Lipid compositions targeting antigen-presenting cells and their use

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 belongs to the technical field of molecular biology and relates to a lipid composition targeting antigen-presenting cells and its use.

Background Art

[0002] Inducing a T cell response against a tumor by expressing a tumor antigen in an antigen-presenting cell (APC) by the encoding mRNA holds great potential in tumor immunotherapy. Negatively charged mRNA molecules cannot directly enter antigen-presenting cells, and in most conventional delivery techniques, it is generally inevitable to deliver a large amount of mRNA to the liver and lungs. For immunotherapy, since the immune response of mRNA in the lungs or liver is generally harmful, it is important to highly selectively deliver mRNA encoding a specific antigen to antigen-presenting cells (APCs) in order to succeed in mRNA immunotherapy.

[0003] The method of encapsulating mRNA using a lipid composition to produce a lipid-RNA composition is one of the commonly used mRNA cell introduction methods in mRNA immunotherapy. Currently, the main lipid systems used in lipid-RNA compositions developed by researchers are as follows. 1. Lipid nanoparticles (Lipid Nanoparticle, LNP) are spherical vesicles composed of one (single layer) or multiple (multilayer) phospholipid bilayers. The LNP delivery technique is to encapsulate mRNA into nanoliposome particles composed of four components: a cationic lipid (e.g., DLin-MC3-DMA, C12-200), a neutral lipid (e.g., DSPC, DOPE), a structural lipid (e.g., cholesterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000). Among them, the cationic lipid includes a permanently cationic lipid (containing a quaternary ammonium group) and an ionizable cationic lipid (containing a primary amine group, a secondary amine group, or a tertiary amine group). Since using a permanently cationic lipid to produce LNP enhances the toxicity to cells, an ionizable cationic lipid is used instead.

[0004] Adding a fifth lipid (selective organ targeting (SORT) lipid) to conventional four-component liver-targeting lipid nanoparticles enables organ targeting of mRNA drugs. For example, CN112996519A describes a composition comprising a therapeutic agent and a lipid nanoparticle composition, of which the lipid nanoparticle composition comprises [1] cationic ionizable lipids, phospholipids (i.e., neutral lipids), steroids (i.e., structural lipids such as cholesterol), pegylated lipids (i.e., polymer-bound lipids such as dimyristoyl-sn-glycerol), and permanent cationic lipids (i.e., liver-targeting SORT lipids), or [2] cationic ionizable lipids, phospholipids (i.e., neutral lipids), steroids (i.e., structural lipids such as cholesterol), pegylated lipids (i.e., polymer-bound lipids such as dimyristoyl-sn-glycerol), and permanent anionic lipids (i.e., spleen-targeting SORT lipids), and further comprises the apparent pK of the lipid nanoparticle composition containing the selective spleen-targeting compound. a By changing this, the targeting effect of the manufactured composition formulation on different organs is adjusted.

[0005] 2. Lipopolyplexes (LPPs) are bilayer structures consisting of a polymer-encapsulated mRNA core and a phospholipid capsule shell. Negatively charged mRNA aggregates inside the positively charged polymer, forming a dense polyplex "core" structure with a diameter of several nanometers to several hundred nanometers. The bilayer nanostructure of LPPs has a better effect on encapsulating and protecting mRNA compared to conventional LNPs, and the mRNA molecule can be gradually released as the polymer degrades. Furthermore, its excellent dendritic cell targeting effect allows for better activation of the T cell immune response through antigen presentation, thereby achieving an ideal immunotherapy effect.

[0006] For example, CN115845040A describes a lipopolyplex mRNA vaccine consisting of a poly-(β-aminoester) 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 the aforementioned shell / core-mRNA vaccine, the polyplex "core" is encapsulated by a hydrophilic phospholipid bilayer "shell," which enhances uptake by dendritic cells and protects the mRNA molecules within the core from degradation by intracellular nucleases. Furthermore, as an effective approach to enhance the uptake of vaccine particles by dendritic cells, it has also been described that affinity moieties (e.g., sugar moieties such as mannose, binding proteins, or antibodies specific to one or more DC-expressed epitopes) may be conjugated onto the surface of the lipid shell of the mRNA vaccine to "functionalize" the vaccine particles by enhancing the interaction between the vaccine particles and the antigen-presenting cells (dendritic cells, macrophages, B cells, etc.) targeted by the vaccine core / shell complex, and / or increasing the binding between them.

[0007] 3. Lipoplexes (LPXs) are complex delivery carriers developed by BioNTech, consisting of cationic lipids and neutral helper lipids, in which mRNA molecules are embedded between bilayer lipids. For example, CN109331176A discloses that they may be formed from permanently cationic (positively charged) liposomes and anionic (negatively charged) nucleic acids, and that neutral lipids may be added as helper lipids. By optimizing the ratio of lipids to RNA in RNA-lipoplexes (RNA-LPXs) and adjusting the net charge of the formulation composition to be neutral or negative, it is possible to accurately target dendritic cells (DC cells) when administered intravenously without the need for targeting improvements to nanoparticles.

[0008] In its simplest form, lipoplexes are spontaneously formed by mixing nucleic acids and liposomes in a specific mixing protocol, although various other protocols may be applied. Electrostatic interactions between positively charged liposomes and negatively charged nucleic acids are the driving force behind lipoplex formation. Besides the lipid composition, the charge ratio between cationic and anionic sites plays a crucial role for efficient condensation and transfection. Generally, an excess positive charge in the lipoplex is considered 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). Since most natural membranes are negatively charged, electrostatic interactions that create an attractive force between positively charged lipoplexes and negatively charged biological membranes can play a crucial role in lipoplex cell binding and uptake. Conversely, with lower excess positive charge, the effectiveness of transfection dramatically drops 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 lipoplex described above has been proven to be transfectable in various organs. The distribution of its expression in specific organs depends on many parameters, including the formulation and administration parameters (lipid composition, size, route of administration). To date, selective expression in designated target organs or cell sites while avoiding expression in non-target organs has not been fully achieved. Transfection in organs such as the lungs, liver, spleen, kidneys, and heart has been reported using luciferase DNA or RNA as a reporter. Avoiding targeting of the lungs and liver is particularly difficult, as targeting of these organs is predominant in many cases. The lungs have a very large surface area and are the first organ through which intravenously administered (iv) compounds pass. The liver is a typical target organ for formulations containing lipophilic compounds, such as liposomes and lipids present in lipoplexes. For RNA-based immunotherapy, targeting the lungs or liver may be harmful due to the risk of immune responses in these organs. Therefore, such therapies require formulations that exhibit high selectivity only to dendritic cells (DCs) in the spleen, for example.

[0010] Certain ligands are thought to improve targeting selectivity. For example, lipids modified by conjugating them with mannose, for instance, are thought to significantly improve their targeting of macrophages. However, when modifying lipids by conjugation, it is necessary to consider interactions in serum and the degradation of RNA in serum. This makes such components more complex in formulations, making actual drug development even more difficult.

[0011] Furthermore, the incubation of RNA with cationic liposomes commonly results in the formation of large aggregates. This aggregate formation significantly increases particle size and reduces stability, which is one of the main obstacles to developing formulations acceptable 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 called receptor-targeted nanocomplexes (RTNs), which consist of cations and anions. This formulation includes 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 siRNA encapsulation and receptor-mediated uptake into cells. When RTNs are intravenously injected into mice, they accumulate mainly in tumor xenografts and are hardly detectable in the liver, lungs, or spleen, demonstrating that specific tumor targeting can be achieved with RTN formulations. Furthermore, because liver clearance is very low, tumor therapy with siRNA-targeted drugs can be realized (see abstract and right column on page 9).

[0013] In "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, 532-542, the study examines factors influencing the induction of IL-1 cytokines, such as monocyte count, inflammasome, and aspartase activity, using an RNA-LPX vaccine encoding a TLR7 / 8 agonist (where the lipid composition of the RNA-LPX includes 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.

[0014] In "Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy," Lena M. Kranz et al., NATURE, volume 534, 396-401 (2016), RNA-LPX containing cationic liposomes composed of DOTMA and DOPE is disclosed, and the effect of positive and negative charges on targeting of antigen-presenting cells in the body is studied by changing the ratio of lipids to RNA. When RNA-LPX is charged with a small amount of positive charge or is close to neutral (positive-negative charge ratio of 2.5:1 to 1.8:2), the RNA-LPX produced is unstable and immediately 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, and as the cationic lipid content decreases, protein expression shifts from the lungs to the spleen. In particles that are nearly neutral or negatively charged (with a charge ratio of 1.7:2 or less), protein expression is transferred to the spleen. Furthermore, it is noted that transfection efficiency gradually decreases as the negative charge increases, which may be a result of an increase in free RNA (see the right column on page 396).

[0015] Given the current limitations of the conventional technologies described above, and the advancements in large-scale cancer genome sequencing and immunogenic tumor mutation prediction, new tumor-related antigens are being identified one after another, presenting an unprecedented opportunity for the development of new and improved vaccines for tumor treatment. There is an urgent need to provide injectable RNA formulations that can guarantee compliance with standards for products administered to patients, as a new method for highly selectively delivering mRNA to antigen-presenting cells. [Disclosure of the Invention] [Problems that the invention aims to solve]

[0016] This invention provides a lipid composition that targets antigen-presenting cells and its use.

[0017] The composition provided by the present invention, upon systemic administration, exhibits high RNA expression in the spleen (particularly within antigen-presenting cells) and low expression in other organs (e.g., liver, lungs), resulting in a favorable targeting effect. Furthermore, administration of the composition induces a strong immune response to the antigen. Generally, excessive positive charge is considered a prerequisite for successful uptake and expression, but the composition having the charge ratio of the present invention remains highly expressed after systemic administration, still providing the therapeutic effect of lipoplex. [Means for solving the problem]

[0018] To achieve the above objectives, the present invention, in a first embodiment, (1) Permanent anionic lipids, (2) Permanent cationic lipids, (3) Provide a lipid composition containing neutral lipids, However, the molar ratio of permanent anionic lipids, permanent cationic lipids, and neutral lipids in the lipid composition is 14-33:40-57:22-40.

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

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

[0021] According to a preferred embodiment of the present invention, the neutral lipid contains 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 substance is 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, and salts thereof.

[0023] According to a preferred embodiment of the present invention, the permanent cationic lipid is It is one or more selected from 1,2-di-O-octadecenyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium propane, and their salts.

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

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

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

[0027] According to the present invention, the lipid composition is the lipid composition described in the first embodiment, and its characteristics have been described above, so a detailed explanation is omitted here.

[0028] In a third aspect, the present invention is as follows: (A) A therapeutic and / or prophylactic agent comprising one or more nucleic acid molecules, small molecule compounds, polypeptides, or proteins, (B) A composition comprising the lipid composition described in the first embodiment is provided, The composition is used to deliver the therapeutic and / or prophylactic agent to antigen-presenting cells in a target organ.

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

[0030] According to a preferred embodiment of the present invention, the antigen is a disease-related antigen, or the nucleic acid molecule or antigen can induce an immune response against a disease-related antigen or cells expressing a disease-related antigen.

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

[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 one or more of dendritic cells, macrophages, and B cells.

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

[0035] According to a preferred embodiment of the present invention, the lipid composition, (1) 14-33 mol% permanent anionic lipids, (2) 40-57 mol% permanent cationic lipids, (3) Contains 22-40 mol% 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-trimethylammoniumpropane and / or a salt thereof, and the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or a salt thereof. The net charge ratio of positive charge to negative charge in the above composition is 1:2 to 1:5.

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

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

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

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

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

[0041] In a fourth embodiment, the present invention provides a method for producing a composition for delivering a therapeutic agent and / or prophylactic agent to antigen-presenting cells in a target organ, the features of which the method is: (a) A step of 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) A step of mixing the lipid solution obtained in step (a) with water to obtain a lipid mixture, (c) A step of mixing the lipid mixture obtained in step (b) with a therapeutic agent and / or prophylactic agent to form the composition, wherein the therapeutic agent and / or prophylactic agent comprises a nucleic acid buffer solution obtained by dissolving nucleic acid molecules in a buffer solution with 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 having a pore size of 100 to 400 nm.

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

[0045] According to a preferred embodiment of the present invention, in step (c), the 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 aqueous HEPES buffer and EDTA.

[0048] According to a preferred embodiment of the present invention, in step (c), the antigen is a disease-related antigen, or the nucleic acid molecule or antigen can induce an immune response against a disease-related antigen or a cell expressing a disease-related antigen.

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

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

[0051] According to a preferred embodiment of the present invention, the neutral lipid contains 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 substance is 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, and salts thereof.

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

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

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

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

[0057] In a sixth aspect, the present invention provides a use in the manufacture of a drug, a lipid composition according to the second aspect, a composition according to the third aspect, or a composition manufactured according to the method according to the fourth aspect, the drug being used to prevent, diagnose, treat or improve a disease, condition, abnormal state or dysfunction in a mammalian subject.

[0058] According to preferred embodiments of the present invention, the disease, condition, abnormal state, or dysfunction is one or more selected from infectious diseases, cancer, proliferative disorders, genetic disorders, autoimmune diseases, neurodegenerative diseases, cardiovascular diseases, renovascular 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, pets, exotic species, livestock, and food animals.

[0060] According to several preferred embodiments of the present invention, compositions having appropriate particle size distribution characteristics can be formed that can satisfy the requirements for intravenous administration to patients, and these compositions are formed by incubation of lipid compositions with RNA by self-assembly / self-combination. As a result, the compositions of the present invention satisfy 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 permanent anionic lipids, has at least the following advantages: [1] It has good particle size and uniform particle distribution. [2] It can significantly improve the protein expression level of the antigen in the spleen. [3] It significantly increases the percentage of cells expressing the antigen among antigen-presenting cells (e.g., B cells, pDC cells, cDC cells, macrophages) in the spleen. Specifically, I. By adding a permanent anionic lipid containing a phosphate group, a composition can be produced that has good particle size (limited to particle size of 240-500 nm) and uniform particle distribution (PDI < 0.5).

[0062] 1. Compositions produced by adding permanent anionic lipids, particularly those containing phosphate groups such as ADOPE, 18PA, DOPG, tetradecylphosphonic acid, farnesyl pyrophosphate, γ,γ-dimethylallyl pyrophosphate, and pA(2'-OMe)mpG, all exhibit good particle size and PDI. Among these, ADOPE is the best, with a particle size of 303.1 nm, a PDI reduced to 0.2455, and 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, and sodium lauryl sulfonate, are unsuitable as mRNA delivery carriers because their particle size exceeds 1000 nm, and solid precipitates form.

[0064] II. Compositions comprising permanent anionic lipids, permanent cationic lipids, and neutral lipids designed in the present invention can significantly improve the protein expression level of antigens in the spleen (corresponding to total radiation intensity), particularly by limiting their mole percentages to (14-33 mol%):(40-57 mol%):(22-40 mol%) and / or the charge ratio to 1:2-1:5. 1. In vivo contrast-enhanced experiments in mice have shown that the protein expression level of antigens delivered by compositions containing permanent anionic lipids designed in this invention is significantly higher in the spleen than in other organs (e.g., liver, lungs), and the total radiance intensity of the protein expressed in the spleen by Fluc-mRNA delivered by the manufactured composition was 1.20 × 10⁻⁶. 7 ~7.38×10 7 It has reached p / s.

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

[0066] For example, the total radiant intensity of the Fluc-mRNA expressed in the spleen by the mRNA composition designed in the present invention is 12.1 times higher than that of the composition without added anionic lipids.

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

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

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

[0070] For example, among antigen-presenting cells, B cells make up approximately 0.1-0.4%, pDC cells approximately 2-6%, cDC cells approximately 2-9%, and macrophages approximately 2.4-7.5%.

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

[0072] For example, when the composition provided by the present invention is administered, the percentage of cells that express eGFP, such as B cells, pDC cells, cDC cells, and macrophages, is 19 times, 2.8 times, 8.1 times, and 11.8 times, respectively, compared to when the composition without added anionic lipids is administered.

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

[0074] For example, when a specific combination of compositions of the present invention (permanent anionic lipid ADOPE, permanent cationic lipid DOTMA, neutral lipid DOPE) is administered, the percentage of cells that express eGFP, such as B cells, pDC cells, cDC cells, and macrophages, is 4.8 times, 5.6 times, 12.1 times, and 11.6 times, respectively, compared to when the pA(2'-OMe)mpG eGFP RNA composition (permanent anionic lipid pA(2'-OMe)mpG, permanent cationic lipid DOTMA, neutral lipid DOPE) is administered.

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

[0076] For example, when the composition of the present invention containing permanent anionic lipids is administered 6 hours and 24 hours after injection, respectively, the serum IFN-α cytokine content is approximately 1.7 to 4 times and 1.5 to 5 times higher than when the LPX-RNA composition without anionic lipids is administered.

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

[0078] For example, administering a specific composition of the present invention containing permanent anionic lipids on the 13th day after injection is used to target OVA antigen-specific CD8 in serum. + T cells are CD8 + The percentage of T cells in the overall population is 1.3 to 1.9 times higher when an LPX-RNA composition without anionic lipids is administered.

[0079] VI. The specific combinations of compositions designed in the present invention have shown in animal experiments to clearly control tumor growth and extend the survival period of tumor-bearing experimental animals compared to LPX-RNA compositions that do not contain anionic lipids.

[0080] For example, after subcutaneous inoculation of B16F10-OVA melanoma cells into mice, compared to a blank lipid control group and a control group administered an LPX-RNA composition without anionic lipids, injecting a specific composition of the present invention containing permanent anionic lipids effectively slowed the rate of tumor growth, resulting in a clear reduction in tumor size. Furthermore, the survival rate of tumor-bearing mice injected with the specific composition of the present invention containing permanent anionic lipids was significantly increased. [Brief explanation of the drawing]

[0081] To more clearly explain 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 shown in the following description relate to some specific embodiments of the present invention and do not constitute limitations on the present invention. [Figure 1a] Figure 1a shows fluorescence images of mice and mouse organs (liver, spleen, lungs) containing a composition that includes Fluc-mRNA corresponding to item 6 in Table 8. [Figure 1b] Figure 1b shows fluorescence images of mice and mouse organs (liver, spleen, lungs) containing a composition that includes Fluc-mRNA corresponding to item 7 in Table 8. [Figure 1c] Figure 1c shows fluorescence images of mice and mouse organs (liver, spleen, lungs) containing a composition that includes Fluc-mRNA corresponding to item 8 in Table 8. [Figure 1d] Figure 1 shows fluorescence images of mice and mouse organs (liver, spleen, lungs) containing a composition that includes Fluc-mRNA corresponding to item 17 in Table 8. [Figure 2a]Figure 2a shows mouse spleen flow cytometry experiments with compositions containing small-particle LPX (number 13 in Table 10) that do not contain anionic lipids, large-particle LPX (number 2 in Table 12) that do not contain anionic lipids, and eGFP-mRNA corresponding to number 1 in Table 12. [Figure 2b] Figure 2b shows mouse spleen flow cytometry experiments with compositions containing small-particle LPX (number 13 in Table 10) that do not contain anionic lipids, large-particle LPX (number 2 in Table 12) that do not contain anionic lipids, and eGFP-mRNA corresponding to number 1 in Table 12. [Figure 3] Figure 3 compares the content of stimulation-produced IFN-α cytokines at different time points for different compositions in groups 1-4 of Table 13. [Figure 4] Figure 4 compares the stimulation and production of antigen-specific cytotoxic T cells 13 days after injection of the different compositions in groups 1-4 of Table 15. [Figure 5] Figure 5 shows a comparison of the changes in tumor volume in tumor-bearing mice after injection of different compositions from groups 1 to 4 in Table 16. [Figure 6] Figure 6 compares the survival of tumor-bearing mice after injecting them with different compositions from groups 1 to 4 in Table 16. [Modes for carrying out the invention]

[0082] The following describes the technical solutions of the present invention more clearly and completely, in conjunction with the drawings of the present invention, so that the object, technical solutions, and advantages of the present invention may be more clearly understood. Needless to say, the specific embodiments described are not all embodiments, but only some embodiments of the present invention. Any other embodiments that a person skilled in the art may obtain without performing an inventive step based on the embodiments of the present invention described are all within the scope of protection of the present invention.

[0083] The present invention may be implemented in other specific forms without departing from the spirit of the invention. It should be understood that, to the extent that no inconsistency arises, other embodiments can be obtained from any or all embodiments of the present invention in combination with the technical features of any or more other embodiments. The present invention includes other embodiments obtained from such combinations.

[0084] All publications and patents referenced herein are incorporated collectively by reference. If any use or terminology in any of the incorporated publications or patents conflicts with the use or terminology in this invention, the use and terminology of this invention shall prevail.

[0085] The section headings described in this invention are for the purpose of constructing the specification and should not be understood as limitations on the subject matter.

[0086] Unless otherwise specified, all technical and scientific terms used in this invention have their common meanings in the art to which the subject matter being protected belongs. Where there are multiple definitions for a particular term, the definitions used in this invention shall prevail.

[0087] Unless otherwise indicated in the examples or otherwise specified, all numerical values ​​in the specification and claims, such as those for dosages, should be understood to be modified in all cases by the term "approximately." Furthermore, it should be understood that all numerical ranges enumerated in this invention are intended to include all subranges within that range and any combination of endpoints within that range or subrange.

[0088] Similar words used in this invention, such as "contains" or "includes," mean that the element preceding the word covers the elements and their equivalents listed after it, and that elements not listed are not excluded. The terms "contains" or "includes" used in this invention may be open, semi-closed, or closed. In other words, the terms include "substantially consisting of" or "consisting of."

[0089] In this invention, the term "pharmaceutically acceptable" means that the compound or composition is chemically and / or toxicologically compatible with the other components of the formulation and / or with the human or mammal to whom the disease or condition is to be prevented or treated.

[0090] In this invention, the terms "subject" or "patient" include mammalian subjects. For example, the mammalian subjects may be selected from one or more species, including humans, non-human primates, pets, exotic species, livestock, and food animals.

[0091] As used in this invention, the term "treatment" refers to administering one or more drugs to a patient or subject suffering from a disease or exhibiting symptoms of the disease, thereby curing, alleviating, reducing, improving, or influencing the disease or its symptoms. In the context of this invention, unless otherwise specifically stated, the term "treatment" may include prevention.

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

[0093] Therefore, the antigen or fragment thereof should be recognized by a T cell receptor. Preferably, if recognized by a T cell receptor, the antigen or fragment can induce clonal proliferation of T cells carrying a T cell receptor that specifically recognizes the antigen or fragment, in the presence of appropriate costimulatory signals. In the context of embodiments of the present invention, the antigen or fragment is preferably presented by cells in an environment with MHC molecules, preferably by antigen-presenting cells and / or pathogenic cells, which leads to an immune response against the antigen or cells expressing the antigen.

[0094] According to the present invention, any suitable antigen can be assumed as a candidate for use in an immune response, provided that the immune response is preferably a cellular immune response.

[0095] The antigen is preferably one that corresponds to or is derived from a naturally occurring antigen species. The naturally occurring antigen may include or be derived from allergens, viruses, bacteria, fungi, parasites, other sources of infection and pathogens, or the antigen may be a tumor antigen. According to the present invention, the antigen may correspond to a naturally occurring substance species, for example, a viral protein or a part thereof.

[0096] The term "pathogen" refers to pathogenic microorganisms such as viruses, bacteria, fungi, single-celled organisms, and parasites. 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). Examples of single-celled organisms include, but are not limited to, malaria parasites, trypanosomes, and amoebas.

[0097] The term "disease-related antigen" refers to any antigen with significant pathogenicity, and also includes "tumor antigens." According to the present invention, it is desirable to induce an immune response against a disease-related antigen, or a cell that expresses a disease-related antigen, preferably a cell that presents a disease-related antigen in an environment containing MHC molecules. Preferably, the disease-related antigen is a naturally occurring antigen. In one embodiment, the disease-related antigen is expressed within a diseased cell and preferably presented by the cell's MHC molecules.

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

[0099] The composition provided by the present invention has an average 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) The present invention provides, in some embodiments, one or more types of lipids having one or more hydrophobic components and permanent anionic groups. One of the anionic groups available for use in permanent anionic lipids is a phosphate group. The phosphate group may be a compound that is deprotonated and has a negative charge at pH 8, 9, 10, 11, 12, 13, or 14 or lower. The hydrophobic component is one or more C6-C6 24 The group may be an alkyl group or an 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 to 33 mol% of the total amount of the lipid composition (i.e., the content of the permanent anionic lipid may be 14 to 33 mol% on a basis of the total amount of the lipid composition). The composition may contain the permanent anionic lipid in an amount of about 14 mol%, about 20 mol%, about 25 mol%, or about 33 mol%, or any range comprising these amounts.

[0102] According to some preferred embodiments of the present invention, the permanent anionic lipid is The substance is 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, and their salts (e.g., sodium salts, chloride salts, etc.).

[0103] (Permanent cationic lipids) In some embodiments, the present invention provides one or more hydrophobic components and one or more lipids having permanent cationic groups. Permanent cationic lipids may contain groups having a positive charge (regardless of pH). One permanent cationic group that can be used in permanent cationic lipids is a quaternary ammonium group.

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

[0105] According to some preferred embodiments of the present invention, the permanent cationic lipid is It is one or more selected from 1,2-di-O-octadecenyl-3-trimethylammoniumpropane, (1,2-dioleoyl-3-trimethylammonium-propane), and their salts (e.g., sodium salts, chloride salts, etc.).

[0106] (neutral lipid) In this invention, neutral lipids refer to lipids that function as helpers, either having no charge or existing in the form of amphoteric ions at a given pH. These neutral lipids promote the phase transition of lipids, thereby controlling the fluidity of nanoparticles and forming a lipid bilayer structure, improving efficiency, and also influencing the specificity of target organs / target cells.

[0107] For example, the neutral lipid may include one or more of the following: phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and their derivatives.

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

[0109] The neutral lipid portion may be selected from an unspecified group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerin, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid portion may be selected from an unspecified 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-natural species, including natural species with modifications and substitutions, are also included, and such modifications and substitutions include branching, oxidation, cyclization, and alkynes. For example, phospholipids may be functionalized with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds) or crosslinked with such one or more alkynes. Under appropriate reaction conditions, copper-catalyzed cycloaddition reactions may occur when the alkynyl groups are exposed to an azide. These reactions may be used to functionalize the lipid bilayer of a composition to enhance membrane permeability or cell recognition, or to couple the composition with useful components such as a targeting or contrast-enhancing portion (e.g., a dye).

[0110] The neutral lipids available for use in these compositions are 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), and 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuxinyl-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-difytanoyl-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 following may be selected from an unspecified 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 includes DSPC. In some embodiments, the neutral lipid includes DOPE. In some embodiments, the neutral lipid includes DSPC and DOPE (simultaneously).

[0112] In some embodiments, the neutral lipids are present in an amount of about 22 to 40 mol% of the total amount of the lipid composition. The composition may contain neutral lipids in an amount of 22 mol%, 25 mol%, 29 mol%, or 40 mol%, or any range comprising these amounts.

[0113] (Therapeutic and / or preventive agents) The compositions of the present invention may comprise 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 the lipid composition or bound to 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 (such as a therapeutic and / or prophylactic agent) is 1:2 to 1:5 (i.e., the charge ratio of the net positive charge to the 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 includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Of these, nucleic acid molecules are preferred.

[0115] For example, the therapeutic and / or prophylactic agent is a vaccine or compound that can induce an immune response. Therefore, 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 can deliver therapeutic and / or prophylactic agents (as carriers) to target cells and / or target organs in mammals, the present invention provides methods for treating mammalian diseases or conditions requiring such treatment, which include administering a composition containing a therapeutic and / or prophylactic agent to a mammal and / or bringing mammalian cells into contact with the composition.

[0117] Therapeutic and / or prophylactic agents contain biologically active substances, which may alternatively be called “activators,” “active ingredients,” etc. Therapeutic and / or prophylactic agents may be substances that, upon delivery 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, the therapeutic and / or prophylactic agent is a small molecule drug used to treat a particular disease, condition, or pathological state. Examples of drugs usable in the composition include antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, streptozotocin) and antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside)Arabinoside, anthracycline, alkylating agents, platinum compounds, antimetabolites, nucleoside analogs (such as methotrexate), purines and pyrimidine analogs), antiinfective agents, local anesthetics (e.g., dibucaine, chlorpromazine), β-adrenergic receptor blockers (e.g., propranolol, timolol, labetalol), antihypertensive agents (e.g., clonidine, hydralazine), antidepressants (e.g., imipramine, amitriptyline, doxepin), antispasmodics (e.g., phenytoin), antihistamines Antibiotics (e.g., diphenhydramine, chlorpheniramine, promethazine), antibiotics / antimicrobials (e.g., gentamycin, ciprofloxacin, cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, amphotericin B) B)) This includes, but is not limited to, antiparasitic drugs, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma drugs, vitamins, sedatives, and contrast agents.

[0118] In some embodiments, therapeutic and / or prophylactic agents are cytotoxins, radioactive ions, chemotherapeutic agents, vaccines, compounds that induce an immune response, and / or other therapeutic and / or prophylactic agents. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emidine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, and dihydroxyanthracine dione. This includes, but is not limited to, dione, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids (such as maytansinol), rachelmycin (CC-1065), and their analogs or congeners. The radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate groups, 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, pertussis, tetanus, epidemic, hepatitis, and pulmonary tuberculosis), which may contain nucleic acid molecules (e.g., mRNA) encoding infectious disease-derived antigens and / or epitopes.The vaccine is a compound and formulation that induces an immune response against cancer cells, and may further include nucleic acid molecules (e.g., mRNA) encoding tumor cell-derived antigens, epitopes and / or neoepitopes. The compound that elicits the immune response may include the vaccine, 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, rashelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibro). This includes, but is not limited to, momannitol, streptozotocin, mitomycin C, cis-dichlorodiammineplatinum(II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin), doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC)), and mitotic inhibitors (e.g., vincristine, vinblastine, taxol, mytansinoids).

[0119] In other embodiments, the therapeutic and / or prophylactic agent is a protein. Therapeutic proteins available for use 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 factor, luteinizing hormone-releasing hormone (LHRH) analogues, 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 used in the present invention include, but are not limited to, one or more deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) (such as messenger mRNA (mRNA) or its hybrids, RNAi inducers, RNAi factors, siRNA, shRNA, miRNA, antisense RNA, ribozyme, catalytic DNA, RNA induced from a triple helix, aptamers, etc.). In some preferred embodiments, the therapeutic and / or prophylactic agent is RNA. The RNA available for use in the compositions and methods described in the present invention may be selected from, but is not limited to, the group consisting of shortmers, antagomirs, antisense RNAs, ribozymes, small interfering RNAs (siRNAs), asymmetric interfering RNAs (aiRNAs), microRNAs (miRNAs), Dicer substrate RNAs (dsRNAs), small hairpin RNAs (shRNAs), transfer RNAs (tRNAs), messenger RNAs (mRNAs), and mixtures thereof. In some embodiments, the RNA is mRNA.

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

[0122] In other embodiments, the therapeutic and / or prophylactic agent is siRNA. siRNA can selectively reduce or downregulate the expression of a target gene. For example, due to the selectivity of siRNA, a composition containing the siRNA may silence genes associated with a specific disease, condition, or pathological state after administration to a subject in need. siRNA may contain a sequence complementary to the mRNA sequence encoding the target gene or protein. In some embodiments, the siRNA may be immunomodulatory siRNA.

[0123] In some embodiments, the therapeutic and / or prophylactic agent is sgRNA and / or cas9 mRNA. sgRNA and / or cas9 mRNA may also be used as a tool for gene editing. For example, the sgRNA-cas9 complex affects the translation of mRNA of a cell's genes.

[0124] In some embodiments, the therapeutic and / or prophylactic agent is shRNA or a carrier or plasmid encoding it. The shRNA may be produced within the target cell after a suitable construct has been delivered into the nucleus. Constructs and mechanisms associated with shRNA are well known in the relevant fields.

[0125] (Disease or medical condition) The composition / carrier of the present invention can deliver therapeutic and / or prophylactic agents to a subject or patient. The therapeutic and / or prophylactic agent includes, but is not limited to, one or more nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Therefore, the composition of the present invention may be used to manufacture nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides, or biopharmaceuticals. As described above, there are many types of therapeutic and / or prophylactic agents, so the composition of the present invention may be used to treat or prevent various diseases or conditions.

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

[0127] The agents, compositions, and methods described in the present invention may be used to treat subjects suffering from a disease (for example, a disease characterized by the appearance of an expressed antigen and diseased cells that present an antigen peptide). Examples of treatable and / or preventable diseases cover all diseases expressing one of the antigens described in the present invention. Particularly preferred diseases are infectious diseases (for example, viral diseases) and cancers. The agents, 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, including infectious diseases and cancer, and in particular, to the forms of infectious diseases and diseases described in the present invention.

[0129] According to the present invention, the target disease for treatment is preferably an antigen-related disease. According to the present invention, “antigen-related disease” or a similar description means that the antigen is expressed in cells of a pathologically altered tissue or organ. Expression in cells of a pathologically altered tissue or organ may be elevated compared to the state in healthy tissue or organ. In one embodiment, expression occurs only in pathologically altered tissue and is suppressed in healthy tissue. According to the present invention, antigen-related diseases include infectious diseases and cancers, where the disease-related antigens are preferably infectious source antigens and tumor antigens, respectively. Preferably, antigen-related diseases are diseases relating to cells that express an antigen and present the antigen in an environment with MHC molecules (particularly MHC class I).

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

[0131] Examples of the aforementioned infectious diseases include: [1] viral infections (e.g., AIDS (HIV), hepatitis A, hepatitis B or C, herpes zoster (varicella), rubella (rubella virus), yellow fever, dengue fever, etc., flavivirus, coronavirus, influenza virus, rabies virus, hemorrhagic infections (Marburg virus or Ebola virus)), [2] bacterial infections (e.g., Legionnaires' disease) [3] Infections caused by prokaryotic pathogens (e.g., malaria, African sleeping sickness, leishmaniasis, toxoplasmosis, i.e., infections caused by Plasmodium, Trypanosoma, Leishmania, Toxoplasma), or [4] Fungal infections (e.g., Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides imitis) This includes immitis (caused by Blastomyces dermatitidis or Candida albicans).

[0132] Cancer, or malignant tumor in medical terms, is a disease in which some cells exhibit uncontrolled proliferation (dividing beyond the normal limit), invasiveness (invading and destroying adjacent tissues), and, in some cases, metastasis (spreading to other parts of the body via the lymphatic system or bloodstream). These three harmful characteristics distinguish cancer from benign tumors, which are self-contained and do not invade or metastasize. Most cancers form tumors, i.e., swellings or pathological changes formed by the abnormal proliferation of cells (called malignant neoplasm cells or tumor cells), but some (such as leukemia) are not of this type. According to the present invention, the term "cancer" includes leukemia, seminomas, melanoma, teratomas, lymphomas, sarcomas, blastomas, neuroblastomas, gliomas, glioblastomas, kidney cancer, adrenal cancer, renal cell carcinoma, thyroid cancer, hematological cancers, skin cancers, brain cancers, cervical cancers, intestinal cancers, liver cancers, colon cancers, stomach cancers, lung cancers, intestinal cancers, head and neck cancers, digestive tract cancers, multiple myelomas, lymph node cancers, esophageal cancers, rectal cancers, bladder cancers, endometrial cancers, pancreatic cancers, ear, nose, and throat (ENT) cancers, breast cancers, uterine cancers, prostate cancers, ovarian cancers, and their metastases.

[0133] Malignant melanoma is a type of severe skin cancer. It arises from the uncontrolled proliferation 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 breast cancer, prostate cancer, lung cancer, and common forms of colon cancer.

[0135] Lymphoma and leukemia are malignant tumors that originate from hematopoietic (blood-forming) cells.

[0136] Sarcoma is a type of cancer that arises from a single transformed cell in some tissues that originate from the fetal mesoderm. Therefore, sarcomas include bone tumors, chondroma, lipoma, muscle tumor, vascular tumor, and hematopoietic tissue tumor.

[0137] A blastic tumor, or blastocyte tumor, is a tumor that resembles immature or fetal tissue (and is generally malignant). Many of these tumors occur in children.

[0138] Gliomas are a type of tumor that originates in the brain or spinal cord. They are called gliomas because they originate from glial cells. The brain is the most common site for gliomas.

[0139] (Other ingredients or adjuvants) The compositions of the present invention may be administered together with auxiliary immunostimulants (e.g., one or more adjuvants), and may also contain one or more immunostimulants to further enhance their efficacy and preferably achieve a synergistic effect of immune stimulation. The term “adjuvant” refers to compounds that prolong, enhance, or accelerate the immune response. In this regard, there may be various different mechanisms depending on the type of adjuvant. For example, compounds that mature DCs (e.g., lipopolysaccharides or CD40 ligands) constitute a suitable adjuvant of the first class. In general, any drug that affects the immune system of the “danger signal” type (e.g., LPS, GP96, dsRNA) or cytokines (e.g., GM-CSF) may be used as adjuvants that can enhance and / or influence the immune response in a controlled manner. As mentioned above, CpG oligodeoxynucleotides may be used selectively in some environments, although they are thought to have side effects in some environments. 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, as well as lipopeptides (e.g., Pam3Cys, Pam3CSK4), glucopyranosyllipid adjuvants (GLA), CpG oligodeoxyribonucleotides (e.g., class A or class B), and poly(I:C) are also suitable adjuvants in the compositions of the present invention.

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

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

[0142] Surface modifiers include anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants (such as dimethyldistearylammonium bromide)), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocysteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, thiopronin, gelsolin, thymosin β4, dornase α) The composition may include, but is not limited to, alfa, neltenexine, erdosteine, and DNase (e.g., rhDNase). The surface modifier may be positioned within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent bonding, or other means).

[0143] The composition may further contain one or more functionalized lipids. For example, the lipids may be functionalized with alkynyl groups that can undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. More precisely, the lipid bilayer may be functionalized with one or more groups that can effectively promote membrane permeability, cell recognition, or contrast enhancement in this manner. The surface of the composition may be coupled with one or more useful antibodies. Functional groups and conjugates used for targeted cell delivery, contrast enhancement, and membrane permeability are well known in the art.

[0144] The composition may contain any substance used in pharmaceutical compositions other than these components. For example, the composition may contain, but is not limited to, one or more pharmaceutically acceptable (i.e., pharmaceutically acceptable) excipients or helper components, such as one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, flow accelerators, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, and colorants.

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

[0146] Suitable buffering agents for use in the compositions of the present invention include salts of acetic acid, citric acid, boric acid, and phosphoric acid.

[0147] As used in the present invention, the term "excipient" means all substances that are not active ingredients and may be present in the pharmaceutical composition of the present invention, such as carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents, or colorants. Excipients include, 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., ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0148] The term "carrier" refers to an organic or inorganic component that possesses natural or synthetic properties and, when used in combination with an active ingredient, promotes, enhances, or enables its administration. According to the present invention, the term "carrier" further includes one or more solid or liquid fillers, diluents, or encapsulating substances that are suitable for administration to a patient.

[0149] Carrier materials used for parenteral administration include, for example, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyalkylene glycol, naphthalene hydride, and particularly biocompatible 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 ester, 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, dried starch, corn starch, powdered sugar, and / or combinations thereof.

[0152] (Dosage form and administration) The compositions of the present invention may be manufactured as solid, semi-solid, liquid, or gaseous formulations, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, or aerosols. The compositions of the present invention may be manufactured by methods well known in the pharmaceutical field. For example, a sterile solution for injection may be manufactured by adding a predetermined amount of the therapeutic or prophylactic agent and each of the other specified components described 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 homogeneous solution or suspension.

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

[0154] (Therapeutic effective dose) The "therapeutic dose" is the amount of therapeutic agent that, when administered to a patient, can improve a disease or symptom. The "preventive dose" is the amount of preventive agent that, when administered to a subject, can prevent a disease or symptom. The amount of therapeutic agent constituting the "therapeutic dose" or the amount of preventive agent constituting the "preventive dose" varies depending on the therapeutic agent and / or preventive agent, the state and severity of the disease, the age and weight of the patient and / or subject receiving treatment and / or prevention, etc. A person skilled in the art may generally determine the therapeutic dose and the preventive dose based on their knowledge and the present invention.

[0155] The compositions of the present invention are administered in therapeutically effective doses, the amount of which varies not only depending on the specific 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, the therapeutic or prophylactic agent may be administered to a mammal (e.g., a human) in doses of about 0.0001 to about 10 mg / kg.

[0156] (antigen presenting cells) Antigen-presenting cells (APCs) are cells that present (i.e., display) antigens in an environment containing the major histocompatibility complex (MHC) on their surface. This includes cases where they present only one or more fragments of the antigen. T cells can recognize this complex with their T cell receptor (TCR). Antigen-presenting cells process the antigen and then present it to T cells.

[0157] Specialized antigen-presenting cells are adept at taking up antibodies (by phagocytosis or receptor-mediated endocytosis) and then displaying antigen fragments that bind to MHC class II molecules on their membranes. 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 characteristic of specialized antigen-presenting cells.

[0158] The main types of specialized antigen-presenting cells are dendritic cells (which have the broadest 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 white blood cells that include plasmacytoid dendritic cells (pDC cells) and classical dendritic cells (cDC cells). They 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 the immune response, and their activation is a crucial step in inducing anti-tumor immunity.

[0160] Nucleic acids encoding peptides or proteins containing the peptides to be presented (e.g., nucleic acids encoding antigens (e.g., RNA)) may be introduced into antigen-presenting cells to load the cells with peptides presented by MHC. Transfecting dendritic cells with mRNA is a promising antigen-loading technique for stimulating strong anti-tumor immunity.

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

[0162] The terms "T cell" and "T lymphocyte" may be used interchangeably in the present invention, and include helper T cells (CD4 + T cells), cytotoxic T cells (CTLs, CD8 + T cells) which are cytolytic T cells.

[0163] T cells belong to a group of white blood cells called lymphocytes and play a central role in cell-mediated immunity. The presence of a special receptor called the T cell receptor (TCR) located on their surface distinguishes them from other types of lymphocytes (e.g., B cells, natural killer cells). The thymus is the main organ involved in the maturation of T cells. Several different T cell subsets with different functions have been discovered.

[0164] Helper T cells coordinate with 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 are also called CD4 + T cells because they express the CD4 protein on their surface. 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 immediately divide and secrete small proteins called cytokines that regulate or assist the active immune response.

[0165] Cytotoxic T cells destroy diseased cells (e.g., infected cells (such as virus-infected cells)) and cancer cells, and are also involved in transplant rejection. These cells express the CD8 glycoprotein on their surface, hence the name CD8. + These cells are also called T cells. They recognize their targets by binding to antigens associated with MHC class I, and MHC class I is present on the surface of almost all cells in the body.

[0166] Most T cells possess a T cell receptor (TCR), which exists as a complex of multiple proteins. The actual T cell receptor consists of two independent peptide chains, called the α-TCR chain and the β-TCR chain, produced by independent 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 consists of one γ chain and one δ chain. This group of T cells is rarer than αβ T cells (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 through cell division, producing a large number of immature thymocytes. Early thymocytes do not express either CD4 or CD8, and are therefore double-negative (CD4). - CD8 - They are classified as ) cells. As they develop, they become double-positive thymocytes (CD4 + CD8 + ) becomes, and finally matures into a single positive (CD4 + CD8 - or CD4 - CD8 + They become thymocytes and are later released from the thymus into peripheral tissues.

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

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

[0170] The following are 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-diyldioleate 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-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-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 synthesis route for 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). Then, HOBt (910 mg, 6.71 mmol) and EDCI (1.28 g, 6.71 mmol) were added to the solution in that order, and the mixture was stirred at room temperature for 20 minutes. After that, acetic acid (403 mg, 6.71 mmol) was added to the mixed solution, and the mixture was stirred at room temperature overnight. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was purified by silica gel chromatography (eluent: dichloromethane / 20% methanol (0.1% trifluoroacetic acid)). The fraction containing the product was spin-dried, dissolved in ethyl acetate, and mixed with saturated NaHCO3 aqueous solution. The mixture was 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. The yield was 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 plasmids were ligated into the pVAX1 vector (purchased from Thermo Fisher Scientific) via restriction enzyme EcoRV digestion to construct the vector. 2) The plasmid constructed in the pVAX1 vector in step 1) was homogeneously mixed with 50 μL of E. coli competent cells Stbl2 (purchased from Thermo Fisher Scientific), then bathed in ice for 30 minutes, subjected to a heat shock at 42°C for 90 seconds, immediately returned to ice, and bathed in ice for 2 minutes. 3) Add 400 μL of LB medium (purchased from Thermo Fisher Scientific) and incubate at 30°C in a shaker for 45-60 minutes with gentle shaking. 4) 50-100 μL of bacterial suspension 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 incubated inverted at 37°C overnight. 5) The obtained monoclonal colony plates were sequenced to verify their accuracy, and the 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 plasmid high-volume purification kit (purchased from Yisheng Biotechnology Co., Ltd). 7) Using restriction enzymes, the extracted plasmid was cut into linear plasmids, which were used as transcription templates. The specific steps of the cutting process are described in steps [1] to [3].

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

[0175] [Table 1]

[0176] In step [2], after the reaction is complete, anhydrous ethanol is added, then sodium acetate is added, 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 Anhydrous ethanol and 3M sodium acetate were added in a volume ratio of 1:3:1, and allowed to stand at -20°C for 1 hour to precipitate. The precipitate was then retained by centrifugation at 12000 rpm. In step [3], the precipitate from step [2] was washed twice with 70% ethanol, the centrifugated material was dried at 55°C for 10 minutes, and then dissolved in 1.7 mL of sterile water for injection. The concentration of linear plasmids in the lysis solution was 500 ng / μL, the linearization rate was over 90%, and the purification and recovery efficiency was 85%.

[0177] B) Preparation of Fluc mRNA, eGFP mRNA, HA mRNA, and OVA mRNA 1) Co-transcription capping reaction Using Fluc DNA, eGFP DNA, or HA DNA and OVA DNA purchased from Suzhou Jinweizhi Biotechnology Co., Ltd. as templates, mRNA was synthesized by T7 RNA polymerase transcription using NTP solution (NTPs) and Cap1 cap analog (catalog number 10678ES80, purchased from Yisheng Biotechnology Co., Ltd.) as starting materials. See Table 2 for details of the reaction system. 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 having an m7G(5')ppp(5')(2'-OMeA)pG structure, 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 DNase I (purchased from Yisheng Biotechnology Co., Ltd.) was added to the co-transcription capping reaction system after completing the reaction in step 1) above, resulting in a final concentration of 1 U / μg linear plasmid. After homogeneous mixing, the system was centrifuged and digested at 37°C for 1 hour to obtain the co-transcription capping product.

[0180] 3) Purification by lithium chloride precipitation method The co-transcription capping product obtained in step 2) above was purified by lithium chloride precipitation, and the method was as follows: Step [1] was the addition of lithium chloride. A lithium chloride solution (purchased from Thermo Fisher Scientific) was added to the product of step 2) above, and the final concentration was 2.8 M, and the mixture was allowed to precipitate at low temperature for 2 hours. Step [2] was precipitation. The precipitate was retained by high-speed centrifugation at 12000 rpm for 15 minutes. Step [3] was washing. The mRNA was washed twice with 75% ethanol and dissolved in sterile water for injection to obtain the mRNA solution. The purified mRNA solution was stored at -80°C.

[0181] C) Preparation of mRNA composition using lipid composition 1) Preparation of lipid composition solution In the standard 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 in 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, stirred for 30 minutes, and then filtered through a polycarbonate membrane with a pore size of 100-400 nm.

[0182] In the conventional 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 in 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 mRNA (prepared in Example 2) was diluted with a HEPES and EDTA solution to obtain an aqueous mRNA solution (concentration 0.5 mg / mL, buffer consisting of 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 taken using a syringe and injected into the mRNA aqueous solution prepared as described above. A lipid complex solution was taken using 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 RNA concentration was 100 μg / mL), which was stored at 4-8°C.

[0184] 3) Measurement of particle size and PDI The mRNA composition was diluted in a 1:5 ratio with a 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 distribution analyzer.

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

[0186] [Table 3] JPEG0007872397000005.jpg130170

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

[0188] The complex solutions containing anionic lipids in Examples 4 to 11 were all prepared using Method 1 of Example 2, and a detailed explanation is omitted.

[0189] [Table 4]

[0190] As is clear from the experimental results, when permanent anionic lipids, particularly permanent anionic lipids containing phosphate groups such as ADOPE, 18PA, and DOPG, are added, their solubility in ethanol is good. 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-purine-9-yl)-3,4-dihydroxytetrahydrofurethane Methyl dihydrogen phosphate (pA(2'-OMe)mpG) has low solubility in ethanol, and compositions produced by both methods had good particle size and PDI. The best of these was ADOPE, with a particle size of 303.1 nm, a PDI reduced to 0.2455, and good particle uniformity.

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

[0192] As can be seen from the above comparison, by adding a permanent anionic lipid containing a phosphate group, it is possible to produce a composition with good particle size (particle size limited to 240-500 nm) and uniform particle distribution (PDI < 0.5).

[0193] Example 4: Effect of lipid content ratio on particle size and PDI of RNA composition An mRNA composition containing the following permanent anionic lipids was prepared by the method of Example 2, where the lipid complex solution was prepared by Method 1 (i.e., dissolving the permanent anionic lipids in ethanol).

[0194] The particle size and PDI were measured by varying the molar ratio of the three lipid complexes—permanent anionic lipid ADOPE, permanent cationic lipid DOTMA, and neutral lipid DOPE—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 lipids, permanent cationic lipids, and neutral lipids were set to (10-33 mol%):(40-60 mol%):(22-40 mol%), and in particular when the ratio of permanent anionic lipids was limited to 10-33 mol%, the particle size of the produced composition (limited to within 240-500 nm) and the PDI were both within a good range (less than 0.5). When the amount of permanent anionic lipids 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 solids were produced.

[0197] Furthermore, as can be seen from the results above, it is possible to produce compositions with acceptable particle size and PDI, such as number 13, without adding permanent anionic lipids.

[0198] Example 5: Effect of particle size and charge ratio on PDI of RNA composition The following Fluc-mRNA composition was prepared using the method of Example 2, where the lipid complex solution was prepared using Method 1 (i.e., permanent anionic lipids dissolved in ethanol).

[0199] The molecular ratios of the three lipid complexes—permanent anionic lipid ADOPE, permanent cationic lipid DOTMA, and neutral lipid DOPE—were kept the same, and the charge ratios were varied to measure particle size and PDI.

[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, it is possible to produce a lipid composition with good particle size (particle size limited to 240 to 500 nm) and uniform particle distribution (PDI < 0.5).

[0202] For example, when the molar percentages of ADOPE, DOTMA, and DOPE in the lipid composition were 25:50:25 and the charge ratio was in 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 further 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 molar percentages of ADOPE, DOTMA, and DOPE in the lipid composition are 20:40:40, and the charge ratio is within the range of 1:5 to 1:2, a composition with good particle size (limited to particle size of 240 to 500 nm) and uniform particle distribution (PDI < 0.5) can be produced. As the charge ratio increases, the particle size and PDI of the composition increase. When the charge ratio is 1:2, the particle size and PDI are 456.2 nm and 0.4144, respectively. When the charge ratio exceeds the range of 1:5 to 1:2, for example, when the charge ratio is 2:3 and 3:4 as in samples 11 and 12, large solid particles precipitated in both cases.

[0204] Furthermore, as can be seen from the results above, it is possible to produce compositions with acceptable particle size and PDI, such as those shown in examples 13 and 14, without adding anionic lipids. Further investigations were then conducted using mouse in vivo protein expression experiments and mouse spleen cell flow cytometry experiments.

[0205] Example 6: Experiment on protein expression in mice The method for producing control LNPs was as follows: Cationic lipid compound YK-009 (or YK-407) was dissolved in ethanol with DSPC (Aveitaku (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (AVT (Shanghai) Pharmaceutical Tech Co., Ltd.), and DMG-PEG2000 in a molar ratio of 49:10:39.5:1.5 to prepare an ethanol lipid solution. The ethanol lipid solution was rapidly added to citrate buffer (pH=4~5) by ethanol injection and vortexed for 30 seconds in preparation for use. Fluc-mRNA was diluted with citrate buffer (pH=4~5) to obtain an aqueous solution of Fluc-mRNA. Liposomes were prepared using a predetermined amount of liposome solution and the aqueous solution of Fluc-mRNA so that the weight ratio of total lipids to Fluc-mRNA was 10:1. Ultrasonics were applied at 25°C for 15 minutes (ultrasonic frequency was 40 kHz and ultrasonic output was 800 W). The obtained liposomes were diluted tenfold with PBS, and ethanol was removed by ultrafiltration using a 300 kDa centrifugal ultrafiltration filter. Further dilution with PBS was performed to obtain LNP formulations in which Fluc-mRNA was encapsulated with cationic lipid 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] A composition containing a permanent anionic lipid containing 20 μg of Fluc-mRNA prepared in Example 2 (or an LNP preparation containing YK-009 (or YK-407) containing 20 μg of Fluc-mRNA prepared by the above method) was injected intracellularly 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 injected intraperitoneally with a fluorescent contrast substrate, and allowed to move freely for 5 minutes. Then, the total radiance intensity of the protein expressed in the mouse body by the mRNA supported on the RNA composition (corresponding to the expression intensity of the fluorescent protein, i.e., the amount of protein expressed) was measured using an IVIS Spectrum small animal imaging instrument. After sampling was completed, the mice were euthanized by cervical dislocation, dissected, and the liver, spleen, and lungs were correctly isolated. The total radiance intensity of proteins expressed with Fluc-mRNA in various organs of mice (corresponding to the expression intensity of the fluorescent protein, i.e., the amount of protein expressed) was measured using the IVIS Spectrum small animal imaging instrument. See Table 8 and Figures 1a to 1d for the results of the in vivo contrast-enhanced measurements in mice.

[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, indicating good targeting of the composition to the spleen.

[0210] For example, when the mole percentages of [1]ADOPE, DOTMA, and DOPE are 25:50:25, the total radiant intensity in mice increases as the charge ratio increases, and when the charge ratio is 1:2, the total radiant intensity in mice is 2.32 × 10⁻⁶. 7When the total radiation intensity of the spleen reaches p / s, and is significantly higher than that of the liver and lungs, and the charge ratio is 2:5, the total radiation intensity of the spleen is 7.38 × 10⁻¹⁰. 7 The p / s ratio was the highest, being 56.77 times and 67.09 times higher than that of the liver and lungs, respectively, indicating that the protein expression level of Fluc-mRNA delivered by the composition in the spleen was significantly higher than that in the liver and lungs.

[0211] [2] When the mole percentages of ADOPE, DOTMA, and DOPE are 20:40:40, the total radiant intensity in mice tends to increase as the charge ratio increases, and when the charge ratio is 1:2, the total radiant intensity in mice is 1.91 × 10⁻⁶. 7 When the total radiation intensity of the spleen reaches p / s, and is significantly higher than that of the liver and lungs, and the charge ratio is 1:2, the total radiation intensity of the spleen is 4.84 × 10⁻¹⁰ 7 The p / s ratio was the highest, and both 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 that in the liver and lungs.

[0212] [3] As is clear from the experimental results of Examples 3 and 4, it is possible to produce compositions with acceptable particle size and PDI even without the addition of anionic lipids. However, as can be seen from the mouse in vivo protein expression experiments, composition No. 17, which was produced without the addition of anionic lipids, had a total radiant intensity of 0.64 × 10⁻¹⁶ in mice. 7 The radiation intensity reached p / s, and the radiation intensity in the liver, spleen, and lungs was 0.10 × 10⁻¹⁰, respectively. 7 p / s, 0.61 × 10 7 p / s, 0.10 × 10 7 The radiation intensity was p / s. This radiation intensity was significantly lower than that of the composition containing the permanent anionic lipids described above, and there was no significant difference in radiation intensity across different organs. This indicates that the protein expression level of Fluc-mRNA delivered by the composition produced without the addition of anionic lipids was significantly reduced in the spleen.

[0213] [4] When the molar percentages of the permanent anionic lipid, permanent cationic lipid, and neutral lipid exceed the range of (14 - 33 mol%):(40 - 57 mol%):(22 - 40 mol%), for example, when the molar percentages of ADOPE, DOTMA, and DOPE are 10 mol%:60 mol%:30 mol%, even if the charge ratio is 1:3 or 1:2 (No. 1 and No. 2), the total radioactivity intensity of the spleen is 0.52×10 7 p / s, 0.59×10 7 p / s only, which is equivalent to the total radioactivity intensity in the spleen of the composition (No. 17) produced without containing anionic lipid. Therefore, there is no significant difference in the protein expression levels in the liver, spleen, and lung for the Fluc-mRNA delivered by the compositions produced by both methods. The targeting of the composition to the spleen is shown to be equivalent to that of the composition (No. 17) produced without containing anionic lipid and not increased.

[0214] (2) When using other permanent anionic lipids, such as 18PA and DOPG (No. 15, No. 16), under the above conditions, the protein expression level of the antigen in the spleen can also be significantly improved, showing a good and significant spleen targeting effect.

[0215] For example, when the molar percentages of the permanent anionic lipid, DOTMA, and DOPE are 25:50:25 and the charge ratio is 1:2, the total radioactivity intensities in mice reach 1.98×10 7 p / s, 2.11×10 7 p / s respectively, and the total radioactivity intensity of the spleen is 6.72×10 7 p / s, 5.99×10 7 p / s respectively, which are 56 times and 54.45 times that of the liver respectively, and 61.09 times and 54.45 times that of the lung respectively.

[0216] (3) Compared with the composition without adding anionic lipid, the composition containing the permanent anionic lipid designed in the present invention has a significantly increased protein expression level in the spleen.

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

[0218] (4) Compared with the LNP according to the prior art (No. 18, No. 19), the composition containing the permanent anionic lipid designed in the present invention shows a significantly increased protein expression level in the spleen and good spleen targeting, but the protein expression levels in the liver and lung are decreased (the radioactivity intensity was about 0.1×10 7 p / s), or it remains in the liver and does not express the target protein.

[0219] For example, in the composition (No. 6) where the molar percentages of ADOPE, DOTMA, and DOPE are 25:50:25 and the charge ratio is 1:3, the total radioactivity intensity in the liver is only 0.09 times that of YK-009-Fluc mRNA-LNP (No. 18), but the total radioactivity intensity in the spleen reaches 6.22 times that of YK-009-Fluc mRNA-LNP.

[0220] In the composition (No. 7) where the molar percentages of ADOPE, DOTMA, and DOPE are 25:50:25 and the charge ratio is 2:5, the total radioactivity intensity in the spleen reached 7.1 times that of YK-407-Fluc mRNA-LNP (No. 19).

[0221] The conclusion is 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 protein expression level of the antigen in the spleen.

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

[0223] 2. Preparation of single cells 2.1 The spleen tissue was crushed and then subjected to a cell strainer to obtain single cells. 2.2 Ten times the volume (4 mL) of red blood cell lysate was added to dissolve and remove red blood cells from the tissue. 2.3 Count the cells and measure 5 × 10⁻⁶ 6 Individual cells were collected in flow cytometry tubes (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 surface antibody mix was added to each single-cell suspension and incubated in the dark at room temperature for 15 minutes (one negative control).

[0225] [Table 9] 3.2 Add 2 mL of PBS and centrifuge at 500 g for 5 minutes, then discard the supernatant. 3.3 Cells were resuspended in 200 μL of PBS (after filtration through a 200-mesh nylon mesh) and loaded into a Cytoflex S flow cytometer 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 ratio in macrophages: CD45 + →F4 / 80 + →GFP +

[0226] [Table 10]

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

[0228] Compositions containing ADOPE (numbers 1-9), where the molar percentages of ADOPE are limited to (14-33 mol%), (40-57 mol%), and the charge ratio is limited to 1:2-1:5, or compositions utilizing other permanent anionic lipids including but not limited to 18PA (number 11) or DOPG (number 12), all significantly increase the percentage of antigen-expressing cells in antigen-presenting cells (e.g., B cells, pDC cells, cDC cells, macrophages) within 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 antigen-presenting cells (B cells, pDC cells, cDC cells, macrophages) in the spleen 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 percentages by 0.15%, 3.38%, 4.55%, and 6.25%, respectively. Composition 4 (ADOPE:DOT Composition number 5 (ADOPE:DOTMA:DOPE = 25 mol%:50 mol%:25 mol%, charge ratio 1:2) was increased by 0.37%, 5.90%, 8.69%, and 7.44%, respectively. Composition number 5 (ADOPE:DOTMA:DOPE = 20 mol%:40 mol%:40 mol%, charge ratio 1:2) was increased by 0.14%, 3.76%, 4.28%, and 4.81%, respectively. Composition number 9 (ADOPE:DOTMA:DOPE = 33 mol%:45 mol%:55 mol%, charge ratio 1:2) was 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 by 0.17%, 2.95%, 4.52%, and 4.67%, respectively, compared to the blank control, while the DOPG composition (No. 12) increased 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, ADOPE compositions (Nos. 1-9) can significantly increase the percentage of antigen-expressing cells in antigen-presenting cells (e.g., B cells, pDC cells, cDC cells, macrophages) within the spleen.

[0231] For example, compared to small-sized LPX-RNA (No. 13) without anionic lipid addition, in the ADOPE composition (No. 2), the percentages of eGFP-positive cells in antigen-presenting cells (B cells, pDC cells, cDC cells, macrophages) in the spleen were 6.5-fold, 1.7-fold, 4.5-fold, and 9.3-fold respectively; in the ADOPE composition (No. 3), they were 8-fold, 1.6-fold, 4.2-fold, and 9.9-fold respectively; in the ADOPE composition (No. 4), they were 19-fold, 2.8-fold, 8.1-fold, and 11.8-fold respectively; in the ADOPE composition (No. 5), they were 7.5-fold, 1.8-fold, 4-fold, and 7.7-fold respectively; in the ADOPE composition (No. 9), they were 10.5-fold, 2.1-fold, 4.3-fold, and 6.7-fold respectively.

[0232] When using other permanent anionic lipids, compared to small-sized LPX-RNA (No. 13) without anionic lipid addition, in the 18PA composition (No. 11), the percentages were 9-fold, 1.4-fold, 4.2-fold, and 7.4-fold respectively; in the DOPG composition (No. 12), they were 7.5-fold, 2.2-fold, 3.5-fold, and 6.5-fold respectively.

[0233] 3. Compared to other combinations of pA(2’-OMe)mpG eGFP RNA compositions (including permanent anionic lipid pA(2’-OMe)mpG, permanent cationic lipid DOTMA, and neutral lipid DOPE), the ADOPE compositions (No. 1 - 9) can significantly increase the percentage of cells expressing antigens in antigen-presenting cells (e.g., B cells, pDC cells, cDC cells, macrophages).

[0234] For example, ADOPE composition (2) increased the percentage of eGFP-positive antigen-presenting cells (B cells, pDC cells, cDC cells, macrophages) in the spleen by 1.6 times, 3.4 times, 6.8 times, and 9.2 times, respectively. ADOPE composition (3) increased it by 2 times, 3.2 times, 6.3 times, and 9.8 times, respectively. ADOPE composition (4) increased it by 4.8 times, 5.6 times, 12.1 times, and 11.6 times, respectively. ADOPE composition (5) increased it by 1.9 times, 3.6 times, 6 times, and 7.5 times, respectively. ADOPE composition (9) increased it by 2.6 times, 4.1 times, 6.4 times, and 6.6 times, respectively.

[0235] When other permanent anionic lipids of the present invention are used, the 18PA composition (No. 11) was 2.3 times, 2.8 times, 6.3 times, and 7.3 times more effective 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), respectively, while the DOPG composition (No. 12) was 1.9 times, 4.2 times, 5.2 times, and 6.4 times more effective.

[0236] 4. When the molar percentages and / or charge ratios of ADOPE, DOTMA, and DOPE exceeded the above range, the percentage of antigen-expressing cells in the spleen (e.g., B cells, pDC cells, cDC cells, macrophages) increased to some extent, but the effect was significantly reduced compared to the above. For example, Composition No. 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 (No. 13) without added anionic lipids, indicating that adding a small amount of ADOPE does not provide a significant effect on targeting antigen-presenting cells.

[0237] The conclusion is as follows: As can be seen from the experimental results above, the composition 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 an RNA composition containing ADOPE and large-particle LPX-RNA As can be seen in the invention patent related to LPX-RNA (authorization publication number CN109331176B), there are differences in luciferase activity in the mouse spleen after injecting luciferase-RNA liposome compositions of different sizes, mainly because the larger liposome composition exhibits higher activity. According to the manufacturing method of CN109331176B, a large-particle LPX-RNA composition can be produced by reducing the amount of ethanol used when producing LPX-RNA by 50%. In this example, the differences between a composition containing ADOPE and a large-particle LPX-RNA composition in mouse in vivo contrast imaging and the percentage of eGFP-positive cells in mouse spleen cells were compared.

[0239] [Table 11]

[0240] The results were analyzed as follows: As shown in Table 11, the total radiance in mice of composition 1 (particle size 379.9 nm) with the permanent anionic lipid ADOPE added was 2.4 times higher than that of composition 2 (particle size 478.5 nm), and the total radiance in the spleen was 3.8 times higher than that of composition 2. The difference in in vivo contrast imaging between the two compositions in mice was mainly due to the addition of ADOPE, and particle size did not have a significant effect. In other words, the composition produced with the permanent anionic lipid added was able to significantly improve the protein expression level of the antigen in the spleen, demonstrating a good and significant spleen targeting effect.

[0241] [Table 12]

[0242] The analysis of the results was as follows: As shown in Table 12 and Figures 2a to 2b, when particle sizes were equivalent, the composition containing the permanent anionic lipid ADOPE (No. 1, particle size 365.8 nm) showed 4.8 times, 2.4 times, 3.7 times, and 2.3 times higher percentages of eGFP-positive cells in antigen-presenting cells such as B cells, pDC cells, cDC cells, and macrophages compared to the LPX-RNA composition without anionic lipids (No. 2, particle size 418.8 nm).

[0243] As is evident from the experimental results, the addition of permanent anionic lipids (e.g., ADOPE) can significantly increase the percentage of antigen-expressing cells in 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 concentrate in the mouse spleen, and the mRNA delivered thereby significantly increases protein expression in the mouse spleen compared to compositions produced without the addition of anionic lipids and conventional delivery techniques, and can also significantly increase the percentage of antigen-expressing cells (e.g., B cells, pDC cells, cDC cells, macrophages).

[0245] Example 9: Stimulation of IFN-α cytokine production by an RNA composition containing ADOPE Interferon-alpha (IFN-α) is an important cytokine that plays a crucial role in the body's 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 when APC cells sense double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA) via endosomal TLR3 and TLR7, respectively, in the event of RNA virus infection, and is therefore extremely important for creating an effective anti-inflammatory and antiviral environment.

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

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

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

[0249] [Table 13]

[0250] The analysis of the results was as follows: As shown in Table 13 and Figure 3, when the particle size was equivalent, the compositions containing the permanent anionic lipid ADOPE (No. 2, particle size 325.7 nm) and (No. 3, particle size 352.5 nm) showed that, compared to the LPX-RNA composition without anionic lipids (No. 4, particle size 384.1 nm), the average serum IFN-α cytokine levels were 1.9 times and 3.1 times higher at 6 hours post-injection compared to the LPX-RNA composition without anionic lipids (No. 4), and at 24 hours post-injection, the average serum IFN-α cytokine levels were 1.8 times and 3.8 times higher than those of the LPX-RNA composition without anionic lipids (No. 4).

[0251] As is evident from the experimental results, by measuring the IFN-α cytokine stimulation production by compositions containing ADOPE, it was demonstrated that the compositions of the present invention can initiate a potent immune stimulation program driven by type I IFN, and that their stimulation 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 strength of the effect of tumor mRNA-lipoplex on T cells is related to antigen-specific CD8 in the serum. + Cytotoxic T cells (CD8 + This can be determined by detecting T cells, which is crucial for the antitumor effect of tumor mRNA delivered by lipid complexes.

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

[0254] On day 13 (5 days after 3 immunizations), approximately 200 μL of whole blood was collected and flow cytometry was performed to determine OVA antigen-specific CD8 + CD8 cells in T cells + The percentage of T cells in the total was measured. The specific procedure was as follows: 1. 100 μL of whole blood was collected in a flow cytometry tube (a blank control was required). 2. Add 100 μL of surface antibody premix, pipette to mix uniformly, and incubate in the dark at room temperature for 15 minutes. The components of the premix are as follows, and should be added in the order shown in Table 14 during preparation.

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

[0256] [Table 15]

[0257] The analysis of the results was as follows: As shown in Table 15 and Figure 4, when particle sizes were equivalent, compositions containing the permanent anionic lipid ADOPE (No. 2, particle size 333.1 nm) and (No. 3, particle size 356.3 nm) showed higher levels of OVA antigen-specific CD8 in serum 13 days after injection compared to the LPX-RNA composition without anionic lipids (No. 4, particle size 373.4 nm). + CD8 cells in T cells + The average percentage across all T cells was approximately 1.5 times and 1.8 times higher than that of the LPX-RNA composition (No. 4, particle size 373.4 nm) that did not contain anionic lipids.

[0258] As is evident from the experimental results, by measuring the stimulation production of antigen-specific cytotoxic T cells by the composition containing ADOPE, it was proven that the composition of the present invention can produce a very potent effect on T cells, and that its stimulation 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) The objective was to establish a B16F10-OVA melanoma mouse model. Before starting the study, female C57BL / 6J mice aged 6-8 weeks were adapted for at least 3 days. The mice were given free access to food and sterile water and were housed at 22°C ± 2°C and 55% ± 15% relative humidity with a 12-hour light-dark cycle. B16F10-OVA cells were cultured in complete medium as described in the instructions at 37°C with 5% CO2. After collecting the cells with 0.25% trypsin-EDTA, they were resuspended in Dulbecco's phosphate-buffered saline (DPBS) and 2 × 10⁶ cells were collected. 5 A subcutaneous B16F10-OVA tumor model was established by subcutaneous (SC) transplantation of 100 μL of cells per mouse into the lateral thorax of female C57BL / 6J mice, with a tumor volume of 100 mm². 3 Vaccination began once the condition had reached a certain level.

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

[0261] [Table 16]

[0262] 3) Tumor size and mouse survival rate were measured. From the 7th day after tumor inoculation, the diameter of the tumor was measured three times a week. Formula: V(mm) 3 ) = x × y 2 The tumor volume of the C57BL / 6J mouse was calculated using the formula / 2, in units of mm, where V represents the tumor volume, x represents the major axis of the tumor, and y represents the minor axis of the tumor. Simultaneously, the weight change of the C57BL / 6J mouse was recorded three times a week using an electronic balance, and the tumor volume was 2000 mm. 3 If the threshold was exceeded, euthanasia was performed on the 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 vaccines were administered on days 10, 13, and 17 after inoculation. On day 10 after inoculation, all mice in each group entered the rapid tumor growth phase. From day 14, tumor growth was clearly slower in the OVA mRNA composition groups (2 and 3) containing ADOPE compared to the blank lipid control group (1), and the tumor size was clearly smaller in all groups than in the blank lipid control group. From day 19, stronger suppression of tumor growth was also observed in the tumor volume of the OVA mRNA composition groups (2 and 3) containing ADOPE compared to LPX-OVA mRNA (4).

[0264] [Table 17]

[0265] As shown in Figure 6 and Table 18, in the blank lipid control group, deaths began to occur from day 15 after tumor inoculation, and all animals died on day 17. In the LPX-OVA mRNA control group (No. 4), deaths began to occur from day 19, and all animals died on day 28. In the OVA mRNA compositions containing ADOPE (Nos. 2 and 3), deaths began to occur from day 21 in both cases, and all animals died on days 31 and 33, 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 LPX-RNA compositions that do not contain anionic lipids, the specific compositions of the present invention that contain permanent anionic lipids can significantly enhance the inhibition of tumor growth and improve the survival rate of mice.

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

[0269] 1. Compositions produced by adding permanent anionic lipids, particularly those containing phosphate groups such as ADOPE, 18PA, DOPG, tetradecylphosphonic acid, farnesyl pyrophosphate, γ,γ-dimethylallyl pyrophosphate, and pA(2'-OMe)mpG, all exhibit good particle size and PDI. Among these, ADOPE is the best, with a particle size of 303.1 nm and a PDI reduced to 0.2455, demonstrating 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, and sodium lauryl sulfonate, are unsuitable as mRNA delivery carriers because their particle size exceeds 1000 nm, and solid precipitates form.

[0271] II. Compositions comprising permanent anionic lipids, permanent cationic lipids, and neutral lipids designed in the present invention can significantly improve the protein expression level of antigens in the spleen (corresponding to total radiation intensity), particularly by limiting the molar percentages of ADOPE, DOTMA, and DOPE to (14-33 mol%):(40-57 mol%):(22-40 mol%), or the charge ratio to 1:2-1:5. 1. In vivo contrast-enhanced experiments in mice have shown that the protein expression level of antigens delivered by compositions containing permanent anionic lipids designed in this invention is significantly higher in the spleen than in other organs (e.g., liver, lungs), and the total radiance intensity of the protein expressed in the spleen by Fluc-mRNA delivered by the manufactured composition was 1.20 × 10⁻⁶. 7 ~7.38×10 7 It has reached p / s.

[0272] 2. Compared to compositions manufactured without the addition of anionic lipids, the protein expression level of antigens delivered by the compositions designed in this invention is significantly improved in the spleen.

[0273] For example, the total radiant intensity of the Fluc-mRNA expressed in the spleen by the mRNA composition designed in this invention is 12.10 times higher than that of a composition produced without the addition of anionic lipids.

[0274] 3. Compared to conventional LNPs, the protein expression level of the antigen delivered by the composition designed in the present invention in the spleen is significantly higher than that of conventional LNPs.

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

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

[0277] For example, among antigen-presenting cells, B cells make up approximately 0.1-0.4%, pDC cells approximately 2-6%, cDC cells approximately 2-9%, and macrophages approximately 2.4-7.5%.

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

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

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

[0281] For example, in a particular combination of compositions of the present invention (permanent anionic lipid ADOPE, permanent cationic lipid DOTMA, neutral lipid DOPE), the percentage of cells expressing eGFP, such as B cells, pDC cells, cDC cells, and macrophages, is 4.8 times, 5.6 times, 12.1 times, and 11.6 times, respectively, compared to the eGFP RNA composition of pA(2'-OMe)mpG (permanent anionic lipid pA(2'-OMe)mpG, permanent cationic lipid DOTMA, neutral lipid DOPE).

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

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

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

[0285] For example, on the 13th day after injection of a specific composition of the present invention containing permanent anionic lipids, OVA antigen-specific CD8 in serum + CD8 cells in T cells +The percentage of T cells in the total is 1.3 to 1.9 times higher when an LPX-RNA composition without anionic lipids is injected.

[0286] VI. The specific combinations of compositions designed in the present invention have shown in animal experiments to clearly control tumor growth and extend the survival period of tumor-bearing experimental animals compared to LPX-RNA compositions that do not contain anionic lipids.

[0287] After subcutaneous inoculation of B16F10-OVA melanoma cells into mice, compared to a blank lipid control group and a control group administered an LPX-RNA composition without anionic lipids, injection administration of a specific composition of the present invention containing permanent anionic lipids effectively slowed the rate of tumor growth, resulting in a clear reduction in tumor size. Furthermore, the survival rate of tumor-bearing mice injected with the specific composition of the present invention containing permanent anionic lipids was significantly increased.

[0288] Although the present invention has been described in detail above, its purpose is to inform those skilled in the art of its contents and encourage them to implement it; this does not limit the scope of protection of the present invention. Any equivalent modifications or alterations made in accordance with the spirit of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A lipid composition, (1) Permanent anionic lipids, (2) Permanent cationic lipids, (3) Neutral lipids, However, the molar ratio of permanent anionic lipids, permanent cationic lipids, and neutral lipids in the lipid composition is 14-33:40-57:22-40. The aforementioned permanent anionic lipid is 2-Acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate, 1,2-dioleoyl-sn-glycero-3-phospho-rac-glycerol, or any one or more selected from salts thereof, The aforementioned permanent cationic lipid is Selected from 1,2-di-O-octadecenyl-3-trimethylammonium propane, or salts thereof, The aforementioned neutral lipids are A lipid composition selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or salts thereof.

2. The lipid composition is (1) 25 mol% permanent anionic lipids, (2) 50 mol% permanent cationic lipid, (3) Consists of 25 mol% neutral lipids, Alternatively, the lipid composition may be: (1) 20 mol% permanent anionic lipids, (2) 40 mol% permanent cationic lipid, (3) Consists of 40 mol% neutral lipids, Alternatively, the lipid composition may be: (1) 14 mol% permanent anionic lipids, (2) 57 mol% permanent cationic lipids, (3) Consists of 29 mol% neutral lipids, Alternatively, the lipid composition may be: (1) 33 mol% permanent anionic lipids, (2) 45 mol% permanent cationic lipid, (3) The lipid composition according to claim 1, comprising 22 mol% of a neutral lipid.

3. A lipid composition for use in preparing reagents to improve targeting of antigen-presenting cells within a target organ, The lipid composition is (1) Permanent anionic lipids, (2) Permanent cationic lipids, (3) Neutral lipids, However, the molar ratio of permanent anionic lipids, permanent cationic lipids, and neutral lipids in the lipid composition is 14-33:40-57:22-40. The aforementioned permanent anionic lipid is 2-Acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate, 1,2-dioleoyl-sn-glycero-3-phospho-rac-glycerol, or any one or more selected from salts thereof, The aforementioned permanent cationic lipid is Selected from 1,2-di-O-octadecenyl-3-trimethylammonium propane, or salts thereof, The aforementioned neutral lipids are Selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, or salts thereof, The target organ is the spleen. The antigen-presenting cells are a lipid composition comprising any one or more of dendritic cells, macrophages, or B cells.

4. A composition, (A) A therapeutic and / or prophylactic agent comprising one or more nucleic acid molecules, small molecule compounds, polypeptides, or proteins, (B) comprising the lipid composition described in claim 1, The composition 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 antigen-presenting cells are a composition comprising one or more of the following: dendritic cells, macrophages, or B cells.

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

6. The nucleic acid molecule can induce an immune response to disease-related antigens. Alternatively, the nucleic acid molecule can induce an immune response against cells expressing disease-related antigens. Alternatively, the composition according to claim 4, wherein the nucleic acid molecule is RNA encoding one or more antigens.

7. The antigen in question is a disease-related antigen, Alternatively, the antigen can induce an immune response to a disease-related antigen. Alternatively, the composition according to claim 5, wherein the antigen can induce an immune response against cells expressing a disease-associated antigen.

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

5.

9. The charge ratio of the net positive charge to the negative charge in the above composition is 1:

2. Alternatively, the charge ratio of the net positive charge to the negative charge in the composition is 2:

5. Alternatively, the charge ratio of the net positive charge to the negative charge in the composition is 1:

3. Alternatively, the composition according to claim 8, wherein the charge ratio of the net positive charge to the negative charge 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 modifiers, functionalized lipids, or cytokines. Alternatively, the composition further comprises one or more auxiliary agents, Alternatively, the composition further comprises one or more pharmaceutically acceptable carriers. Alternatively, the composition according to claim 4, wherein the composition further comprises one or more diluents.

11. A method for producing a composition for delivering a therapeutic agent and / or prophylactic agent to antigen-presenting cells in a target organ, (a) A step of 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 to 33: 40 to 57: 22 to 40. The aforementioned permanent anionic lipid is 2-Acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate, 1,2-dioleoyl-sn-glycero-3-phospho-rac-glycerol, or any one or more selected from salts thereof, The aforementioned permanent cationic lipid is Selected from 1,2-di-O-octadecenyl-3-trimethylammonium propane, or salts thereof, The aforementioned neutral lipids are A step selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or a salt thereof, (b) A step of mixing the lipid solution obtained in step (a) with water to obtain a lipid mixture, (c) A method comprising the step of mixing the lipid mixture obtained in step (b) with a therapeutic agent and / or prophylactic agent to form the composition, wherein the therapeutic agent and / or prophylactic agent comprises a nucleic acid buffer solution obtained by dissolving nucleic acid molecules in a buffer solution with 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 with a pore size of 100 to 400 nm. And / or, in step (c), the buffer comprises aqueous HEPES buffer, The method according to claim 11, wherein 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 contains 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 can induce an immune response to a disease-related antigen, Alternatively, the method according to claim 11 or 12, wherein in step (c), the nucleic acid molecule can induce an immune response against cells expressing a disease-associated antigen.

14. In step (c), the antigen is a disease-related antigen, Alternatively, the antigen can induce an immune response to a disease-related antigen. Alternatively, the method according to claim 12, wherein the antigen can induce an immune response against cells expressing a disease-associated antigen.

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

5.

16. A lipid composition according to claim 1 or 2 for preventing, treating, or improving a disease or medical condition in a mammalian subject.

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

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

Images