652; Zhdanov, RI et al., (2002) Bioelectrochemistry 58(1):53-64; Templeton, NS (2003) Current Medicinal Chemistry 10(14):1279-1287). Because most natural membranes are negatively charged, electrostatic interactions that create an attractive force between positively charged lipoplexes and negatively charged biological membranes may play an important role in cellular binding and uptake of lipoplexes. Conversely, with a lower excess of positive charges, transfection efficacy drops dramatically to virtually zero. However, positively charged liposomes and lipoplexes have been reported to be highly cytotoxic, which may pose problems for their use in pharmaceuticals.

[0009] The lipoplexes described above have been proven to transfect various organs. Their expression distribution in specific organs depends on many parameters, including the formulation and administration parameters (lipid composition, size, and administration route). To date, selective expression in a given target organ or cellular site while avoiding expression in non-target organs has not been fully achieved. Transfection of organs such as the lung, liver, spleen, kidney, and heart using luciferase DNA or RNA as a reporter has been reported. Avoiding lung and liver targeting is particularly difficult because, in many cases, lung and liver targeting predominates. The lung has a very large surface area and is the first organ through which intravenously injected compounds pass after administration. The liver is a typical target organ for formulations containing lipophilic compounds, such as lipids present in liposomes and lipoplexes. For RNA-based immunotherapy, targeting the lung or liver can be harmful due to the risk of an immune response against these organs. Therefore, for such treatments, formulations with high selectivity for DCs, for example, in the spleen, are needed.

[0010] It is believed that certain ligands can improve targeting selectivity.For example, it is believed that the lipid modified by conjugating mannose or the like to lipid can significantly improve targeting to macrophage.However, when conjugating and modifying lipid, it is necessary to consider the interaction in serum and the degradation of RNA in serum, so this component makes formulation more complicated, and makes actual drug development more difficult.

[0011] Furthermore, the formation of large aggregates is commonly observed upon incubation of RNA with cationic liposomes, which significantly increases particle size and reduces stability, and is one of the major obstacles to developing acceptable composition formulations for intravenous or subcutaneous administration.

[0012] Integrin-Targeted, Short Interfering RNA Nanocomplexes for Neuroblastoma Tumor-Specific Delivery Achieve MYCN Silencing with Improved Survival, Adv. Funct. Mater. 2021, 31, 2104843, discloses a multifunctional siRNA nanoparticle formulation consisting of cations and anions, called receptor-targeted nanocomplexes (RTN), which contains a lipid composition (including the permanent anionic lipid DOPG, the permanent cationic lipid DOTMA, the neutral lipid DOPE, and DPPE-PEG2000) and a polypeptide used for encapsulating siRNA and receptor-mediated uptake into cells. When RTN was intravenously injected into mice, it accumulated mainly in tumor xenografts and was barely detectable in the liver, lungs, or spleen, indicating that the RTN formulation can achieve specific tumor targeting. It also has very low liver clearance, making tumor treatment with siRNA-targeted drugs possible (see the abstract and the right column on page 9).

[0013] IL-1 and IL-1ra are key regulators of the inflammatory response to RNA vaccines, Siri Tahtinen et al., NATURE IMMUNOLOGY, VOL 23, April 2022, pp. 532-542, use an RNA-LPX vaccine encoding a TLR7 / 8 agonist (here, the lipid composition of RNA-LPX contains DOTMA and DOPE, with a (+):(-) charge ratio of 1.3:2; see the third paragraph in the left column on page 543) as an example to study factors that affect the induced production of the IL-1 cytokine, such as monocyte count, inflammasome, and aspartase activity.

[0014] "Systemic RNA delivery to dendritic cells exploits antiviral defense for cancer immunotherapy," Lena M. Kranz et al., NATURE, volume 534, 396-401 (2016), describes RNA-LPX containing cationic liposomes composed of DOTMA and DOPE. By varying the lipid-to-RNA ratio, the researchers investigated the effect of positive and negative charges on targeting to antigen-presenting cells in the body. When RNA-LPX is slightly positively charged or nearly neutral (positive / negative charge ratio of 2.5:1 to 1.8:2), the resulting RNA-LPX is unstable and readily forms large aggregates. When Luc-RNA-LPX is positively charged (positive / negative charge ratio of 5:1), its protein expression is concentrated in the lungs of mice, with less expression in the spleen. As the cationic lipid content decreases, protein expression shifts from the lungs to the spleen. Near-neutral or negatively charged particles (charge ratio of 1.7:2 or less) transferred protein expression to the spleen. It was also reported that as the negative charge increased, transfection efficiency gradually decreased, possibly due to an increase in free RNA (see the right column on page 396).

[0015] Due to the current limitations of the above conventional technologies and the rapid identification of new tumor-associated antigens due to large-scale cancer genome sequencing efforts and technological advances in predicting immunogenic tumor mutations, unprecedented opportunities are being offered for the development of new and improved vaccines for tumor therapy. As a new method for highly selectively delivering mRNA to antigen-presenting cells, there is an urgent need to provide injectable RNA formulations that can be guaranteed to meet the standards for products administered to patients. DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0016] The present invention provides lipid compositions and uses thereof that target antigen-presenting cells.

[0017] When the composition provided by the present invention is administered systemically, the RNA is expressed in large amounts in the spleen (particularly in antigen-presenting cells) and at low levels in other organs (e.g., the liver and lungs), demonstrating good targeting effects. Furthermore, administration of the composition also induces a strong immune response against the antigen. While excess positive charge is generally considered a prerequisite for successful uptake and expression, compositions with the charge ratio of the present invention are still expressed at such a high level that they can provide the therapeutic effects of lipoplexes after systemic administration. [Means for solving the problem]

[0018] In order to achieve the above object, the present invention provides, in a first aspect, (1) a permanent anionic lipid; (2) a permanent cationic lipid; (3) a lipid composition comprising a neutral lipid; However, the molar ratio of the permanent anionic lipid, the permanent cationic lipid, and the neutral lipid in the lipid composition is 14-33:40-57:22-40.

[0019] According to a preferred embodiment of the invention, the permanent anionic lipid comprises a phosphate group.

[0020] According to a preferred embodiment of the present invention, the permanent cationic lipid comprises a quaternary ammonium group.

[0021] According to a preferred embodiment of the invention, the neutral lipid comprises a phosphate group and / or a quaternary ammonium group.

[0022] According to a preferred embodiment of the present invention, the permanent anionic lipid is The ester may be any one or more selected from the group consisting of 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate, (Z)-(R)-3-(phosphonooxy)propane-1,2-diyldiolate, 1,2-dioleoyl-sn-glycero-3-phospho-rac-glycerol, and salts thereof.

[0023] According to a preferred embodiment of the present invention, the permanent cationic lipid is The ammonium salt is at least one selected from the group consisting of 1,2-di-O-octadecenyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium propane, and salts thereof.

[0024] According to a preferred embodiment of the present invention, the neutral lipid is It is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or distearoylphosphatidylcholine, and salts thereof.

[0025] According to a preferred embodiment of the present invention, the lipid composition comprises: (1) 25 mol% permanent anionic lipid; (2) 50 mol% permanent cationic lipid; (3) 25 mol% neutral lipids, Or, the lipid composition is (1) 20 mol% permanent anionic lipid; (2) 40 mol% permanent cationic lipid; (3) 40 mol% neutral lipids, Or, the lipid composition is (1) 14 mol% permanent anionic lipid; (2) 57 mol% permanent cationic lipid; (3) 29 mol% neutral lipids, Or, the lipid composition is (1) 33 mol% permanent anionic lipid; (2) 45 mol% permanent cationic lipid; (3) Contains 22 mol% neutral lipids.

[0026] In a second aspect, the present invention provides use of a lipid composition in improving targeting to antigen-presenting cells in a target organ, the lipid composition comprising: (1) a permanent anionic lipid; (2) a permanent cationic lipid; (3) neutral lipids, However, the molar ratio of the permanent anionic lipid, the permanent cationic lipid, and the neutral lipid in the lipid composition is 14-33:40-57:22-40.

[0027] According to the present invention, the lipid composition is the lipid composition according to the first aspect, and since its characteristics have been described above, detailed description thereof will be omitted here.

[0028] In a third aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (A) a therapeutic and / or prophylactic agent comprising one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein; (B) a lipid composition according to the first aspect, The composition is used to deliver the therapeutic and / or prophylactic agent to antigen-presenting cells within a target organ.

[0029] According to a preferred embodiment of the present invention, the therapeutic and / or prophylactic agent is a nucleic acid molecule capable of encoding one or more antigens.

[0030] According to a preferred embodiment of the invention, said antigen is a disease-associated antigen, or said nucleic acid molecule or antigen is capable of eliciting an immune response against the disease-associated antigen or against cells expressing a disease-associated antigen.

[0031] According to a preferred embodiment of the present invention, the target organ is one or more selected from the group consisting of the spleen, the liver, and the lung.

[0032] According to a preferred embodiment of the present invention, the target organ is the spleen.

[0033] According to a preferred embodiment of the present invention, the antigen-presenting cells include any one or more of dendritic cells, macrophages, and B cells.

[0034] According to a preferred embodiment of the present invention, the charge ratio of net positive charges to net negative charges in the composition is 1:2 to 1:5 depending on the amounts of the therapeutic agent and / or prophylactic agent and lipid composition used.

[0035] According to a preferred embodiment of the present invention, the lipid composition comprises: (1) 14 to 33 mol% of permanent anionic lipids; (2) 40 to 57 mol% of a permanent cationic lipid; (3) 22 to 40 mol% of neutral lipids, the permanent anionic lipid is 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate and / or a salt thereof, the permanent cationic lipid is 1,2-di-O-octadecenyl-3-trimethylammonium propane and / or a salt thereof, and the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or a salt thereof; The charge ratio of net positive charges to net negative charges in the composition is 1:2 to 1:5.

[0036] According to a preferred embodiment of the present invention, the charge ratio of net positive to negative charges in the composition is 1:2; or the composition has a net positive to negative charge ratio of 2:5; or the composition has a net positive to negative charge ratio of 1:3; Alternatively, the composition has a net positive to negative charge ratio of 1:5.

[0037] According to a preferred embodiment of the invention, the nucleic acid molecule is RNA encoding one or more antigens.

[0038] According to a preferred embodiment of the invention, the composition further comprises at least one helper component or adjuvant.

[0039] According to a preferred embodiment of the invention, the composition further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

[0040] According to a preferred embodiment of the invention, the composition further comprises one or more hydrophobic small molecules, permeability enhancing molecules, carbohydrates, polymers, surface-altering agents, functionalized lipids or cytokines.

[0041] In a fourth aspect, the present invention provides a method for producing a composition for delivering a therapeutic agent and / or a prophylactic agent to antigen-presenting cells in a target organ, the method comprising: (a) dissolving permanent anionic lipids, permanent cationic lipids, and neutral lipids in an organic solvent to form a lipid solution, wherein the molar ratio of the permanent anionic lipids, permanent cationic lipids, and neutral lipids is 14-33:40-57:22-40; (b) mixing the lipid solution obtained in step (a) with water to obtain a lipid mixture; (c) mixing the lipid mixture obtained in step (b) with a therapeutic agent and / or a prophylactic agent to form the composition, wherein the therapeutic agent and / or the prophylactic agent comprises a nucleic acid buffer solution obtained by dissolving a nucleic acid molecule in a buffer solution having a pH of 6.8 to 7.6.

[0042] According to a preferred embodiment of the present invention, in step (a), the organic solvent is an alcohol solvent.

[0043] According to a preferred embodiment of the present invention, in step (b), the lipid mixture can pass through a polycarbonate membrane with a pore size of 100 to 400 nm.

[0044] According to a preferred embodiment of the present invention, in step (c), the buffer comprises an aqueous HEPES buffer.

[0045] According to a preferred embodiment of the present invention, in step (c), said nucleic acid molecule is a nucleic acid molecule capable of encoding one or more antigens.

[0046] According to a preferred embodiment of the present invention, in step (a), the organic solvent comprises an alcohol having 1 to 4 carbon atoms.

[0047] According to a preferred embodiment of the present invention, in step (c), the buffer comprises an aqueous HEPES buffer and EDTA.

[0048] According to a preferred embodiment of the invention, in step (c), said antigen is a disease-associated antigen, or said nucleic acid molecule or antigen is capable of eliciting an immune response against the disease-associated antigen or against cells expressing a disease-associated antigen.

[0049] According to a preferred embodiment of the invention, the permanent anionic lipid comprises a phosphate group.

[0050] According to a preferred embodiment of the present invention, the permanent cationic lipid comprises a quaternary ammonium group.

[0051] According to a preferred embodiment of the invention, the neutral lipid comprises a phosphate group and / or a quaternary ammonium group.

[0052] According to a preferred embodiment of the present invention, the permanent anionic lipid is The ester may be any one or more selected from the group consisting of 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate, (Z)-(R)-3-(phosphonooxy)propane-1,2-diyldiolate, 1,2-dioleoyl-sn-glycero-3-phospho-rac-glycerol, and salts thereof.

[0053] According to a preferred embodiment of the present invention, the permanent cationic lipid is The compound is any one or more selected from 1,2-di-O-octadecenyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium propane, and salts thereof.

[0054] According to a preferred embodiment of the present invention, the neutral lipid is It is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or distearoylphosphatidylcholine, and salts thereof.

[0055] According to a preferred embodiment of the present invention, in step (c), the lipid mixture and the therapeutic and / or prophylactic agent are used in such amounts that the charge ratio of net positive charges to net negative charges in the resulting composition is 1:2 to 1:5.

[0056] In a fifth aspect, the present invention provides the use of a lipid composition according to the first aspect, a composition according to the third aspect, or a composition produced according to the method according to the fourth aspect, in the manufacture of a medicament capable of inducing a cellular immune response.

[0057] In a sixth aspect, the present invention provides the use of a lipid composition according to the second aspect, a composition according to the third aspect, or a composition produced according to the method according to the fourth aspect, in the manufacture of a medicament for use in preventing, diagnosing, treating or ameliorating a disease, condition, abnormal state or dysfunction in a mammalian subject.

[0058] According to a preferred embodiment of the present invention, the disease, pathology, abnormal condition or dysfunction is any one or more selected from infectious diseases, cancer, proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0059] According to a preferred embodiment of the present invention, the mammalian subject is one or more species selected from humans, non-human primates, companion animals, exotic species, livestock and food-producing animals.

[0060] According to some preferred embodiments of the present invention, a composition having suitable particle size distribution characteristics that can meet the requirements for intravenous administration to patients can be formed by incubating a lipid composition with RNA through self-assembly / self-organization, thereby satisfying the important requirements for pharmaceutical formulations administered to patients in terms of particle size distribution characteristics and stability. [Effects of the Invention]

[0061] The composition of the present invention produced by adding a permanent anionic lipid has at least the following advantages: [1] The particle size is good and the particle distribution is uniform. [2] The amount of antigen protein expression in the spleen can be significantly improved. [3] The percentage of antigen-expressing cells (e.g., B cells, pDC cells, cDC cells, macrophages) in the spleen can be significantly increased. Specifically, I. By adding a permanent anionic lipid containing a phosphate group, a composition can be produced with good particle size (particle size limited to 240-500 nm) and uniform particle distribution (PDI<0.5).

[0062] 1. By adding permanent anionic lipids, especially those containing phosphate groups such as ADOPE, 18PA, DOPG, tetradecylphosphonic acid, farnesyl pyrophosphate, γ,γ-dimethylallyl pyrophosphate, and pA(2'-OMe)mpG, the compositions prepared all have good particle size and PDI. Among them, the best is ADOPE, with a particle size of 303.1 nm and a PDI of 0.2455, showing good particle uniformity.

[0063] 2. Conversely, compositions utilizing other types of anionic lipids that do not contain phosphate groups, such as oleic acid, sodium bis(laureth-7) citrate, or sodium lauryl sulfonate, are unsuitable as mRNA delivery carriers due to their large particle size (over 1000 nm) and the resulting solid precipitate.

[0064] II. The composition containing the permanent anionic lipid, permanent cationic lipid, and neutral lipid designed in the present invention can significantly improve the protein expression level of the antigen in the spleen (corresponding to the total radiation intensity), particularly by limiting the molar percentages to (14 to 33 mol%):(40 to 57 mol%):(22 to 40 mol%) and / or the charge ratio to 1:2 to 1:5. 1. Mouse in vivo imaging experiments showed that the protein expression level of the antigen delivered by the composition containing the permanent anionic lipid designed in the present invention in the spleen was significantly higher than that in other organs (e.g., liver, lung), and the total radiation intensity of the expressed protein in the spleen of Fluc-mRNA delivered by the prepared composition was 1.20 × 10 7 ~7.38×10 7 p / s has been reached.

[0065] 2. Compared to a composition prepared without the addition of anionic lipids, the protein expression level in the spleen of the antigen delivered by the composition designed in the present invention is significantly higher than that of a composition prepared without the addition of anionic lipids.

[0066] For example, the total radiation intensity of the expressed protein in the spleen of Fluc-mRNA delivered by the mRNA composition designed in the present invention was 12.1 times that of the composition without added anionic lipid.

[0067] 3. The protein expression level in the spleen of the antigen delivered by the lipid composition designed in the present invention is significantly higher than that of the LNPs of the prior art.

[0068] For example, the total radiation intensity of the expressed protein in the spleen of the composition designed in the present invention was 9.46 times that of YK-009-mRNA-LNP, a conventional LNP, and 7.1 times that of YK-407-mRNA-LNP.

[0069] III. The compositions designed in the present invention significantly increase the percentage of antigen-expressing antigen-presenting cells (eg, B cells, pDC cells, cDC cells, macrophages) in the spleen. 1. Mouse spleen cell flow cytometry experiments show that compositions containing permanent anionic lipids designed in the present invention significantly increase the percentage of antigen-expressing antigen-presenting cells (e.g., B cells, pDC cells, cDC cells, macrophages) in the spleen.

[0070] For example, among antigen-presenting cells, the percentage of B cells is about 0.1 to 0.4%, the percentage of pDC cells is about 2 to 6%, the percentage of cDC cells is about 2 to 9%, and the percentage of macrophages is about 2.4 to 7.5%.

[0071] 2. Compared to a composition without added anionic lipids, the compositions designed in the present invention significantly increase the percentage of antigen-expressing antigen-presenting cells in the spleen.

[0072] For example, when a composition provided by the present invention is administered, the percentage of cells that are eGFP-expressing B cells, pDC cells, cDC cells, and macrophages is 19-fold, 2.8-fold, 8.1-fold, and 11.8-fold higher, respectively, than when a composition without added anionic lipid is administered.

[0073] 3. The specific combination of compositions designed in the present invention significantly increases the percentage of antigen-expressing cells in the antigen-presenting cells in the spleen compared to other combinations of compositions.

[0074] For example, administration of a particular combination composition of the present invention (permanent anionic lipid ADOPE, permanent cationic lipid DOTMA, neutral lipid DOPE) results in 4.8-fold, 5.6-fold, 12.1-fold, and 11.6-fold higher percentages of cells that are eGFP-expressing B cells, pDC cells, cDC cells, and macrophages, respectively, than administration of the pA(2'-OMe)mpG eGFP RNA composition (permanent anionic lipid pA(2'-OMe)mpG, permanent cationic lipid DOTMA, neutral lipid DOPE).

[0075] IV. The specific combination compositions designed in the present invention have significantly enhanced ability to stimulate the production of IFN-α cytokine compared to LPX-RNA compositions that do not contain anionic lipids, demonstrating that the compositions of the present invention can initiate a potent immune stimulatory program driven by type I IFN.

[0076] For example, when the composition of the present invention containing a permanent anionic lipid is administered 6 hours and 24 hours after injection, respectively, the content of IFN-α cytokine in the serum is approximately 1.7 to 4 times and 1.5 to 5 times higher than when an LPX-RNA composition not containing an anionic lipid is administered.

[0077] V. The specific combination compositions designed in the present invention have significantly enhanced ability to stimulate antigen-specific cytotoxic T cells compared to LPX-RNA compositions that do not contain anionic lipids, demonstrating that the compositions of the present invention can produce very potent effects on T cells.

[0078] For example, administration of a particular composition of the present invention containing a permanent anionic lipid on day 13 after injection increases serum OVA antigen-specific CD8 + T cells are CD8 + The percentage of T cells in total is 1.3 to 1.9 times higher than when an LPX-RNA composition not containing anionic lipids is administered.

[0079] VI. The specific combination composition designed in this invention has been shown in animal experiments to significantly control tumor growth and extend the survival time of tumor-bearing experimental animals compared to an LPX-RNA composition that does not contain anionic lipids.

[0080] For example, after subcutaneous inoculation of B16F10-OVA melanoma cells into mice, the rate of tumor growth was effectively slowed by injecting a specific composition of the present invention containing a permanent anionic lipid, compared to a blank lipid control group and a control group administered an LPX-RNA composition not containing an anionic lipid, with a clear reduction in tumor size and a significant increase in the survival rate of tumor-bearing mice injected with a specific composition of the present invention containing a permanent anionic lipid. [Brief explanation of the drawings]

[0081] In order to more clearly describe the technical solutions of the embodiments of the present invention, the drawings of the present invention will be briefly described below. Needless to say, the drawings appearing in the following description are related to some specific embodiments of the present invention and do not constitute limitations on the present invention. [Figure 1a] FIG. 1a shows fluorescence images of a composition containing Fluc-mRNA corresponding to No. 6 in Table 8 in a mouse and its organs (liver, spleen, lung). [Figure 1b] FIG. 1b shows fluorescence images of a composition containing Fluc-mRNA corresponding to No. 7 in Table 8 in mice and their organs (liver, spleen, lung). [Figure 1c] FIG. 1c shows fluorescence images of a composition containing Fluc-mRNA corresponding to No. 8 in Table 8 in mice and their organs (liver, spleen, lung). [Figure 1d] FIG. 1 shows fluorescence images of a composition containing Fluc-mRNA corresponding to No. 17 in Table 8 in mice and their organs (liver, spleen, lung). [Figure 2a]Figure 2a shows a mouse spleen flow cytometry experiment of small-particle LPX without anionic lipids (No. 13 in Table 10), large-particle LPX without anionic lipids (No. 2 in Table 12), and a composition containing eGFP-mRNA corresponding to No. 1 in Table 12. [Figure 2b] Figure 2b shows a mouse spleen flow cytometry experiment of small-particle LPX without anionic lipids (No. 13 in Table 10), large-particle LPX without anionic lipids (No. 2 in Table 12), and a composition containing eGFP-mRNA corresponding to No. 1 in Table 12. [Figure 3] FIG. 3 shows a comparison of the IFN-α cytokine content produced by different compositions in groups 1 to 4 of Table 13 at different times after stimulation. [Figure 4] FIG. 4 shows a comparison of the stimulated production of antigen-specific cytotoxic T cells by different compositions in groups 1 to 4 of Table 15 on day 13 after injection of the pharmaceutical composition. [Figure 5] FIG. 5 shows a comparison of the changes in tumor volume in tumor-bearing mice after inoculation with different compositions of Groups 1 to 4 in Table 16. [Figure 6] FIG. 6 shows a comparison of mouse survival after inoculation of tumor-bearing mice with different compositions of groups 1 to 4 in Table 16. DETAILED DESCRIPTION OF THE INVENTION

[0082] In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described more clearly and completely below in conjunction with the drawings of the present invention. Needless to say, the specific embodiments described are not all embodiments, but only some embodiments of the present invention. Other embodiments that a person skilled in the art can obtain based on the described embodiments of the present invention without inventive steps all belong to the protection scope of the present invention.

[0083] The present invention may be embodied in other specific forms without departing from the spirit of the present invention. It should be understood that, unless a contradiction arises, any or all of the embodiments of the present invention may be combined with technical features of any other embodiment or embodiments to obtain another embodiment. The present invention includes the other embodiment obtained from such a combination.

[0084] All publications and patents referred to in this application are hereby incorporated by reference in their entirety. In the event that the application or terminology used in any publication or patent incorporated by reference conflicts with the application or terminology used in this application, the application and terminology of this application shall control.

[0085] The section headings provided herein are for the purpose of organization and are not to be construed as limitations on the subject matter.

[0086] Unless otherwise specified, all technical and scientific terms used herein have their ordinary meaning within the field to which the claimed subject matter belongs. In the event that there is a plurality of definitions for a particular term, the definition in this invention shall prevail.

[0087] Except in the examples or where otherwise indicated, all numerical values ​​appearing in the specification and claims regarding quantitative properties, such as dosages, are understood to be modified in all instances by the term "about." It is also to be understood that all numerical ranges recited herein are intended to include all subranges within that range and any combination of the endpoints of that range or subrange.

[0088] As used herein, the terms "comprise" or "contain" or similar terms mean that the elements appearing before the term cover the elements listed after the term and their equivalents, and do not exclude elements not listed. The terms "comprise" or "comprise" as used herein may be open, semi-closed, or closed. In other words, the terms include "consisting essentially of" or "consisting of."

[0089] As used herein, the term "pharmaceutically acceptable" refers to a compound or composition that is chemically and / or toxicologically compatible with other ingredients constituting the formulation and / or with the human or mammal in which it is used to prevent or treat a disease or condition.

[0090] As used herein, the term "subject" or "patient" includes mammalian subjects, which may be selected from one or more of the following: humans, non-human primates, companion animals, exotic species, livestock, and food-producing animals.

[0091] The term "treatment" as used herein refers to the administration of one or more drugs to a patient or subject suffering from a disease or having symptoms of said disease, thereby curing, alleviating, ameliorating, or affecting said disease or symptoms of said disease. In the context of the present invention, the term "treatment" may also include prevention, unless specifically stated to the contrary.

[0092] In the present invention, the term "antigen" includes any molecule, preferably a peptide or protein, containing at least one epitope capable of inducing and / or being the subject of an immune response. Preferably, in the context of the present invention, an antigen is a molecule that, after optional processing, preferably induces a specific immune response against said antigen or a cell expressing said antigen. In particular, "antigen" refers to a molecule that, after optional processing, is presented by an MHC molecule and reacts specifically with T lymphocytes (T cells).

[0093] Thus, the antigen or fragment thereof must be recognized by a T cell receptor. Preferably, if recognized by a T cell receptor, the antigen or fragment is capable of inducing, in the presence of an appropriate costimulatory signal, the clonal expansion of T cells bearing a T cell receptor that specifically recognizes the antigen or fragment. In the context of embodiments of the present invention, the antigen or fragment is preferably presented by cells, preferably antigen-presenting cells and / or pathological cells, in the context of MHC molecules, which leads to an immune response against the antigen or cells expressing the antigen.

[0094] According to the present invention, any suitable antigen is contemplated as a candidate for use in eliciting an immune response, although it is preferred that the immune response is a cellular immune response.

[0095] Preferably, the antigen corresponds to or is derived from a naturally occurring antigenic species. Such naturally occurring antigens may include or be derived from allergens, viruses, bacteria, fungi, parasites, other infectious agents and pathogens, or the antigen may be a tumor antigen. According to the present invention, the antigen may correspond to a naturally occurring species, for example, a viral protein or part thereof.

[0096] The term "pathogen" refers to a pathogenic microorganism, such as a virus, a bacterium, a fungus, a single-celled organism, or a parasite. Examples of pathogenic viruses include, but are not limited to, human immunodeficiency virus (HIV), cytomegalovirus (CMV), herpesvirus (HSV), hepatitis A virus (HAV), HBV, HCV, papillomavirus, and human T-lymphotrophic virus (HTLV). Single-celled organisms include, but are not limited to, malaria parasites, trypanosomes, and amoebas.

[0097] The term "disease-associated antigen" refers to any antigen with significant pathogenicity, and includes "tumor antigens." According to the present invention, it is desirable to induce an immune response against the disease-associated antigen or against cells that express the disease-associated antigen, preferably presenting the disease-associated antigen in the context of MHC molecules. Preferably, the disease-associated antigen is a naturally occurring antigen. In one embodiment, the disease-associated antigen is expressed in diseased cells and preferably presented by the cells' MHC molecules.

[0098] The antigen encoded by the RNA (i.e., the therapeutic and / or prophylactic agent) contained in the (lipid composition) nanoparticles of the present invention induces an immune response against the target disease-associated antigen or against cells expressing the target disease-associated antigen. Thus, the antigen encoded by the RNA contained in the nanoparticles of the present invention may correspond to or comprise a disease-associated antigen or one or more immunogenic fragments thereof, such as one or more MHC-binding peptides of the disease-associated antigen. Thus, the antigen encoded by the RNA contained in the nanoparticles of the present invention may be a recombinant antigen.

[0099] The composition provided by the present invention has an average particle size of 240 to 500 nm and a polydispersity index of 0.5 or less, preferably 0.2 or less, and more preferably 0.1 or less.

[0100] (permanent anionic lipids) In some embodiments, the present invention provides one or more lipids having one or more hydrophobic moieties and a permanent anionic group. One of the anionic groups available in the permanent anionic lipid is a phosphate group. The phosphate group may be deprotonated and may be a compound that has a negative charge at a pH of 8, 9, 10, 11, 12, 13, or 14 or less. The hydrophobic moiety may be one or more C6-C 24 It may be an alkyl or alkenyl group. The compound may have one hydrophobic group, two hydrophobic groups or three hydrophobic groups.

[0101] In some embodiments, the permanent anionic lipid is present in an amount of about 14-33 mol% of the total lipid composition (i.e., the permanent anionic lipid content may be 14-33 mol% based on the total lipid composition). The composition may contain about 14 mol%, about 20 mol%, about 25 mol%, or about 33 mol%, or any range thereof, of permanent anionic lipid.

[0102] According to some preferred embodiments of the invention, the permanent anionic lipid is The hydroxybenzoate may be any one or more selected from the group consisting of 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl)phosphate, (Z)-(R)-3-(phosphonooxy)propane-1,2-diyldiolate, 1,2-dioleoyl-sn-glycero-3-phospho-rac-glycerol, and salts thereof (e.g., sodium salts, chloride salts, etc.).

[0103] (permanent cationic lipids) In some embodiments, the present invention provides one or more lipids having one or more hydrophobic components and a permanent cationic group. The permanent cationic lipid may contain a group that has a positive charge (regardless of pH). One permanent cationic group available for permanent cationic lipids is a quaternary ammonium group.

[0104] In some embodiments, the permanent cationic lipid is present in an amount of about 40-57 mol% of the total lipid composition, and the composition may contain about 40 mol%, about 45 mol%, about 50 mol%, or about 57 mol% permanent cationic lipid, or any range thereof.

[0105] According to some preferred embodiments of the present invention, the permanent cationic lipid is The compound may be any one or more selected from 1,2-di-O-octadecenyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium propane, and salts thereof (for example, sodium salts, chloride salts, etc.).

[0106] (neutral lipid) In the present invention, the term "neutral lipid" refers to a lipid that functions as a helper and is uncharged or exists in the form of a zwitterion at a certain pH. The neutral lipid can promote the phase transition of lipids and control the fluidity of nanoparticles, thereby forming a lipid bilayer structure and improving the efficiency, and also affecting the specificity of target organs / target cells.

[0107] For example, the neutral lipid may comprise one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0108] The carrier component of the composition containing a cationic lipid may also contain one or more neutral lipids-phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids can be organized into one or more lipid bilayers. Generally, the phospholipids may contain a phospholipid moiety and one or more fatty acid moieties.

[0109] The neutral lipid moiety may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-naturally occurring species, including naturally occurring species with modifications and substitutions, including branching, oxidation, cyclization, and alkynes, are also included. For example, phospholipids may be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, copper-catalyzed cycloaddition reactions can occur when alkynyl groups are exposed to azides. These reactions may be used to functionalize the lipid bilayer of the composition to promote membrane permeability or cellular recognition, or to couple the composition to useful components such as targeting moieties or imaging moieties (e.g., dyes).

[0110] Neutral lipids available for these compositions include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16).0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerin) sodium salt (DOPG), dipalmitoylphosphatidylglycerin (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl- The phosphatidylcholine may be selected from the non-limiting group consisting of ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0111] In some embodiments, the neutral lipid comprises DSPC. In some embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises DSPC and DOPE (simultaneously).

[0112] In some embodiments, the neutral lipid is present in an amount of about 22-40 mol% of the total lipid composition, and the composition may contain 22 mol%, 25 mol%, 29 mol%, or 40 mol% neutral lipid, or any range thereof.

[0113] (Therapeutic and / or preventive agents) The compositions of the present invention may include one or more therapeutic and / or prophylactic agents. The lipid compositions of the present invention may be used to deliver active ingredients, such as therapeutic and / or prophylactic agents. The active ingredients may be encapsulated by or associated with the lipid composition. In one embodiment, the charge ratio of the net positive charge in the lipid composition to the negative charge in the active ingredient (e.g., therapeutic and / or prophylactic agent) is 1:2 to 1:5 (i.e., the charge ratio of the net positive charge to the net negative charge in the compositions provided by the present invention is 1:2 to 1:5). In some embodiments, the charge ratio is preferably 1:2, 2:5, 1:3, or 1:5.

[0114] The therapeutic and / or prophylactic agent may include, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein, of which a nucleic acid molecule is preferred.

[0115] For example, the therapeutic and / or prophylactic agent may be a vaccine or a compound capable of eliciting an immune response. Thus, in some preferred embodiments, the therapeutic and / or prophylactic agent may be a nucleic acid molecule capable of encoding one or more antigens.

[0116] Because the lipid compositions of the present invention (as carriers) are capable of delivering therapeutic and / or prophylactic agents to target cells and / or target organs in mammals, the present invention provides methods for treating a disease or condition in a mammal in need thereof, which methods comprise administering to the mammal a composition comprising the therapeutic and / or prophylactic agent and / or contacting mammalian cells with the composition.

[0117] Therapeutic and / or prophylactic agents include biologically active substances that may alternatively be referred to as "active agents," "active ingredients," etc. Therapeutic and / or prophylactic agents may be substances that, when delivered to a cell or organ, cause a desired change within that cell or organ or in other tissues or systems. Such substances may be used to treat one or more diseases, conditions, or pathological states. In some embodiments, therapeutic and / or prophylactic agents are small molecule drugs used to treat specific diseases, conditions, or pathological states. Examples of drugs that can be used in the composition include antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, streptozotocin), antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside), and the like.arabinoside, anthracycline, alkylating agents, platinum compounds, antimetabolites, nucleoside analogues (such as methotrexate), purine and pyrimidine analogues), anti-infectives, local anesthetics (e.g., dibucaine, chlorpromazine), β-adrenergic receptor blockers (e.g., propranolol, timolol, labetalol), antihypertensives (e.g., clonidine, hydralazine), antidepressants (e.g., imipramine, amitriptyline, doxepin), antispasmodics (e.g., phenytoin), antihistamines Antibiotics (e.g., diphenhydramine, chlorpheniramine, promethazine), antibiotics / antibacterials (e.g., gentamycin, ciprofloxacin, cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, amphotericin B), B)), including, but not limited to, antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, sedatives and contrast media.

[0118] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and / or another therapeutic and / or prophylactic agent. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emidine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, and the like. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. Vaccines include compounds and preparations that can provide immunity against one or more pathological conditions associated with infectious diseases (such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, whooping cough, tetanus, plague, hepatitis, and pulmonary tuberculosis), and may include nucleic acid molecules (e.g., mRNA) encoding antigens and / or epitopes from the infectious disease.Vaccines may further include compounds and formulations that induce an immune response against cancer cells, which may include nucleic acid molecules (e.g., mRNA) encoding tumor cell-derived antigens, epitopes, and / or neoepitopes. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other substances. Other therapeutic and / or prophylactic agents include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, racemicin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromocyclododecanoate, and the like). Anti-mitotic agents include, but are not limited to, cyclohexyl methyl ...

[0119] In another embodiment, the therapeutic and / or prophylactic agent is a protein. Therapeutic proteins that can be used in the nanoparticles of the present invention include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), VIR factors, luteinizing hormone-releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0120] In some embodiments, the therapeutic and / or prophylactic agent may be a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term "polynucleotide" in its broadest sense includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in the present invention include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (messenger mRNA (mRNA) or hybrids thereof, RNAi inducers, RNAi factors, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, triple helix-derived RNA, aptamers, etc.). In some preferred embodiments, the therapeutic and / or prophylactic agent is RNA. RNA that can be used in the compositions and methods described herein may be selected from the group consisting of, but is not limited to, shortmers, antagomirs, antisense RNA, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the RNA is mRNA.

[0121] In some embodiments, the therapeutic and / or prophylactic agent is an mRNA. The mRNA can encode a polypeptide of any use, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0122] In other embodiments, the therapeutic and / or prophylactic agent is an siRNA. The siRNA can selectively reduce or downregulate the expression of a gene of interest. For example, the selectivity of the siRNA may result in silencing of a gene associated with a particular disease, condition, or pathological state after administration of a composition containing the siRNA to a subject in need thereof. The siRNA may comprise a sequence complementary to an mRNA sequence encoding a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0123] In some embodiments, the therapeutic and / or prophylactic agent is an sgRNA and / or a cas9 mRNA. The sgRNA and / or the cas9 mRNA may be used as a gene editing tool. For example, the sgRNA-cas9 complex affects the translation of mRNA of a cellular gene.

[0124] In some embodiments, the therapeutic and / or prophylactic agent is an shRNA or a carrier or plasmid encoding the same. The shRNA may be produced inside the target cell after delivery of an appropriate construct into the nucleus. The constructs and mechanisms associated with shRNA are well known in the relevant fields.

[0125] (disease or condition) The compositions / carriers of the present invention can deliver therapeutic and / or prophylactic agents to a subject or patient. The therapeutic and / or prophylactic agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Therefore, the compositions of the present invention may be used to produce nucleic acid pharmaceuticals, genetic vaccines, small molecule drugs, polypeptides, or biopharmaceuticals. Due to the wide variety of therapeutic and / or prophylactic agents available as described above, the compositions of the present invention may be used to treat or prevent a variety of diseases or conditions.

[0126] In one embodiment, the disease or condition is characterized by a malfunction or abnormal activity of a protein or polypeptide.

[0127] The medicaments, compositions and methods described in the present invention may be used to treat a subject suffering from a disease (e.g., a disease in which an expressed antigen appears and which is characterized by pathological cells presenting an antigenic peptide). Examples of diseases that can be treated and / or prevented cover all diseases that express one of the antigens described in the present invention. Particularly preferred diseases are infectious diseases (e.g., viral diseases) and cancer diseases. The medicaments, compositions and methods described in the present invention may be used for immunization or vaccination to prevent the diseases described in the present invention.

[0128] According to the present invention, the term "disease" refers to any pathological condition such as infectious diseases, cancer diseases, etc., in particular those forms of infectious diseases and diseases described in the present invention.

[0129] According to the present invention, the disease to be treated is preferably an antigen-related disease. According to the present invention, an "antigen-related disease" or similar term means that the antigen is expressed in the cells of a tissue or organ with pathological changes. Expression in the cells of a tissue or organ with pathological changes may be elevated compared to the state in healthy tissue or organ. In one embodiment, expression occurs only in tissue with pathological changes, and expression is suppressed in healthy tissue. According to the present invention, antigen-related diseases include infectious diseases and cancer diseases, where the disease-associated antigen is preferably an antigen of an infectious source or a tumor antigen, respectively. Preferably, antigen-related diseases are diseases related to cells that express the antigen and present the antigen in the presence of MHC molecules (particularly MHC class I).

[0130] For example, the disease or condition is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renovascular diseases, metabolic diseases.

[0131] Examples of the infectious diseases include: [1] viral infections (e.g., AIDS (HIV), hepatitis A, hepatitis B, or hepatitis C, shingles (chickenpox), rubella (rubella virus), yellow fever, dengue fever, flaviviruses, coronaviruses, influenza viruses, rabies viruses, and hemorrhagic infections (Marburg virus or Ebola virus)); [2] bacterial infections (e.g., Legionnaire's disease) infections caused by bacteria (e.g., malaria, African sleeping sickness, leishmaniasis, toxoplasmosis, i.e., infections caused by Plasmodium, Trypanosoma, Leishmania, Toxoplasma); or fungal infections (e.g., Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis). immitis, Blastomyces dermatitidis, or Candida albicans).

[0132] Cancer, or carcinoma (the medical term for cancer, is malignant tumor), is a disease in which some cells exhibit uncontrollable proliferation (dividing beyond normal limits), invasiveness (invading and destroying adjacent tissues), and in some cases metastasis (spreading to other parts of the body via the lymphatics or blood). These three harmful characteristics distinguish cancer from benign tumors, which are self-limited and do not invade or metastasize. Most cancers result in the formation of tumors, i.e., swellings or pathological changes caused by the abnormal proliferation of cells (called malignant neoplastic cells or tumor cells), but some (such as leukemia) are not of this category. According to the present invention, the term "cancer" includes leukemia, seminoma, melanoma, teratoma, lymphoma, sarcoma, blastoma, neuroblastoma, glioma, glioblastoma, kidney cancer, adrenal cancer, renal cell carcinoma, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, lung cancer, bowel cancer, head and neck cancer, digestive cancer, multiple myeloma, lymph node cancer, esophageal cancer, rectal cancer, bladder cancer, endometrial cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, uterine cancer, breast cancer, prostate cancer, ovarian cancer, and metastases thereof.

[0133] Malignant melanoma is a severe form of skin cancer that arises from the uncontrolled growth of pigment cells called melanocytes.

[0134] According to the present invention, "epithelial cancer" is a malignant tumor derived from epithelial cells. This includes the most common cancers, such as common forms of breast, prostate, lung, and colon cancer.

[0135] Lymphomas and leukemias are malignant tumors that originate in hematopoietic (blood-forming) cells.

[0136] Sarcomas are cancers that arise from transformed cells in one of the tissues that originate from the fetal mesoderm, and therefore include tumors of bone, cartilage, fat, muscle, blood vessels, and blood-forming tissue.

[0137] A blastic tumor, or blastoma, is a tumor that resembles immature or fetal tissue (and is generally malignant). Most of these tumors are found in children.

[0138] Gliomas are a type of tumor that begins in the brain or spine. They are called gliomas because they arise from glial cells. The brain is the most common location for gliomas.

[0139] (Other ingredients or adjuvants) The compositions of the present invention may be administered with supplemental immune-enhancing substances (e.g., one or more adjuvants) or may contain one or more immune-enhancing substances that further enhance their efficacy and preferably achieve a coordinated immune-stimulating effect. The term "adjuvant" refers to a compound that prolongs, enhances, or accelerates an immune response. Different types of adjuvants may have different mechanisms in this regard. For example, compounds that mature DCs (e.g., lipopolysaccharides or CD40 ligand) constitute a first class of suitable adjuvants. In general, any agent that influences the immune system of the "danger signal" type (e.g., LPS, GP96, dsRNA, etc.) or cytokines (e.g., GM-CSF) may be used as an adjuvant that can enhance and / or influence the immune response in a controlled manner. As mentioned above, CpG oligodeoxynucleotides may optionally be used in some circumstances, although they may have side effects in those circumstances. Particularly preferred adjuvants are cytokines (e.g., monokines, lymphokines, interleukins, or chemokines (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFα, INF-γ, GM-CSF, LT-α)) or growth factors (e.g., hGH). Other known adjuvants include aluminum hydroxide, Freund's adjuvant, or oils. For example, Montanide®, preferably Montanide® ISA51, lipopeptides (e.g., Pam3Cys, Pam3CSK4), glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxyribonucleotides (e.g., class A or class B), and poly(I:C) are also suitable as adjuvants in the compositions of the present invention.

[0140] The composition may include one or more ingredients other than those described in the above sections. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0141] The composition may further comprise one or more permeability enhancing molecules, carbohydrates, polymers, surface-altering agents, or other components. Permeability enhancing molecules may be, for example, molecules described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include monosaccharides (e.g., glucose), polysaccharides (e.g., glycogen and its derivatives and analogs).

[0142] Surface-altering agents include anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants (such as dimethyl distearyl ammonium bromide)), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, poloxamer), mucolytic agents (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase α), and the like. alfa), neltenexine, erdosteine), DNase (e.g., rhDNase). The surface-altering agent may be disposed within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent bonding, or other methods).

[0143] The composition may further comprise one or more functionalized lipids.For example, lipids may be functionalized with alkynyl groups, which can undergo cycloaddition reaction when exposed to azide under suitable reaction conditions.Accurately, lipid bilayers may be functionalized with one or more groups that can effectively promote membrane permeability, cell recognition or imaging in this way.The surface of the composition may be coupled with one or more useful antibodies.The functional groups and conjugates used for targeted cell delivery, imaging and membrane permeability are well known in the art.

[0144] In addition to these ingredients, the composition may contain any substance used in pharmaceutical compositions. For example, the composition may contain one or more pharmaceutically acceptable (i.e., medicament-usable) excipients or helper ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonicity agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc.

[0145] The term "pharmaceutical acceptable" refers to a material that is non-toxic and does not affect the action of the active ingredient of the pharmaceutical composition. Non-pharmaceutical acceptable ingredients may be used to prepare pharmaceutical acceptable ingredients and are included in the present invention.

[0146] Suitable buffers for use in the compositions of the present invention include acetic acid in a salt form, citric acid in a salt form, boric acid in a salt form, and phosphoric acid in a salt form.

[0147] As used herein, the term "excipient" refers to any substance other than an active ingredient that may be present in the pharmaceutical composition of the present invention, such as a carrier, binder, lubricant, thickener, surfactant, preservative, emulsifier, buffer, flavoring agent, or coloring agent. Excipients are, for example, starch, lactose, or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st ed., A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0148] The term "carrier" refers to an organic or inorganic ingredient, of natural or synthetic nature, that is combined with an active ingredient to facilitate, enhance or enable administration. According to the present invention, the term "carrier" further includes one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration to a patient.

[0149] Carrier materials used for parenteral administration are, for example, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalene, and, particularly, biologically compatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0150] Suitable preservatives for use in the compositions of the present invention include benzalkonium chloride, chlorobutanol, parahydroxybenzoic acid esters, and thimerosal.

[0151] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof.

[0152] (Dosage Form and Administration) The compositions of the present invention may be prepared as solid, semisolid, liquid, or gaseous preparations, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injectables, and aerosols. The compositions of the present invention may be prepared by methods well known in the pharmaceutical arts. For example, a sterile injectable solution may be prepared by adding a predetermined amount of a therapeutic or prophylactic agent and the other ingredients specified above to a suitable solvent, such as sterile distilled water, followed by filtration and sterilization. Furthermore, a surfactant may be added to promote the formation of a uniform solution or suspension.

[0153] For example, the compositions of the present invention may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In one embodiment, the compositions are administered intravenously or subcutaneously.

[0154] (therapeutically effective amount) A "therapeutically effective amount" is an amount of a therapeutic agent that, when administered to a patient, ameliorates a disease or condition. A "prophylactically effective amount" is an amount of a prophylactic agent that, when administered to a subject, prevents a disease or condition. The amount of a therapeutic agent that constitutes a "therapeutically effective amount" or the amount of a prophylactic agent that constitutes a "prophylactically effective amount" varies depending on the therapeutic and / or prophylactic agent, the disease state and its severity, and the age, weight, etc., of the patient and / or subject receiving treatment and / or prevention. Those skilled in the art may generally determine therapeutically effective amounts and prophylactically effective amounts based on their knowledge and the present invention.

[0155] The compositions of the present invention are administered in a therapeutically effective amount, which will vary not only depending on the particular agent selected, but also on the route of administration, the characteristics of the disease being treated, and the age and condition of the patient, and may ultimately be determined at the discretion of the attending physician or clinician. For example, a dose of about 0.0001 to about 10 mg / kg of the therapeutic or prophylactic agent may be administered to a mammal (e.g., a human).

[0156] (antigen presenting cells) Antigen-presenting cells (APCs) are cells that present (i.e., display) antigens in the context of major histocompatibility complexes (MHC) on their surface. This can involve presenting only one fragment of an antigen or multiple fragments. T cells can recognize this complex with their T cell receptors (TCRs). Antigen-presenting cells process antigens and present them to T cells.

[0157] Professional antigen-presenting cells are adept at ingesting antibodies (by phagocytosis or receptor-mediated endocytosis) and then displaying antigen fragments in their membranes that bind to MHC class II molecules. T cells recognize and interact with the antigen-MHC class II molecule complex on the membrane of the antigen-presenting cell. The antigen-presenting cell then produces additional costimulatory signals to activate the T cell. The expression of costimulatory molecules is a typical feature of professional antigen-presenting cells.

[0158] The main types of professional antigen-presenting cells are dendritic cells (which have the widest antigen-presenting range and may be the most important antigen-presenting cells), macrophages, B cells, and some activated epithelial cells.

[0159] Dendritic cells are a group of leukocytes, including plasmacytoid dendritic cells (pDC cells) and classical dendritic cells (cDC cells), that present antigens captured in peripheral tissues to T cells via two antigen presentation pathways: MHC class II and class I. Dendritic cells are potent inducers of immune responses, and activation of these cells is a critical step for inducing antitumor immunity.

[0160] Antigen-presenting cells may be loaded with peptides presented by MHC by transducing them with nucleic acids encoding peptides or proteins containing the peptides to be presented (e.g., nucleic acids (e.g., RNA) encoding antigens). Transfection of dendritic cells with mRNA is a promising antigen-loading technique for stimulating potent antitumor immunity.

[0161] The term "immunogenicity" relates to the relative efficiency of an antigen in inducing an immune response.

[0162] The terms "T cells" and "T lymphocytes" may be used interchangeably herein, and include helper T cells (CD4 + T cells), cytolytic T cells (CTL, CD8 + T cells).

[0163] T cells belong to a group of white blood cells called lymphocytes and play a central role in cell-mediated immunity. They are distinguished from other types of lymphocytes (e.g., B cells, natural killer cells) by the presence of specialized receptors called T cell receptors (TCRs) located on their surface. The thymus is the main organ responsible for the maturation of T cells. Several different T cell subsets have been discovered, each with distinct functions.

[0164] Helper T cells synchronize other white blood cells in the immune process, such as maturing B cells into plasma cells and activating cytotoxic T cells and macrophages. These cells express the CD4 protein on their surface, and thus are CD4 + Also called T cells, helper T cells are activated when MHC class II molecules expressed on the surface of antigen-presenting cells (APCs) present peptide antigens to them. After activation, they rapidly divide and secrete small proteins called cytokines that regulate or support active immune responses.

[0165] Cytotoxic T cells destroy diseased cells (e.g., infected cells (e.g., virus-infected cells)), cancer cells, and are involved in transplant rejection. These cells express the CD8 glycoprotein on their surface and are therefore CD8 + Also called T cells, these cells recognize their targets by binding antigens associated with MHC class I, which is present on the surface of almost every cell in the body.

[0166] Most T cells have a T cell receptor (TCR) that exists as a complex of multiple proteins. The actual T cell receptor is composed of two independent peptide chains, called the α-TCR chain and the β-TCR chain, which are produced by separate T cell receptor α and β (TCRα and TCRβ) genes. γδ T cells are a small subtype of T cells that have a unique T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR is composed of one γ chain and one δ chain. This group of T cells is rarer than αβ T cells (representing 2% of all T cells).

[0167] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells reside in the thymus and proliferate by cell division to produce large numbers of immature thymocytes. Early thymocytes express neither CD4 nor CD8, and are therefore double-negative (CD4 - CD8 - ) cells. As they develop, they become double-positive thymocytes (CD4 + CD8 + ) and finally mature into single-positive (CD4 + CD8 - or CD4 - CD8 + ) into thymocytes, which are later released from the thymus into peripheral tissues.

[0168] The initial signal for T cell activation is provided by the binding of the T cell receptor to a short peptide presented by the major histocompatibility complex (MHC) on another cell. This ensures that only T cells with a TCR specific for that peptide are activated. The partner cell is generally a professional antigen-presenting cell (APC), typically a dendritic cell in the primary response, but B cells and macrophages can also be important APCs. MHC class I molecules bind to CD8 + Peptides presented to T cells are 8–10 amino acids in length and are bound to CD4 by MHC class II molecules. + Peptides presented to T cells are longer due to the open end of the binding cleft of the MHC class II molecule.

[0169] (Example) The present invention will be further described below with reference to examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples may be further adjusted based on different specific requirements in their use, and unless specified, the implementation conditions are normal conditions in the art. All raw materials used in the specific examples of the present invention are commercially available. Unless otherwise specified, percentages in the context are percentages by weight, and all temperatures are in degrees Celsius. The technical features of each embodiment of the present invention may be combined unless they are inconsistent with each other.

[0170] Below are the abbreviations and the reagents they represent. ADOPE: 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate sodium salt DOTMA: 1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine 18PA: (Z)-(R)-3-(phosphonooxy)propane-1,2-diyldioleic acid monosodium salt DOPG: 1,2-dioleoyl-sn-glycero-3-phospho-rac-glycerol sodium salt pA(2'-OMe)mpG: ((2R,3S,4S,5R)-3-(((((2R,3R,4S,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-methoxytetrahydrofuran-2-yl)methyl dihydrogen phosphate DSPC: Distearoylphosphatidylcholine DOTAP: (2,3-dioleoxypropyl)trimethylammonium chloride HOBt: 1-hydroxybenzotriazole EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0171] Example 1: Synthesis of ADOPE The synthetic route of 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate sodium salt (ADOPE) was as follows.

[0172] [ka] 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (1.0 g, 1.34 mmol) was dissolved in dichloromethane (3 mL). To the solution were then added HOBt (910 mg, 6.71 mmol) and EDCI (1.28 g, 6.71 mmol), in that order, and the mixture was stirred at room temperature for 20 minutes to react. Then, acetic acid (403 mg, 6.71 mmol) was added to the mixture, and the mixture was stirred at room temperature overnight to react. After the reaction was completed, the solvent was removed by rotary evaporation and purified by silica gel chromatography (eluent: dichloromethane / 20% methanol (0.1% trifluoroacetic acid)). The product-containing fraction was spin-dried, dissolved in ethyl acetate, added with saturated aqueous NaHCO3 and stirred for 5 minutes, separated, dried over anhydrous sodium sulfate, and the organic phase was spin-dried to obtain 1.03 g of a pale yellow oily compound with a yield of 95.1%. 1 H NMR(400 MHz,CDCl3)δ 5.37-5.15(m,4H),4.16-3.81(m,4H),δ 3.51-3.10(m,4H),2.52(s,2H),2.29(q,J=7.2 Hz,4H),2.08-1.91(m,10H),1.67-1.50(m,4H),1.38-1.14(m,40H),3.62-3.49(m,2H),0.93-0.80(m,6H).C 43 H 80 NO9P, MS(ES): m / z (M-Na - )784.04.

[0173] Example 2: Method for producing mRNA lipid composition A) Preparation of Fluc DNA and eGFP DNA templates 1) Luciferase (luciferase protein CDS) or green fluorescent protein (GFP) circular plasmid was constructed by ligating it into the pVAX1 vector (purchased from Thermo Fisher Scientific) via restriction enzyme EcoRV digestion. 2) The plasmid constructed in the pVAX1 vector in step 1) was uniformly mixed with 50 μL of E. coli competent cells Stbl2 (purchased from Thermo Fisher Scientific), then placed in an ice bath for 30 minutes, heat-shocked at 42°C for 90 seconds, immediately returned to ice, and placed in an ice bath for 2 minutes. 3) 400 μL of LB medium (purchased from Thermo Fisher Scientific) was added, and the mixture was cultured at 30°C with gentle shaking in a shaker for 45 to 60 minutes. 4) 50-100 μL of the bacterial solution was spread onto LB solid medium containing the antibiotic kanamycin (100 μg / mL, purchased from Yisheng Biotechnology Co., Ltd., hereinafter referred to as Yisheng Biotechnology Co., Ltd.), and cultured upside down at 37°C overnight. 5) The obtained monoclonal colony plates were sequenced to verify accuracy, and monoclonal colonies with correct sequencing results were picked and cultured overnight at 30°C with slow shaking in a shaker. 6) Plasmid extraction was performed using an endotoxin-free large-scale plasmid purification kit (purchased from Yisheng Biotechnology Co., Ltd.). 7) Using restriction enzymes, cut the extracted plasmid into a linear plasmid, which was then used as a transcription template. The specific steps of the cutting process were as follows: [1] - [3].

[0174] In step [1], 1 mg of luciferase circular plasmid was cleaved (enzyme BspQ I, purchased from Yisheng Biotechnology Co., Ltd.) at 37°C for 4 hours to obtain a linear DNA transcription template (see Table 1 for the cleavage system).

[0175] [Table 1]

[0176] In step [2], after the reaction is complete, absolute ethanol is added, followed by sodium acetate, and V 切断反応生成物 :V無水エタノール :V 3M酢酸ナトリウム Absolute ethanol and 3M sodium acetate were added in a volume ratio of 1:3:1, and the mixture was left to stand at −20° C. for 1 hour to precipitate, and then centrifuged at 12,000 rpm to retain the precipitate. In step [3], the precipitate from step [2] was washed twice with 70% ethanol, and the resulting centrifuged material was dried at 55°C for 10 minutes, after which 1.7 mL of water for injection was added and dissolved. The concentration of the linearized plasmid in the lysate was 500 ng / μL, the linearization rate was 90% or more, and the purification recovery efficiency was 85%.

[0177] B) Preparation of FlueC mRNA, eGFP mRNA, HA mRNA, and OVA mRNA 1) Co-transcriptional capping reaction Using the Fluc DNA and eGFP DNA prepared in Example 1(A) or HA DNA and OVA DNA purchased from Suzhou Jinweizhi Biotechnology Co., Ltd. as templates, NTP solution (NTPs), and Cap1 cap analog (catalog number 10678ES80, purchased from Yisheng Biotechnology Co., Ltd.) as starting materials, mRNA was synthesized by T7 RNA polymerase transcription. The details of the reaction system are shown in Table 2. The prepared reaction system was placed in a 37°C incubator and incubated with shaking for 3 hours. The Cap1 cap analog is Cap1-GAG, which has the structure m7G(5')ppp(5')(2'-OMeA)pG, and its molecular formula is C. 32 H 43 N 15 O 24 It was P4.

[0178] [Table 2]

[0179] 2) Digestion of template DNA After completing step 1 above, DNase I (purchased from Yisheng Biotechnology Co., Ltd.) was added to the co-transcriptional capping reaction system to a final concentration of 1 U / μg of linearized plasmid. The mixture was mixed uniformly, centrifuged, and digested at 37°C for 1 hour to obtain the co-transcriptional capping product.

[0180] 3) Purification by lithium chloride precipitation method The co-transcriptional capping product obtained in step 2) above was purified by lithium chloride precipitation, as follows. Step [1] was the addition of lithium chloride. Lithium chloride solution (purchased from Thermo Fisher Scientific) was added to the product of step [2] above to a final concentration of 2.8 M, and the mixture was allowed to precipitate at low temperature for 2 hours. Step [2] was precipitation: high-speed centrifugation at 12,000 rpm for 15 minutes, and the precipitate was retained. Step [3] was washing. The mRNA solution was obtained by washing twice with 75% ethanol and dissolving in water for injection. The purified mRNA solution was stored at -80°C.

[0181] C) Preparation of mRNA Compositions Using Lipid Compositions 1) Preparation of lipid composition solution In general method 1, each component of the lipid composition (including permanent anionic lipids, permanent cationic lipids, and / or neutral lipids, or various combinations thereof) was dissolved in ethanol at a predetermined molar ratio to prepare an ethanol-lipid complex solution (total lipid concentration was 100-600 mM). The ethanol-lipid complex solution was rapidly added to RNase-free water by ethanol injection, and after stirring for 30 minutes, the resulting lipid composition solution was filtered through a polycarbonate membrane with a pore size of 100-400 nm.

[0182] In standard method 2, permanent anionic lipids or compounds were dissolved in RNase-free water to obtain RNase-free water containing permanent anionic lipids. Permanent cationic lipids and neutral lipids were dissolved in ethanol at a predetermined molar ratio to prepare an ethanol-lipid complex solution (total lipid concentration was 100-600 mM). The ethanol-lipid complex solution was rapidly added to the RNase-free water containing permanent anionic lipids by ethanol injection, and after stirring for 30 minutes, the resulting lipid composition solution was filtered through a polycarbonate membrane with a pore size of 100-400 nm.

[0183] 2) mRNA composition The mRNA (prepared in Example 2) was diluted with a HEPES and EDTA solution to obtain an mRNA aqueous solution (concentration: 0.5 mg / mL, buffer: 10 mM HEPES and 0.1 mM EDTA, with EDTA added as a chelating agent to aqueous HEPES). A sodium chloride solution (0.9% w / w in water) was obtained with a syringe and injected into the mRNA aqueous solution prepared above. The lipid complex solution was obtained with a syringe and injected into the mRNA and sodium chloride solution, vortexed for 30 seconds, and incubated at room temperature for 10 minutes to obtain the mRNA composition (final concentration of RNA was 100 μg / mL), which was then stored at 4-8°C.

[0184] 3) Measurement of particle size and PDI The mRNA composition was diluted in a 1:5 ratio with sodium chloride solution (0.9% w / w in water), and the particle size and polydispersity index (PDI) were measured using dynamic light scattering with a Malvern Panalytical laser diffraction particle size analyzer.

[0185] Example 3: Effect of type of anionic lipid on particle size and PDI of mRNA composition

[0186] [Table 3] JPEG2025160111000005.jpg130170

[0187] The following mRNA compositions were prepared using the method of Example 2. Here, lipid complex solutions corresponding to numbers 1 to 3 were prepared using Method 1 (i.e., anionic lipids were dissolved in ethanol), lipid complex solutions corresponding to numbers 4 to 7 were prepared using Method 2 (i.e., anionic lipids were dissolved in water), and lipid complex solutions corresponding to numbers 8 to 10 were prepared using Method 1. The molar ratio of the permanent anionic lipid, the permanent cationic lipid DOTMA, and the neutral lipid DOPE in the different complexes was limited to 1:2:1, and the particle size and PDI were measured.

[0188] The anionic lipid-containing complex solutions of Examples 4 to 11 were all prepared by Method 1 of Example 2, and detailed explanations thereof will be omitted.

[0189] [Table 4]

[0190] As is clear from the experimental results, the addition of permanent anionic lipids, especially the permanent anionic lipids containing phosphate groups, ADOPE, 18PA, and DOPG, resulted in good solubility in ethanol, and the addition of tetradecylphosphonic acid, farnesyl pyrophosphate, γ,γ-dimethylallyl pyrophosphate, and ((2R,3S,4S,5R)-3-((((2R,3R,4S,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran resulted in good solubility in ethanol. The solubility of (5-(6-amino-9H-purin-9-yl)-4-methoxytetrahydrofuran-2-yl) methyl dihydrogenphosphate (pA(2'-OMe)mpG) in ethanol is low. Both compositions prepared by the two methods had good particle sizes and PDIs. The best was ADOPE, with a particle size of 303.1 nm and a PDI of 0.2455.

[0191] Conversely, compositions utilizing other anionic lipids that do not contain phosphate groups, such as oleic acid, sodium bis(laureth-7) citrate, or sodium lauryl sulfonate, showed a significant increase in particle size, exceeding 1000 nm, and solid precipitation, making them unsuitable as mRNA delivery carriers.

[0192] As can be seen from the above comparison, the addition of permanent anionic lipids containing phosphate groups allows the production of compositions with good particle size (particle size limited to 240-500 nm) and uniform particle distribution (PDI<0.5).

[0193] Example 4: Effect of lipid formulation ratio on particle size and PDI of RNA compositions The following mRNA compositions containing permanent anionic lipids were prepared according to the method of Example 2, where the lipid complex solution was prepared according to Method 1 (ie, dissolving the permanent anionic lipids in ethanol).

[0194] The particle size and PDI were measured by changing the molar ratio of the permanent anionic lipid ADOPE, the permanent cationic lipid DOTMA, and the neutral lipid DOPE in the lipid complex solution, and the amount of liposome solution used.

[0195] [Table 5]

[0196] As is clear from the experimental results, when the molar percentages of the combination of permanent anionic lipid, permanent cationic lipid, and neutral lipid were (10-33 mol%): (40-60 mol%): (22-40 mol%), and in particular, the ratio of permanent anionic lipid was limited to 10-33 mol%, the particle size (particle size was limited to within 240-500 nm) and PDI of the produced composition were both in a good range (less than 0.5). If the amount of permanent anionic lipid added was too high, exceeding the range of 10-33 mol%, for example, at 40 mol%, the system became unstable during preparation, and a large amount of particulate solid was produced.

[0197] Furthermore, as can be seen from the above results, it is possible to produce a composition with acceptable particle size and PDI, such as No. 13, without adding a permanent anionic lipid.

[0198] Example 5: Effect of charge ratio on particle size and PDI of RNA compositions The following Fluc-mRNA compositions were produced according to the method of Example 2, where the lipid complex solution was prepared according to Method 1 (ie, dissolving permanent anionic lipids in ethanol).

[0199] The lipid complex solution was limited to the same molar ratio of the permanent anionic lipid ADOPE, the permanent cationic lipid DOTMA, and the neutral lipid DOPE, and the particle size and PDI were measured by changing the charge ratio.

[0200] [Table 6]

[0201] As is clear from the experimental results, when the charge ratio of the lipid composition is limited to the range of 1:5 to 1:2, the lipid composition has a good particle size (particle size is limited to 240 to 500 nm) and a composition with a uniform particle distribution (PDI<0.5) can be produced.

[0202] For example, when the mole percentage of ADOPE, DOTMA, and DOPE in the lipid composition was 25:50:25 and the charge ratio was within the range of 1:5 to 1:2, the particle size increased as the charge ratio increased, and when the charge ratio reached 1:2, the particle size was 462.7 nm. When the charge ratio was subsequently increased, for example, to 2:3 and 3:4 as in samples 5 and 6, the system became unstable during preparation, resulting in the formation of large solid particles, and in both cases, large solid particles precipitated.

[0203] Similarly, when the mole percentage of ADOPE, DOTMA, and DOPE in the lipid composition is 20:40:40, a charge ratio within the range of 1:5 to 1:2 results in a lipid composition with good particle size (limited to 240-500 nm) and a uniform particle distribution (PDI<0.5). As the charge ratio increases, the particle size and PDI of the composition increase. At a charge ratio of 1:2, the particle size and PDI are 456.2 nm and 0.4144, respectively. When the charge ratio exceeds the 1:5 to 1:2 range, for example, at charge ratios of 2:3 and 3:4 (as in samples 11 and 12), large solid particles precipitate.

[0204] Furthermore, as can be seen from the above results, it is possible to prepare compositions with acceptable particle size and PDI, such as Nos. 13 and 14, without adding anionic lipids. Based on this, further investigations were carried out later in mouse in vivo protein expression experiments and mouse splenocyte flow cytometry experiments.

[0205] Example 6: Protein expression experiment in mice The control LNP was prepared as follows. The cationic lipid compound YK-009 (or YK-407) was dissolved in ethanol with DSPC (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (AVT (Shanghai) Pharmaceutical Tech Co., Ltd.), and DMG-PEG2000 at a molar ratio of 49:10:39.5:1.5 to prepare an ethanolic lipid solution. The ethanolic lipid solution was rapidly added to citrate buffer (pH 4-5) by ethanol injection and vortexed for 30 seconds before use. The Fluc-mRNA solution was diluted with citrate buffer (pH 4-5) to obtain an aqueous Fluc-mRNA solution. Liposomes were prepared using the designated amounts of liposome solution and Fluc-mRNA solution at a total lipid to Fluc-mRNA weight ratio of 10:1. The mixture was sonicated at 25°C for 15 minutes (ultrasonic frequency: 40 kHz, ultrasonic output: 800 W). The resulting liposomes were diluted 10-fold with PBS and then ultrafiltered with a 300 kDa centrifugal ultrafilter to remove ethanol. The volume was then adjusted to volume with PBS to obtain LNP formulations encapsulating Fluc-mRNA in cationic lipids YK-009 (or YK-407) / DSPC / cholesterol / DMG-PEG2000 (mol percent 49:10:39.5:1.5). The structures of YK-009 and YK-407 were as follows:

[0206] [Table 7]

[0207] The composition containing permanent anionic lipids containing 20 μg of Fluc-mRNA prepared in Example 2 (or the LNP formulation containing YK-009 (or YK-407) containing 20 μg of Fluc-mRNA prepared by the above method) was injected into the tail vein of female BALB / c albino mice, aged 4-6 weeks and weighing 17-19 g. Six hours after administration, the mice were intraperitoneally injected with a fluorescent contrast substrate and allowed to move freely for 5 minutes. The total emission intensity of the protein expressed in the mice by the mRNA encapsulated in the RNA composition (corresponding to the fluorescent protein expression intensity, i.e., the amount of protein expressed) was measured using an IVIS Spectrum small animal imaging device. After sampling was completed, the mice were euthanized by cervical dislocation, dissected, and the liver, spleen, and lungs were properly isolated. The total radiant intensity (corresponding to the expression intensity of the fluorescent protein, i.e., the amount of protein expressed) of the protein expressed by Fluc-mRNA in each organ of the mouse was measured using an IVIS Spectrum small animal imaging device. The results of the in vivo imaging measurements in mice are shown in Table 8 and Figures 1a to 1d.

[0208] [Table 8]

[0209] The analysis of the results was as follows. (1) When the molar percentages of permanent anionic lipids, permanent cationic lipids, and neutral lipids were limited to (14-33 mol%):(40-57 mol%):(22-40 mol%) and the charge ratio was 1:2-1:5 (numbers 3-14), the protein expression level of the antigen in the spleen was significantly improved, and the composition was well targeted to the spleen.

[0210] For example, when the mole percentage of [1]ADOPE, DOTMA, and DOPE is 25:50:25, as the charge ratio increases, the total radiation intensity in the mouse becomes higher and higher. When the charge ratio is 1:2, the total radiation intensity in the mouse is 2.32 × 10 7p / s, and the total radiant intensity of the spleen was significantly higher than that of the liver and lungs. When the charge ratio was 2:5, the total radiant intensity of the spleen was 7.38 × 10 7 p / s was the highest, being 56.77-fold and 67.09-fold higher than those in the liver and lung, respectively, indicating that the protein expression level of Fluc-mRNA delivered by the composition in the spleen was significantly higher than those in the liver and lung.

[0211] [2] When the mole percent of ADOPE, DOTMA, and DOPE is 20:40:40, as the charge ratio increases, the total radiant intensity in the mouse also tends to increase. When the charge ratio is 1:2, the total radiant intensity in the mouse is 1.91 x 10 7 p / s, and the total radiant intensity of the spleen was significantly higher than that of the liver and lungs. When the charge ratio was 1:2, the total radiant intensity of the spleen was 4.84 × 10 7 p / s, both of which were 48.40 times higher than those in the liver and lungs, indicating that the protein expression level of Fluc-mRNA delivered by the composition in the spleen was significantly higher than those in the liver and lungs.

[0212] [3] As is clear from the experimental results of Examples 3 and 4, it is possible to prepare a composition with acceptable particle size and PDI without adding anionic lipids. However, as can be seen from the in vivo protein expression experiment in mice, composition No. 17, which was prepared without adding anionic lipids, had a total radiation intensity of 0.64 × 10 in mice. 7 p / s, and the radiation intensity in the liver, spleen, and lungs was 0.10×10 7 p / s, 0.61 × 10 7 p / s, 0.10 × 10 7 p / s. This radiation intensity was significantly lower than that of the composition of the present invention containing the permanent anionic lipid, and there was no significant difference in radiation intensity between different organs, indicating that the protein expression level in the spleen of Fluc-mRNA delivered by the composition produced without the addition of anionic lipid was significantly lower.

[0213] [4] When the mole percentages of permanent anionic lipids, permanent cationic lipids, and neutral lipids are outside the range of (14-33 mol%):(40-57 mol%):(22-40 mol%), for example, when the mole percentages of ADOPE, DOTMA, and DOPE are 10 mol%:60 mol%:30 mol%, even if the charge ratio is 1:3 or 1:2 (numbers 1 and 2), the total radiation intensity in the spleen is 0.52 × 10 7 p / s, 0.59 × 10 7 The total radiation intensity in the spleen for p / s alone was equivalent to that of the composition (No. 17) prepared without anionic lipid, indicating that there was no significant difference in the protein expression levels in the liver, spleen, and lungs for the Fluc-mRNA delivered by both compositions, and that the targeting of the composition to the spleen was equivalent to, and not increased by, that of the composition (No. 17) prepared without anionic lipid.

[0214] (2) When other permanent anionic lipids, such as 18PA and DOPG (nos. 15 and 16), were used, the above conditions also significantly improved the protein expression level of the antigen in the spleen, demonstrating a good and significant spleen targeting effect.

[0215] For example, when the mole percentages of the permanent anionic lipids, DOTMA and DOPE, are 25:50:25 and the charge ratio is 1:2, the total radiant intensity in mice is 1.98 x 10 7 p / s, 2.11 × 10 7 p / s, and the total radiation intensity of the spleen was 6.72 × 10 7 p / s, 5.99 × 10 7 p / s were 56 and 54.45 times those of the liver, and 61.09 and 54.45 times those of the lung, respectively.

[0216] (3) Compared to a composition without added anionic lipids, a composition containing a permanent anionic lipid designed according to the present invention significantly increases the amount of protein expressed in the spleen.

[0217] For example, a composition (No. 7) with a molar percentage of ADOPE, DOTMA, and DOPE of 25:50:25 and a charge ratio of 2:5 had a total radiation intensity in the spleen that was 12.10 times that of a composition without added anionic lipids (No. 17 small particle size LPX-RNA).

[0218] (4) Compared with LNPs (Nos. 18 and 19) based on conventional technology, the composition containing permanent anionic lipids designed in the present invention significantly increased the protein expression level in the spleen, demonstrating good spleen targeting, but the protein expression levels in the liver and lungs were reduced (radiation intensity was approximately 0.1 × 10 7 p / s), or remained in the liver and did not express the target protein.

[0219] For example, the composition (No. 6) with a molar percentage of ADOPE, DOTMA, and DOPE of 25:50:25 and a charge ratio of 1:3 had a total emission intensity in the liver that was only 0.09 times that of YK-009-Fluc mRNA-LNP (No. 18), but a total emission intensity in the spleen that was 6.22 times that of YK-009-Fluc mRNA-LNP.

[0220] The composition (No. 7) with a molar percentage of ADOPE, DOTMA, and DOPE of 25:50:25 and a charge ratio of 2:5 had a total radiation intensity in the spleen that was 7.1 times that of YK-407-Fluc mRNA-LNP (No. 19).

[0221] The conclusions are as follows: As can be seen from the above experimental results, the composition of the present invention produced by adding a permanent anionic lipid can significantly improve the amount of antigen protein expressed in the spleen.

[0222] Example 7: Mouse spleen cell flow cytometry experiments 1. The composition containing ADOPE containing 80 μg of eGFP-mRNA prepared in Example 2 was injected into the body of female C57BL / 6 mice aged 4 to 6 weeks and weighing 17 to 19 g via the tail vein. 24 hours after administration, the mice were euthanized by cervical dislocation, dissected, and their spleens were properly isolated.

[0223] 2. Single Cell Preparation 2.1 The spleen tissue was crushed and passed through a cell strainer to obtain single cells. 2.2 A 10-fold volume (4 mL) of red blood cell lysis solution was added to lyse and remove red blood cells in the tissue. 2.3 Count the cells and measure 5 x 10 6 Cells were collected into tubes for flow cytometry (ensuring that each sample had the same number of cells).

[0224] 3. Detection of spleen tissue immune cells (8-color surface staining) 3.1 100 μL of each surface antibody mix was added to the single cell suspension and incubated in the dark at room temperature for 15 minutes (one negative control).

[0225] [Table 9] 3.2 2 mL of PBS was added, and the mixture was centrifuged at 500 g for 5 minutes, and the supernatant was discarded. 3.3 The cells were resuspended in 200 μL of PBS (after filtering through a 200-mesh nylon mesh) and loaded onto a flow cytometer, Cytoflex S, for detection. The percentage content of GFP in each cell was analyzed. The detection order for each cell type was as follows: GFP ratio in T cells:CD45 + →CD3 + →GFP + GFP ratio in B cells:CD45 + →CD3 - CD19 + →GFP + GFP ratio in NK cells:CD45 + →NK1.1+ →GFP + GFP ratio in cDC cells:CD45 + →F4 / 80 - CD11c + →GFP + GFP ratio in pDC cells:CD45 + →F4 / 80 - CD11c int CD317 + →GFP + GFP:CD45 ratio in macrophages + →F4 / 80 + →GFP +

[0226] [Table 10]

[0227] The analysis of the results was as follows: Antigen-presenting cells refer to immune cells that can present lymphocytes to antigens, including dendritic cells (DC cells), B cells, and macrophages, which play a very important role in the human body, mainly playing the roles of immune recognition, immune response, and immune regulation.

[0228] Compositions (Nos. 1-9) containing ADOPE, DOTMA, and DOPE in mole percentages of (14-33 mol%):(40-57 mol%):(22-40 mol%) with a charge ratio limited to 1:2-1:5, or compositions utilizing other permanent anionic lipids, including but not limited to 18PA (No. 11) or DOPG (No. 12), all significantly increase the percentage of antigen-expressing antigen-presenting cells (e.g., B cells, pDC cells, cDC cells, macrophages) in the spleen. For example, 1. Compared to the blank control, composition 2 (ADOPE:DOTMA:DOPE = 25 mol%:50 mol%:25 mol%, charge ratio 1:3) increased the percentage of eGFP-positive cells among splenic antigen-presenting cells (B cells, pDC cells, cDC cells, macrophages) by 0.12%, 3.62%, 4.86%, and 5.86%, respectively. Composition 3 (ADOPE:DOTMA:DOPE = 25 mol%:50 mol%:25 mol%, charge ratio 2:5) increased the percentage of eGFP-positive cells among splenic antigen-presenting cells (B cells, pDC cells, cDC cells, macrophages) by 0.15%, 3.38%, 4.55%, and 6.25%, respectively. The concentrations of the compositions (ADOPE:DOTMA:DOPE = 25 mol%:50 mol%:25 mol%, charge ratio 1:2) increased by 0.37%, 5.90%, 8.69%, and 7.44%, respectively; the concentrations of the compositions (ADOPE:DOTMA:DOPE = 20 mol%:40 mol%:40 mol%, charge ratio 1:2) increased by 0.14%, 3.76%, 4.28%, and 4.81%, respectively; and the concentrations of the compositions (ADOPE:DOTMA:DOPE = 33 mol%:45 mol%:55 mol%, charge ratio 1:2) increased by 0.20%, 4.37%, 4.63%, and 4.20%, respectively.

[0229] When other permanent anionic lipids were used, the 18PA composition (No. 11) increased the solubility by 0.17%, 2.95%, 4.52%, and 4.67%, respectively, compared to the blank control, and the DOPG composition (No. 12) increased the solubility by 0.14%, 4.47%, 3.73%, and 4.11%, respectively.

[0230] 2. Compared to small-particle LPX-RNA (No. 13) without added anionic lipids, the ADOPE compositions (Nos. 1-9) can significantly increase the percentage of antigen-expressing antigen-presenting cells (e.g., B cells, pDC cells, cDC cells, macrophages) in the spleen.

[0231] For example, compared to small-particle LPX-RNA (No. 13) without added anionic lipid, ADOPE composition (No. 2) increased the percentage of eGFP-positive cells among splenic antigen-presenting cells (B cells, pDC cells, cDC cells, and macrophages) by 6.5, 1.7, 4.5, and 9.3 times, respectively; ADOPE composition (No. 3) increased the percentage of eGFP-positive cells by 8, 1.6, 4.2, and 9.9 times, respectively; ADOPE composition (No. 4) increased the percentage of eGFP-positive cells by 19, 2.8, 8.1, and 11.8 times, respectively; ADOPE composition (No. 5) increased the percentage of eGFP-positive cells by 7.5, 1.8, 4, and 7.7 times, respectively; and ADOPE composition (No. 9) increased the percentage of eGFP-positive cells by 10.5, 2.1, 4.3, and 6.7 times, respectively.

[0232] When other permanent anionic lipids were used, the 18PA composition (No. 11) was 9, 1.4, 4.2, and 7.4 times larger than the small particle size LPX-RNA (No. 13) without added anionic lipid, and the DOPG composition (No. 12) was 7.5, 2.2, 3.5, and 6.5 times larger, respectively.

[0233] 3. Compared with other combinations of pA(2'-OMe)mpG eGFP RNA compositions (containing the permanent anionic lipid pA(2'-OMe)mpG, the permanent cationic lipid DOTMA, and the neutral lipid DOPE), the ADOPE compositions (numbers 1-9) can significantly increase the percentage of antigen-expressing cells in antigen-presenting cells (e.g., B cells, pDC cells, cDC cells, and macrophages).

[0234] For example, ADOPE Composition (No. 2) increased the percentage of eGFP-positive cells among splenic antigen-presenting cells (B cells, pDC cells, cDC cells, macrophages) by 1.6-fold, 3.4-fold, 6.8-fold, and 9.2-fold, respectively; ADOPE Composition (No. 3) increased the percentage by 2-fold, 3.2-fold, 6.3-fold, and 9.8-fold, respectively; ADOPE Composition (No. 4) increased the percentage by 4.8-fold, 5.6-fold, 12.1-fold, and 11.6-fold, respectively; ADOPE Composition (No. 5) increased the percentage by 1.9-fold, 3.6-fold, 6-fold, and 7.5-fold, respectively; and ADOPE Composition (No. 9) increased the percentage by 2.6-fold, 4.1-fold, 6.4-fold, and 6.6-fold, respectively.

[0235] When other permanent anionic lipids of the present invention were used, the 18PA composition (No. 11) was 2.3-fold, 2.8-fold, 6.3-fold, and 7.3-fold higher than other combinations of pA(2'-OMe)mpG eGFP RNA compositions (containing the permanent anionic lipid pA(2'-OMe)mpG, the permanent cationic lipid DOTMA, and the neutral lipid DOPE), and the DOPG composition (No. 12) was 1.9-fold, 4.2-fold, 5.2-fold, and 6.4-fold higher, respectively.

[0236] 4. When the molar percentage and / or charge ratio of ADOPE, DOTMA, and DOPE exceeds the above range, the percentage of antigen-expressing cells (e.g., B cells, pDC cells, cDC cells, macrophages) in the spleen increases to some extent, but the effect is significantly reduced compared to the above. For example, Composition 10 (ADOPE:DOTMA:DOPE = 10 mol%:60 mol%:30 mol%, charge ratio 4:9) tended to increase the percentage of eGFP-positive antigen-presenting cells, such as B cells, pDC cells, cDC cells, and macrophages, in the spleen compared to the blank control. However, there was no significant difference in the percentage of eGFP-positive antigen-presenting cells, such as B cells, pDC cells, cDC cells, and macrophages, in the spleen compared to small-particle LPX-RNA (composition 13) without added anionic lipid, indicating that the addition of a small amount of ADOPE does not have a significant effect on targeting to antigen-presenting cells.

[0237] The conclusions are as follows: As can be seen from the above experimental results, the compositions of the present invention prepared by adding permanent anionic lipids, permanent cationic lipids, and neutral lipids can significantly increase the percentage of antigen-expressing cells (e.g., B cells, pDC cells, cDC cells, macrophages) in the spleen.

[0238] Example 8: Comparative experiment between RNA composition containing ADOPE and large particle size LPX-RNA As can be seen in the invention patent related to LPX-RNA (grant publication number CN109331176B), there are differences in luciferase activity in the spleen of mice after injection of luciferase-RNA liposome compositions of different sizes, mainly due to the fact that larger liposome compositions have higher activity. Following the manufacturing method of CN109331176B, a large-sized LPX-RNA composition can be produced by reducing the amount of ethanol used in LPX-RNA production by 50%. In this example, the differences in in vivo imaging of mice and the percentage of eGFP-positive cells in mouse spleen cells were compared between a composition containing ADOPE and a large-sized LPX-RNA composition.

[0239] [Table 11]

[0240] The results were analyzed as follows. As shown in Table 11, compared to the large-particle LPX-RNA composition (No. 2, particle size 478.5 nm) without anionic lipid, the composition containing the permanent anionic lipid ADOPE (No. 1, particle size 379.9 nm) had a total radiation intensity in mice that was 2.4 times that of No. 2, and a total radiation intensity in the spleen that was 3.8 times that of No. 2. This indicates that the difference in in vivo imaging between the two compositions is mainly due to the addition of ADOPE, and that particle size has no significant effect. In other words, the composition prepared with the addition of permanent anionic lipid significantly improved the protein expression level of the antigen in the spleen, demonstrating a good and significant spleen targeting effect.

[0241] [Table 12]

[0242] The results were analyzed as follows: As shown in Table 12 and Figures 2a and 2b, when the particle sizes were comparable, the percentage of eGFP-positive cells in antigen-presenting cells such as B cells, pDC cells, cDC cells, and macrophages was 4.8-fold, 2.4-fold, 3.7-fold, and 2.3-fold higher in the LPX-RNA composition containing the permanent anionic lipid ADOPE (No. 1, particle size 365.8 nm) than in the LPX-RNA composition not containing an anionic lipid (No. 2, particle size 418.8 nm), respectively.

[0243] Experimental results show that the addition of permanent anionic lipids (e.g., ADOPE) can significantly increase the percentage of antigen-expressing antigen-presenting cells (e.g., B cells, pDC cells, cDC cells, macrophages).

[0244] In summary, the above results further demonstrate that mRNA compositions containing permanent anionic lipids designed in the present invention can be concentrated in the mouse spleen, and the delivered mRNA significantly increases the protein expression level in the mouse spleen compared to compositions prepared without the addition of anionic lipids or conventional delivery techniques, and can significantly increase the percentage of antigen-expressing cells (e.g., B cells, pDC cells, cDC cells, macrophages) that express the antigen.

[0245] Example 9: Stimulation of IFN-α cytokine production by RNA compositions containing ADOPE Interferon α (IFN-α) is an important cytokine that plays a key role in the immune system. It has various functions, including antiviral, antitumor, hematopoietic cell proliferation inhibition, and immunomodulation, and has therapeutic effects on various diseases. IFN-α is generally produced by APC cells when infected with RNA viruses, in response to the detection of double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA) by endosomal TLR3 and TLR7, respectively, and is crucial for an effective anti-inflammatory and anti-viral environment.

[0246] C57BL / 6J mice were injected via the tail vein with 40 μg of LPX-HA mRNA (mRNA vaccine for influenza prevention) prepared according to Example 2 or 40 μg of an HA mRNA composition containing ADOPE. Blood was then collected 6 and 24 hours later, and the IFN-α content in the serum was measured by ELISA. Parallel groups were set up with three mice per group, and mice injected with an equal amount of blank lipid solution were also set up as a blank group.

[0247] The animals used were C57BL / 6 mice provided by Jackson Laboratory. Qualified female mice aged 8–10 weeks were used throughout the experimental process.

[0248] For ELISA assays, mouse IFN-α (PBL) was measured in mouse serum using a standard ELISA according to the manufacturer's instructions.

[0249] [Table 13]

[0250] The results were analyzed as follows: As shown in Table 13 and Figure 3, when the particle sizes were comparable, the compositions containing the permanent anionic lipid ADOPE (No. 2, particle size 325.7 nm) and (No. 3, particle size 352.5 nm) had mean serum IFN-α cytokine levels 1.9-fold and 3.1-fold higher than those of the LPX-RNA composition without anionic lipid (No. 4, particle size 384.1 nm) at 6 hours after injection, and 1.8-fold and 3.8-fold higher than those of the LPX-RNA composition without anionic lipid (No. 4) at 24 hours after injection.

[0251] As is clear from the experimental results, by measuring the stimulated production of IFN-α cytokine by a composition containing ADOPE, it was proven that the composition of the present invention can initiate a powerful immune stimulation program driven by type I IFN, and that the stimulated production was significantly higher than that of LPX-RNA.

[0252] Example 10: Stimulation of antigen-specific cytotoxic T cells by an RNA composition containing ADOPE The potency of tumor mRNA-lipoplexes on T cells was determined by serum antigen-specific CD8 + Cytotoxic T cells (CD8 + T cells), which is crucial for the antitumor effect of tumor mRNA delivered by lipid complexes.

[0253] C57BL / 6J mice were administered a vaccine (40 μg) containing LPX-ovalbumin (OVA) mRNA or ADOPE prepared according to Example 2 via tail vein injection on days 0, 3, and 8, respectively (each injection contained 40 μg of the therapeutic agent OVA-mA). At the same time, mice were administered the same amount of diluted blank lipid solution as the control group, and parallel groups were set up with 3 mice per group.

[0254] On day 13 (day 5 after the third immunization), approximately 200 μL of whole blood was collected and analyzed by flow cytometry to detect OVA antigen-specific CD8 + CD8 on T cells + The percentage of T cells in the total population was measured. The specific procedure was as follows. 1. 100 μL of whole blood was taken into a flow cytometry tube (a blank control was required). 2. 100 μL of surface antibody premix was added, mixed uniformly by pipetting, and incubated in the dark at room temperature for 15 minutes. The components of the premix are as follows, and when preparing, add them in the order shown in Table 14.

[0255] [Table 14] 3. Add 2 mL of 1x RBC Lysis Buffer, leave in the dark for 10 minutes, centrifuge at 500 g for 5 minutes, and discard the supernatant. 4. 2 mL of 1x RBC Lysis Buffer was added, the mixture was centrifuged at 500 g for 5 minutes, and the supernatant was discarded. 5. Add 200 μL of PBS, transfer to a clean numbered EP tube, load and detect. Before loading, correct with one-color microspheres. The detection order was as follows: CD3 + →CD8 + →APC anti-mouse H-2Kb bound to SIINFEKL

[0256] [Table 15]

[0257] The results were analyzed as follows. As shown in Table 15 and Figure 4, when the particle sizes were the same, the compositions containing the permanent anionic lipid ADOPE (No. 2, particle size 333.1 nm) and (No. 3, particle size 356.3 nm) had a significantly higher serum OVA antigen-specific CD8 activity on day 13 after injection than the LPX-RNA composition without an anionic lipid (No. 4, particle size 373.4 nm). + CD8 on T cells + The average percentage of total T cells was approximately 1.5-fold and 1.8-fold higher than that of the LPX-RNA composition (No. 4, particle size 373.4 nm) that did not contain anionic lipids.

[0258] As is clear from the experimental results, by measuring the stimulated production of antigen-specific cytotoxic T cells by a composition containing ADOPE, it was proven that the composition of the present invention has a very strong effect on T cells, and that the stimulated production was significantly higher than that of LPX-RNA.

[0259] Example 11: Therapeutic effect of a composition containing ADOPE in a tumor-bearing mouse model 1) Establishment of the B16F10-OVA melanoma mouse model. Before the study began, 6- to 8-week-old female C57BL / 6J mice were allowed to adapt for at least 3 days. Mice were provided with free access to food and sterile water and housed at 22°C ± 2°C and 55% ± 15% relative humidity under a 12-h light-dark cycle. B16F10-OVA cells were cultured in complete medium as described in the manufacturer's instructions at 37°C with 5% CO2. Cells were harvested with 0.25% trypsin-EDTA, resuspended in Dulbecco's phosphate-buffered saline (DPBS), and cultured at 2 × 10 5 Establish a subcutaneous B16F10-OVA tumor model by subcutaneously (SC) implanting cells into the lateral chest of female C57BL / 6J mice at 100 cells / 100 µL / mouse, with a tumor volume of 100 mm. 3 Once the level reached a certain level, vaccinations began.

[0260] 2) Vaccination. The C57BL / 6J mice were administered the LPX-OVA mRNA or ADOPE-containing OVA mRNA composition (40 μg) prepared according to Example 3 via tail vein injection on days 10, 13, and 17 after cell injection (each injection contained 40 μg of the therapeutic agent mRNA-OVA vaccine). At the same time, mice were administered with an equal amount of blank lipid solution as a control group, and parallel groups were set up with 8 mice per group.

[0261] [Table 16]

[0262] 3) Measurement of tumor size and mouse survival rate. Starting from day 7 after tumor inoculation, tumor diameter was measured three times a week. Formula: V (mm 3 )=x×y 2 The tumor volume of the C57BL / 6J mice was calculated using a 2-step method (unit: mm), where V represents the tumor volume, x represents the long diameter of the tumor, and y represents the short diameter of the tumor. At the same time, the weight changes of the C57BL / 6J mice were recorded three times a week using an electronic balance. When the tumor volume reached 2000 mm, the tumor volume was increased to 2000 mm. 3 If the number exceeded 100, euthanasia was performed on surviving animals, and the survival rate was calculated.

[0263] The results were analyzed as follows: As shown in Figure 5 and Table 17, B16F10-OVA melanoma cells were subcutaneously inoculated on day 0, and the vaccine was administered on days 10, 13, and 17 after tumor inoculation. On day 10 after tumor inoculation, all mice in each group entered a rapid tumor growth phase. From day 14, tumor growth was significantly delayed in the ADOPE-containing OVA mRNA composition groups (numbers 2 and 3) compared with the blank lipid control group (number 1), and tumor size was significantly smaller than that of the blank lipid control group. From day 19, tumor volume in the ADOPE-containing OVA mRNA composition groups (numbers 2 and 3) showed stronger tumor growth inhibition than that of the LPX-OVA mRNA group (number 4).

[0264] [Table 17]

[0265] As shown in Figure 6 and Table 18, in the blank lipid control group, animals began to die 15 days after tumor inoculation, and all had died by the 17th day; in the LPX-OVA mRNA control group (No. 4), animals began to die 19 days after tumor inoculation, and all had died by the 28th day; and in the OVA mRNA compositions containing ADOPE (Nos. 2 and 3), animals began to die from the 21st day, and all had died by the 31st and 33rd days, respectively.

[0266] [Table 18]

[0267] As can be seen from the results in Tables 17 and 18, compared to the blank lipid control group and the LPX-RNA composition not containing anionic lipids, certain compositions of the present invention containing permanent anionic lipids can significantly enhance tumor growth inhibition and improve mouse survival rates.

[0268] The conclusions are as follows: The present invention provides a composition comprising RNA encoding one or more antigens as a therapeutic agent and a lipid composition, wherein the lipid composition comprises permanent anionic lipids, permanent cationic lipids, and neutral lipids. The composition prepared by adding permanent anionic lipids: [1] has good particle size and uniform particle distribution; [2] can significantly improve the protein expression level of antigens in the spleen; and [3] significantly increases the percentage of antigen-expressing cells in the spleen (e.g., B cells, pDC cells, cDC cells, macrophages). Specifically, I. By adding a permanent anionic lipid containing a phosphate group, a composition can be produced with good particle size (particle size limited to 240-500 nm) and uniform particle distribution (PDI<0.5).

[0269] 1. By adding permanent anionic lipids, especially those containing a phosphate group such as ADOPE, 18PA, DOPG, tetradecylphosphonic acid, farnesyl pyrophosphate, γ,γ-dimethylallyl pyrophosphate, and pA(2'-OMe)mpG, the compositions prepared all have good particle size and PDI, with ADOPE being the best, with a particle size of 303.1 nm and a PDI reduced to 0.2455, and good particle uniformity.

[0270] 2. Conversely, compositions utilizing other anionic lipids that do not contain phosphate groups, such as oleic acid, sodium bis(laureth-7) citrate, or sodium lauryl sulfonate, are unsuitable as mRNA delivery carriers because of their large particle size (over 1000 nm) and the resulting solid precipitates.

[0271] II. The composition containing the permanent anionic lipid, permanent cationic lipid, and neutral lipid designed in the present invention can significantly improve the protein expression level of antigens in the spleen (corresponding to the total radiation intensity), particularly by limiting the molar percentages of ADOPE, DOTMA, and DOPE to (14 to 33 mol%):(40 to 57 mol%):(22 to 40 mol%) or the charge ratio to 1:2 to 1:5. 1. Mouse in vivo imaging experiments showed that the protein expression level of the antigen delivered by the composition containing the permanent anionic lipid designed in the present invention in the spleen was significantly higher than that in other organs (e.g., liver, lung), and the total radiation intensity of the expressed protein in the spleen of Fluc-mRNA delivered by the prepared composition was 1.20 × 10 7 ~7.38×10 7 p / s has been reached.

[0272] 2. The protein expression level in the spleen of the antigen delivered by the composition designed according to the present invention is significantly improved compared to a composition prepared without the addition of anionic lipids.

[0273] For example, the total radiation intensity of the expressed protein in the spleen of Fluc-mRNA delivered by the mRNA composition designed in the present invention was 12.10 times that of the composition prepared without adding anionic lipids.

[0274] 3. Compared to prior art LNPs, the protein expression levels in the spleen of the antigens delivered by the compositions designed in the present invention are significantly higher than those of prior art LNPs.

[0275] For example, the total radiation intensity of the expressed protein in the spleen of the composition designed in the present invention is 9.46 times that of YK-009-mRNA-LNP, a conventional LNP, and 7.10 times that of YK-407-mRNA-LNP.

[0276] III. The compositions designed in the present invention significantly increase the percentage of antigen-expressing antigen-presenting cells (eg, B cells, pDC cells, cDC cells, macrophages) in the spleen. 1. Mouse spleen cell flow cytometry experiments show that compositions containing permanent anionic lipids designed in the present invention significantly increase the percentage of antigen-expressing antigen-presenting cells (e.g., B cells, pDC cells, cDC cells, macrophages) in the spleen.

[0277] For example, among antigen-presenting cells, the percentage of B cells is about 0.1 to 0.4%, the percentage of pDC cells is about 2 to 6%, the percentage of cDC cells is about 2 to 9%, and the percentage of macrophages is about 2.4 to 7.5%.

[0278] 2. Compared to compositions prepared without the addition of anionic lipids, the compositions designed according to the present invention significantly increase the percentage of antigen-expressing antigen-presenting cells in the spleen.

[0279] For example, the percentage of cells that are B cells, pDC cells, cDC cells, and macrophages expressing the eGFP of the present invention is 19-fold, 2.8-fold, 8.1-fold, and 11.8-fold, respectively, compared to that of compositions produced without the addition of anionic lipids.

[0280] 3. The specific combination of compositions designed in the present invention significantly increases the percentage of antigen-expressing cells in the antigen-presenting cells in the spleen compared to other combinations of compositions.

[0281] For example, a particular combination composition of the present invention (permanent anionic lipid ADOPE, permanent cationic lipid DOTMA, neutral lipid DOPE) has 4.8-fold, 5.6-fold, 12.1-fold, and 11.6-fold higher percentages of cells that are eGFP-expressing B cells, pDC cells, cDC cells, and macrophages than the eGFP RNA composition pA(2'-OMe)mpG (permanent anionic lipid pA(2'-OMe)mpG, permanent cationic lipid DOTMA, neutral lipid DOPE), respectively.

[0282] IV. The specific combination compositions designed in the present invention have significantly enhanced ability to stimulate the production of IFN-α cytokine compared to LPX-RNA compositions that do not contain anionic lipids, demonstrating that the compositions of the present invention can initiate a potent immune stimulatory program driven by type I IFN.

[0283] For example, at 6 hours and 24 hours after injection, the composition of the present invention containing a permanent anionic lipid has an IFN-α cytokine content in the serum that is approximately 1.7 to 4 times and 1.5 to 5 times that of an LPX-RNA composition that does not contain anionic lipid.

[0284] V. The specific combination compositions designed in the present invention have significantly enhanced ability to stimulate antigen-specific cytotoxic T cells compared to LPX-RNA compositions that do not contain anionic lipids, demonstrating that the compositions of the present invention can produce very potent effects on T cells.

[0285] For example, on day 13 after injection of a particular composition of the present invention containing a permanent anionic lipid, serum OVA antigen-specific CD8 + CD8 on T cells +The percentage of total T cells is 1.3 to 1.9 times higher than when an LPX-RNA composition not containing anionic lipids is injected.

[0286] VI. The specific combination composition designed in this invention has been shown in animal experiments to significantly control tumor growth and extend the survival time of tumor-bearing experimental animals compared to an LPX-RNA composition that does not contain anionic lipids.

[0287] After subcutaneous inoculation of B16F10-OVA melanoma cells into mice, the rate of tumor growth was effectively slowed by injecting a specific composition of the present invention containing a permanent anionic lipid, compared to the blank lipid control group and the control group administered an LPX-RNA composition not containing anionic lipid, with a clear reduction in tumor size and a significant increase in the survival rate of tumor-bearing mice injected with a specific composition of the present invention containing a permanent anionic lipid.

[0288] Although the present invention has been described in detail above, the purpose is to inform those skilled in the art of the contents of the present invention and to enable them to practice the present invention, and the scope of protection of the present invention is not limited thereby. Any equivalent modifications or alterations made based on the spirit of the present invention shall also be included in the scope of protection of the present invention.

Claims

1. 1. A lipid composition comprising: (1) a permanent anionic lipid; (2) a permanent cationic lipid; and (3) consisting of neutral lipids; provided that the molar ratio of the permanent anionic lipid, the permanent cationic lipid, and the neutral lipid in the lipid composition is 14 to 33:40 to 57:22 to 40; The permanent anionic lipid is any one or more selected from 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl)phosphate, (Z)-(R)-3-(phosphonooxy)propane-1,2-diyldiolate, 1,2-dioleoyl-sn-glycero-3-phospho-rac-glycerol, or salts thereof; The permanent cationic lipid is 1,2-di-O-octadecenyl-3-trimethylammonium propane, or a salt thereof; The neutral lipid is A lipid composition selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, or a salt thereof.

2. The lipid composition comprises: (1) 25 mol % of a permanent anionic lipid; (2) 50 mol % of a permanent cationic lipid; (3) Consists of 25 mol% neutral lipids; Or, the lipid composition is (1) 20 mol % of a permanent anionic lipid; (2) 40 mol% of a permanent cationic lipid; (3) Consists of 40 mol% neutral lipids; Or, the lipid composition is (1) 14 mol% of a permanent anionic lipid; (2) 57 mol% permanent cationic lipid; (3) Consists of 29 mol% neutral lipids; Or, the lipid composition is (1) 33 mol% permanent anionic lipid; (2) 45 mol% permanent cationic lipid; (3) The lipid composition of claim 1, consisting of 22 mol% neutral lipids.

3. 1. Use of a lipid composition in the preparation of a reagent for improving targeting to antigen-presenting cells in a target organ, comprising: The lipid composition comprises: (1) a permanent anionic lipid; (2) a permanent cationic lipid; and (3) consisting of neutral lipids; provided that the molar ratio of the permanent anionic lipid, the permanent cationic lipid, and the neutral lipid in the lipid composition is 14 to 33:40 to 57:22 to 40; The permanent anionic lipid is any one or more selected from 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl)phosphate, (Z)-(R)-3-(phosphonooxy)propane-1,2-diyldiolate, 1,2-dioleoyl-sn-glycero-3-phospho-rac-glycerol, or salts thereof; The permanent cationic lipid is 1,2-di-O-octadecenyl-3-trimethylammonium propane, or a salt thereof; The neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, or a salt thereof; the target organ is the spleen; The antigen-presenting cells comprise any one or more of dendritic cells, macrophages, or B cells.

4. 1. A composition comprising: (A) a therapeutic and / or prophylactic agent comprising one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein; (B) the lipid composition according to claim 1 or 2, is used to deliver the therapeutic agent and / or prophylactic agent to antigen-presenting cells in a target organ, the target organ is the spleen; The composition, wherein the antigen-presenting cells comprise one or more of dendritic cells, macrophages, or B cells.

5. The composition of claim 4 , wherein the therapeutic and / or prophylactic agent is a nucleic acid molecule capable of encoding one or more antigens.

6. the nucleic acid molecule is capable of eliciting an immune response against a disease-associated antigen; or the nucleic acid molecule is capable of eliciting an immune response against cells expressing a disease-associated antigen; Alternatively, the composition according to claim 4 or 5, wherein the nucleic acid molecule is RNA encoding one or more antigens.

7. the antigen is a disease-associated antigen; or the antigen is capable of eliciting an immune response against a disease-associated antigen; Alternatively, the antigen is capable of eliciting an immune response against cells expressing a disease-associated antigen.

8. The composition according to claim 4, wherein the therapeutic and / or prophylactic agent and lipid composition are used in amounts such that the charge ratio of net positive charges to net negative charges in the composition is 1:2 to 1:

5.

9. the composition has a net positive to negative charge ratio of 1:2; or the charge ratio of net positive to negative charges in the composition is 2:5; or the composition has a net positive to negative charge ratio of 1:3; Alternatively, the composition of claim 4 or 8, wherein the charge ratio of net positive to negative charges in the composition is 1:

5.

10. The composition further comprises at least one adjuvant; and / or the composition further comprises one or more excipients; and / or the composition further comprises one or more hydrophobic small molecules, permeability enhancing molecules, carbohydrates, polymers, surface-altering agents, or cytokines; Alternatively, the composition further comprises one or more adjuvants; Alternatively, the composition further comprises one or more pharmaceutically acceptable carriers; Alternatively, the composition of claim 4, wherein the composition further comprises one or more diluents.

11. 1. A method for producing a composition for delivering a therapeutic and / or prophylactic agent to antigen-presenting cells in a target organ, comprising: (a) dissolving a permanent anionic lipid, a permanent cationic lipid, and a neutral lipid in an organic solvent to form a lipid solution, wherein the molar ratio of the permanent anionic lipid, the permanent cationic lipid, and the neutral lipid is 14-33:40-57:22-40; The permanent anionic lipid is any one or more selected from 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl)phosphate, (Z)-(R)-3-(phosphonooxy)propane-1,2-diyldiolate, 1,2-dioleoyl-sn-glycero-3-phospho-rac-glycerol, or salts thereof; The permanent cationic lipid is 1,2-di-O-octadecenyl-3-trimethylammonium propane, or a salt thereof; The neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, or a salt thereof; (b) mixing the lipid solution obtained in step (a) with water to obtain a lipid mixture; (c) mixing the lipid mixture obtained in step (b) with a therapeutic agent and / or a prophylactic agent to form the composition, wherein the therapeutic agent and / or prophylactic agent comprises a nucleic acid buffer solution obtained by dissolving a nucleic acid molecule in a buffer solution having a pH of 6.8 to 7.

6.

12. In step (a), the organic solvent is an alcohol solvent; and / or, in step (b), the lipid mixture can pass through a polycarbonate membrane having a pore size of 100 to 400 nm; and / or, in step (c), the buffer comprises an aqueous HEPES buffer; And / or, in step (c), the nucleic acid molecule is a nucleic acid molecule capable of encoding one or more antigens.

13. In step (a), the organic solvent comprises an alcohol having 1 to 4 carbon atoms; and / or in step (c), the buffer comprises aqueous HEPES buffer and EDTA; and / or, in step (c), the nucleic acid molecule is capable of eliciting an immune response against a disease-associated antigen; Alternatively, the method of claim 11 or 12, wherein in step (c), the nucleic acid molecule is capable of eliciting an immune response against cells expressing a disease-associated antigen.

14. In step (c), the antigen is a disease-associated antigen; or the antigen is capable of eliciting an immune response against a disease-associated antigen; Alternatively, the antigen is capable of eliciting an immune response against cells expressing a disease-associated antigen.

15. The method of claim 11, wherein in step (c), the lipid mixture and the therapeutic and / or prophylactic agent are used in amounts such that the resulting composition has a net positive to negative charge ratio of 1:2 to 1:

5.

16. Use of a lipid composition described in claim 1 or 2, a composition described in any one of claims 4, 5, 7, 8 and 10, or a composition produced by a method described in any one of claims 11, 12, 14 and 15 in the manufacture of a drug used to prevent, treat or ameliorate a disease or condition in a mammalian subject.

17. 17. The use according to claim 16, wherein the disease or condition is one or more selected from infectious diseases, cancer, proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

18. 17. The use according to claim 16, wherein the mammalian subject is one or more species selected from humans and non-human primates.

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