RNA transporter preparations in vivo and methods for preparing these preparations.

Abstract

The invention relates to a preparation for the delivery of liposome-containing mRNA based on cationic lipids. The mRNA delivery preparation of the invention exhibits very good storage stability and high in vivo expression and transport rates, and thus can enhance the stability and efficacy of mRNA vaccines used for cancer therapy or mRNA vaccines used for the treatment of viral infections.

Description

Composition for intravital delivery of RNA and method for producing the same

[0001] The present invention relates to a composition for intracellular delivery of mRNA, and more particularly, to a composition for intracellular delivery of mRNA comprising a cationic lipid-based liposome and a method for preparing the same.

[0002] This patent application claims priority to Republic of Korea Patent Application No. 10-2021-0029929, filed with the Korean Intellectual Property Office on March 8, 2021, the disclosure of which is incorporated herein by reference.

[0003] Gene therapy and genetic vaccines are already proven and commonly applied technologies in the medical field, and can be used to treat not only genetic diseases but also autoimmune diseases, infectious diseases, cancer or tumor-related diseases, and inflammatory diseases.

[0004] Genetic vaccines began to be developed when it was reported that when DNA and RNA encoding target genes were directly injected into animals, the target genes were expressed in living animals, and immunity was possible through this expression (Wolff JA et al. Science, 247:1465-8, 1990).

[0005] Genetic vaccination allows for the induction of a desired immune response against selected antigens, such as characteristic components of bacterial surfaces, viral particles, and tumor antigens. Broadly speaking, vaccination is one of the pivotal achievements of modern medicine. However, effective vaccines are currently available for only a limited number of diseases. Consequently, infections that cannot be prevented by vaccination still affect millions of people each year.

[0006] In gene therapy or genetic vaccination, DNA and RNA can be used as nucleic acid molecules for gene administration, and DNA is known to be relatively stable and easy to handle compared to RNA.

[0007] However, in the case of DNA, potential risks may arise if the DNA fragment administered into the patient's genome is inserted into an unintended location, resulting in genetic damage. Additionally, unwanted anti-DNA antibodies may develop. Another problem is the limited expression level of peptides or proteins produced by DNA administration and subsequent transcription / translation. The presence or absence of specific transcription factors that regulate DNA transcription significantly influences the expression level of the administered DNA. In the absence of specific transcription factors, sufficient RNA is not produced through DNA transcription, resulting in limited levels of peptides or proteins produced through translation.

[0008] On the other hand, when RNA is used as a tool for gene delivery, RNA does not require transcription and can synthesize proteins directly within the cytoplasm without the need for entry into the nucleus like DNA, eliminating the risk of RNA incorporation into cellular chromosomes and causing unwanted genetic damage. Furthermore, its shorter half-life compared to DNA means it does not induce long-term genetic alterations (Sayour EJ et al., J Immunother Cancer 2015;3:13, 2015). When delivered into cells, typical RNA vaccines are activated for a short period of time to express target proteins, and are destroyed within a few days by enzymatic reactions, while a specific immune response to the expressed target antigen (protein) remains.

[0009] Furthermore, when using RNA as a gene delivery tool, it works by passing only through the cell membrane, without the need for nuclear membrane passage. Therefore, even with smaller amounts, the same amount of target protein can be expressed as with DNA. Furthermore, RNA possesses inherent immune-enhancing properties, allowing for the same immune effects with smaller doses compared to DNA.

[0010] By using RNA instead of DNA for genetic vaccination, the risk of unwanted genome integration and the formation of anti-DNA antibodies is minimized or prevented. However, RNA is considered a highly unstable molecular species that can be easily degraded by ubiquitous RNases.

[0011] Despite significant advances over the past several years, there remains a need for methods that do not significantly impair delivery due to inefficient translation of mRNA, resulting in premature antigen degradation or inefficient mRNA release from cells. Furthermore, there is a critical need to reduce the dose of mRNA vaccines to mitigate potential safety concerns and make them affordable in developing countries. Nucleic acid-based therapeutics, such as vaccines, hold tremendous promise, but to realize this potential, more efficient delivery of nucleic acids to the appropriate site within cells or organisms remains a critical need.

[0012] However, the use of nucleic acids for therapeutic and prophylactic purposes currently faces two challenges. First, free RNA is vulnerable to nuclease digestion in plasma. Second, free RNA has limited access to intracellular compartments where relevant translational machinery resides. Lipid nanoparticles formed from cationic lipids and other lipid components, such as neutral lipids, cholesterol, PEG, PEGylated lipids, and nucleic acids, are being attempted to block RNA degradation in plasma and promote cellular uptake of oligonucleotides.

[0013] When using lipid-based delivery vehicles such as liposomes or lipid nanoparticles, mRNA is typically either adsorbed externally or encapsulated internally. In particular, when mRNA is adsorbed externally, it is generally known to exist not as a single liposome, but as an aggregate of liposome and nucleic acid. Adsorption capacity and stability vary depending on the combination of lipids, the state of the nucleic acid, and the ratio of nucleic acid to liposome, and therefore optimization is necessary.

[0014] Furthermore, even if the expression of a carrier has been verified in vitro, there is a problem that the results of in vivo expression may differ in terms of mRNA expression ability. In vivo, there are many factors that reduce the efficiency of delivery into cells, such as a decrease in half-life and bio-distribution due to the aggregation of plasma proteins, differences in cell uptake methods depending on the lipid composition, and barriers by the extracellular matrix (ECM), depending on the physicochemical properties of the lipid composition and the carrier. Therefore, unlike in vitro delivery, carrier optimization is essential for in vivo delivery.

[0015] The present inventors have conducted extensive research to develop a delivery system improvement technology that can induce stable protein expression by stably delivering mRNA into the body and increasing intracellular expression efficiency. As a result, they completed the present invention by manufacturing a cationic liposome-based [liposome + mRNA complex] through a specific process and demonstrating that the [liposome + mRNA complex] manufactured through this process can exhibit high intracellular delivery and expression rates in vivo.

[0016] Accordingly, an object of the present invention is to provide a composition for mRNA delivery comprising cationic lipid-based liposomes having high in vivo delivery and expression rates.

[0017] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease selected from the group consisting of cancer, tumor, autoimmune disease, genetic disease, inflammatory disease, viral infection, and bacterial infection, containing the mRNA delivery composition as an active ingredient.

[0018] Another object of the present invention is to provide a vaccine containing the above mRNA delivery composition as an active ingredient.

[0019] Another object of the present invention is to provide a functional cosmetic composition containing the mRNA delivery composition as an active ingredient.

[0020] Another object of the present invention is to provide a method for preparing a composition for mRNA delivery.

[0021] The present inventors have conducted extensive research to develop a delivery system improvement technology that can induce stable protein expression by stably delivering mRNA into the body and increasing intracellular expression efficiency. As a result, we manufactured a cationic liposome-based [liposome + mRNA complex] through a specific process and demonstrated that the [liposome + mRNA complex] manufactured through this process can exhibit high intracellular delivery and expression rates in vivo.

[0022] The present invention relates to an mRNA delivery composition comprising a cationic lipid-based liposome, a pharmaceutical composition for preventing or treating a disease selected from the group consisting of cancer, viral infection, and bacterial infection, containing the mRNA delivery composition as an active ingredient, a vaccine containing the mRNA delivery composition as an active ingredient, a functional cosmetic composition containing the mRNA delivery composition as an active ingredient, and a method for producing the mRNA delivery composition.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0024] In the present invention, in the case of intracellular gene transfer using nucleic acid, a method for preventing or treating a disease by inducing a desired immune response to a selected antigen such as a characteristic component of the surface of a virus or bacteria, a viral particle, or a tumor antigen, and inducing expression of a therapeutic protein was developed, and a method for increasing the efficiency of intracellular expression of mRNA to exhibit a stable effect was sought.

[0025] Accordingly, a composition for mRNA delivery using cationic lipid-based liposomes was prepared, and it was confirmed that the composition for mRNA delivery exhibited a high intracellular delivery rate and expression rate in vivo.

[0026] In the composition for mRNA delivery using the cationic lipid-based liposome of the present invention, the mRNA may exist in the form of a complex with the cationic lipid-based liposome, and accordingly, the composition for mRNA delivery using the cationic lipid-based liposome and [complex of liposome + mRNA] are used interchangeably.

[0027] Hereinafter, the present invention will be described in more detail.

[0028]

[0029] According to one aspect of the present invention, the present invention provides a composition for mRNA delivery comprising a cationic lipid-based liposome.

[0030] In the present invention, 'cationic lipid' includes a lipid that continuously has cationic properties without being affected by pH changes or an ionic lipid that is converted to cationic properties by pH changes.

[0031] The cationic lipids are 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), dimethyldioctadecylammonium bromide (DDA), 3β-[N-(N',N'-dimethylaminoethane) carbamoyl cholesterol (DC-Chol), 1,2-dioleoyloxy-3-dimethylammoniumpropane (DODAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 Etyle PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 Ethyl PC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 Ethyl PC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholin (18:0 Ethyl PC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 Ethyl PC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 Ethyl PC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholin (12:0 Ethyl PC),N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP), 1,2-distearoyl-3-dimethylammonium-propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 1,2-stearoyl-3-trimethylammoniumpropane (18:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TA), 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP) and / or N4-cholesteryl-spermine (GL67), preferably 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) or C12-200, but is not limited thereto.

[0032] Specifically, the above 'DOTAP (1,2-Dioleoyl-3-trimethylammonium propane)' is a cationic emulsifier having a structure of the following chemical formula 1, is used as a fabric softener, and has recently been used as a nucleic acid carrier forming liposomes.

[0033]

[0034] The composition for mRNA delivery comprising the cationic lipid-based liposome of the present invention may additionally include a neutral lipid.

[0035] In the present invention, 'neutral lipid' includes a lipid that is continuously neutral without being affected by a pH change or an ionic lipid that is converted to neutral by a pH change.

[0036] The neutral lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), It may be 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), phosphatidylserine (PS), phosphoethanolamine (PE), phosphatidylglycerol (PG), phosphoric acid (PA) and / or phosphatidylcholine (PC), and preferably 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), but is not limited thereto.

[0037] Specifically, the above 'DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine)' has a structure represented by the following chemical formula 2 and is used as an auxiliary lipid for cationic liposomes.

[0038]

[0039] In the present invention, the weight ratio of the cationic lipid and the neutral lipid may be 1:9 to 9.5:0.5, 2:8 to 9:1, 3:7 to 8:2, or 4:6 to 7:3, but is not limited thereto.

[0040] If the above weight ratio is exceeded, the mRNA delivery efficiency may be significantly reduced.

[0041] Additionally, the composition for mRNA delivery comprising the cationic lipid-based liposome of the present invention may additionally contain cholesterol.

[0042] In the present invention, the weight ratio of the cationic lipid and cholesterol may be, but is not limited to, 6:1 to 1:3, 4:1 to 1:2.5, 3:1 to 1:2, or 2:1 to 1:1.5.

[0043] In addition, when the liposome according to the present invention includes all of cationic lipid, neutral lipid, and cholesterol, the weight ratio of the cationic lipid, neutral lipid, and cholesterol may be 1 to 9.5:9 to 0.5:0.05 to 3, 3 to 8:7 to 1:0.45 to 7.0, or 1 to 3.5:1 to 3.5:0.5 to 3, but is not limited thereto.

[0044] If the above weight ratio is exceeded, the mRNA delivery efficiency may be significantly reduced.

[0045] For example, when cholesterol is additionally included, liposomes can be prepared by mixing cholesterol in a weight ratio of 0.2 to 0.85, or 0.5 to 0.85, for DOTAP:DOPE=1:1.

[0046] Furthermore, the mRNA delivery composition comprising the cationic lipid-based liposome of the present invention may additionally include one or more delivery factors such as protamine, albumin, transferrin, PTD (protein transduction domains), CPP (cell penetrating peptide), and Macrophage targeting moiety.

[0047] Furthermore, the composition for mRNA delivery comprising the cationic lipid-based liposome of the present invention may additionally include an immunostimulant.

[0048] The above-mentioned immune enhancer may be, but is not limited to, a group of substances that respond to a pathogen-associated molecular pattern (PAMP) and a pattern recognition receptor (PRR), detoxified lipooligosaccharide (dLOS), CpG DNA, lipoprotein, flagella, poly I:C, saponin, squalene, tricaprin, and / or 3D-MPL.

[0049] Specifically, the non-toxic lipo-oligosaccharide (dLOS) may be, but is not limited to, a material disclosed in Korean Patent No. 1509456 or Korean Patent No. 2042993.

[0050] In the mRNA delivery composition of the present invention, the mixing ratio of the mRNA delivery agent represented by liposomes and mRNA can be expressed as the N:P ratio, and the expression and stability of the delivery agent are affected depending on the N:P ratio.

[0051] The N:P ratio of the liposome and mRNA may be, but is not limited to, 0.23 to 1.39:1, 0.3 to 1.3:1, 0.4 to 1.2:1, 0.5 to 1.1:1, 0.6 to 1.0:1, 0.6 to 0.9:1, 0.6 to 0.8:1, or 0.7:1.

[0052] In the mRNA delivery composition of the present invention, the mRNA may be characterized by encoding a peptide or protein that can act as an immunogen.

[0053] The mRNA may typically be an mRNA having at least one open reading frame (ORF) that can be translated by a cell or organism provided with the mRNA. The product of this translation is an antigen, preferably a peptide or protein that can act as an immunogen. The product may also be a fusion protein comprising two or more immunogens, for example, a fusion protein comprising two or more epitopes, peptides, or proteins derived from the same or different viral proteins, wherein the epitopes, peptides, or proteins may be linked by a linker sequence.

[0054] Furthermore, the mRNA may be understood as an artificial mRNA, i.e., an mRNA molecule that does not occur naturally. An artificial mRNA molecule may be understood as a non-natural mRNA molecule. Such mRNA molecules may be non-natural due to individual sequences (that do not occur naturally) and / or other non-natural modifications, such as structural modifications of nucleotides. An artificial mRNA molecule may be designed and / or produced by genetic engineering methods corresponding to a desired artificial nucleotide sequence (a heterologous sequence).

[0055] Furthermore, the mRNA may exhibit modifications that increase its resistance to in vivo degradation (e.g., degradation by exo- or endo-nucleases) and / or ex vivo degradation (e.g., during the manufacturing process prior to vaccine administration, e.g., during the preparation of the vaccine solution to be administered). Stabilization of the RNA may be achieved, for example, by providing a 5'-CAP structure, a poly-A tail, or any other UTR modification. Stabilization of the RNA may also be achieved by chemical modification or by altering the G / C content of the nucleic acid. Various other methods are known in the art and may be applied to the present invention.

[0056]

[0057] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a disease selected from the group consisting of cancer, tumor, autoimmune disease, genetic disease, inflammatory disease, viral infection, and bacterial infection, containing the above-described mRNA delivery composition as an active ingredient.

[0058] In the present invention, ‘prevention’ means any act of suppressing or delaying the progression of the above-described disease by administering a pharmaceutical composition according to the present invention.

[0059] In the present invention, 'treatment' means all acts in which the symptoms of the above-described disease are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

[0060] The pharmaceutical composition of the present invention may be combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. The pharmaceutical composition may include buffers, such as neutral buffered saline, phosphate buffered saline, citric acid buffered solution, and the like; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0061] The pharmaceutical composition of the present invention can be administered orally or parenterally, and can be administered by, for example, intravenous administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intratumoral administration, intracerebral administration, intracranial administration, intrapulmonary administration, and rectal administration, but is not limited thereto.

[0062] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The effective amount may be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route, and excretion rate, treatment duration, concurrent medications, and other factors well known in the medical field.

[0063] The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with the minimum amount possible without causing side effects. This can be readily determined by those skilled in the art.

[0064] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption rate, inactivation rate and excretion rate of the active ingredient in the body, type of disease, and concomitantly administered drugs.

[0065] Since the pharmaceutical composition of the present invention contains the above-described mRNA delivery composition as an active ingredient, description of overlapping content is omitted to avoid excessive complexity of the present specification.

[0066]

[0067] According to another aspect of the present invention, the present invention provides a method for preventing or treating cancer, tumor, autoimmune disease, inflammatory disease, viral infection, and bacterial infection, comprising administering to a patient in need of prevention or treatment of cancer, tumor, autoimmune disease, inflammatory disease, viral infection, and bacterial infection, the method comprising administering to the patient an mRNA delivery composition of the present invention.

[0068] The dosage of the mRNA is 1 to 5 μg, 5 to 10 μg, 10 to 15 μg, 15 to 20 μg, 10 to 25 μg, 20 to 25 μg, 20 to 50 μg, 30 to 50 μg, 40 to 50 μg, 40 to 60 μg, 60 to 80 μg, 60 to 100 μg, 50 to 100 μg, 80 to 120 μg, 40 to 120 μg, 40 to 150 μg, 50 to 150 μg, 50 to 200 μg, 80 to 200 μg, 100 to 200 μg, 120 to 250 μg, 150 to 250 μg, 180 to 280 μg, 200-300 μg, 50 to It may be, but is not limited to, 300 ㎍, 80 to 300 ㎍, 100 to 300 ㎍, 40 to 300 ㎍, 50 to 350 ㎍, 100 to 350 ㎍, 200 to 350 ㎍, 300 to 350 ㎍, 320 to 400 ㎍, 40 to 380 ㎍, 40 to 100 ㎍, 100 to 400 ㎍, 200 to 400 ㎍, or 300 to 400 ㎍.

[0069] The above vaccine may be a vaccine against viruses that can cause infection in humans and animals, including, but not limited to, influenza, coronavirus, shingles, human papillomavirus, Zika virus, herpes virus, AIDS virus, SFTS virus, measles virus, chickenpox virus, Ebola virus, MERS virus, hepatitis virus, avian influenza, rabies virus, and foot-and-mouth disease virus.

[0070] The vaccine comprises at least one mRNA having an open reading frame encoding a polypeptide of at least one viral antigen or an immunogenic fragment thereof.

[0071] Since the treatment method of the present invention includes the above-described pharmaceutical composition as an effective ingredient, description of overlapping content is omitted to avoid excessive complexity of this specification.

[0072]

[0073] According to another aspect of the present invention, the present invention provides a vaccine containing the above-described mRNA delivery composition as an active ingredient.

[0074] The above vaccine may include the above-described mRNA delivery composition at an appropriate concentration considering the weight, age, dietary stage, and / or immunity of the administration subject, within the scope of the purpose of preventing a disease caused by a peptide or protein that can act as an immunogen.

[0075] The above vaccine may further include one or more selected from the group consisting of carriers, diluents, excipients, and adjuvants. The carrier is not particularly limited in type, but may include any and all solvents, dispersion media, coatings, stabilizers, preservatives, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, etc.

[0076] The above vaccine may be administered orally, parenterally, subcutaneously, intramuscularly, intradermally, sublingually, transdermally, rectally, transmucosally, superficially via inhalation, buccal administration, or a combination thereof.

[0077] The vaccine may be administered once or multiple times, or intermittently, for example, daily for several days, weeks, or months, in the same or different doses, depending on the desired duration and effectiveness of the vaccination or treatment. Injections may be administered by injection, subcutaneously, or by nasal spray, or alternatively, by continuous infusion.

[0078] Since the vaccine of the present invention contains the above-described mRNA delivery composition as an active ingredient, description of overlapping content is omitted to avoid excessive complexity of this specification.

[0079]

[0080] According to another aspect of the present invention, the present invention provides a functional cosmetic composition containing the above-described mRNA delivery composition as an active ingredient.

[0081] The cosmetic composition according to the present invention may include components commonly used in cosmetic compositions, such as a metal ion sequestering agent, an active ingredient (e.g., Sodium Hyaluronate and Tocopheryl Acetate), a preservative, a thickening agent, a fragrance, and conventional auxiliary agents, and a carrier.

[0082] In addition, the cosmetic composition according to the present invention can be manufactured in the form of a formulation common in the art, such as an emulsified formulation or a solubilized formulation. Examples of emulsified formulations include nourishing toners, creams, and essences, and examples of solubilized formulations include emollient toners. In addition, the cosmetic composition of the present invention can be manufactured in the form of an adjuvant for topical or systemic application commonly used in the field of dermatology by containing a dermatologically acceptable medium or base, in addition to cosmetics.

[0083] Suitable cosmetic formulations may be, for example, solutions, gels, solid or paste anhydrous products, emulsions obtained by dispersing an oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) or non-ionic vesicular dispersions, creams, toners, lotions, powders, ointments, sprays or concealer sticks. They may also be prepared in the form of foams or aerosol compositions further containing compressed propellants.

[0084] In addition, the cosmetic composition of the present invention may further contain adjuvants commonly used in the fields of cosmetology or dermatology, such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredients commonly used in cosmetics. In addition, the above ingredients may be introduced in amounts commonly used in the field of dermatology.

[0085] Products to which the cosmetic composition of the present invention can be added include, for example, cosmetics such as astringent toners, emollient toners, nourishing toners, various creams, essences, packs, foundations, and cleansing products, facial cleansers, soaps, treatments, and beauty solutions.

[0086] Specific formulations of the cosmetic composition of the present invention include formulations such as skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, pressed powder, loose powder, patch, spray, etc.

[0087] Since the cosmetic composition of the present invention contains the above-described mRNA delivery composition as an active ingredient, description of overlapping content is omitted to avoid excessive complexity of the present specification.

[0088]

[0089] According to another aspect of the present invention, the present invention provides a method for preparing a composition for mRNA delivery, comprising a step of mixing mRNA and liposomes.

[0090] The mRNA and / or liposomes may be provided in a lyophilized powder form, or may be provided dissolved in an appropriate solution or buffer. If the mRNA and / or liposomes are provided in a lyophilized form, they may be used by dissolving them in an appropriate solution or buffer.

[0091] In a specific embodiment of the present invention, the inventors analyzed the characteristics of mRNA-liposome complexes prepared by controlling the mixing order of liposomes and mRNA, and confirmed in vivo mRNA expression according to the mixing order of liposomes and mRNA.

[0092] In the present invention, the mRNA and liposome may be mixed by introducing them in the order of mRNA → liposome.

[0093] By mixing mRNA and liposomes in the above order, the in vivo mRNA delivery and expression rate of the manufactured mRNA-liposome complex can be increased.

[0094] The method for preparing the mRNA delivery composition of the present invention may additionally include a step of mixing an immunostimulant.

[0095] The above-mentioned immunostimulant may be provided in the form of a lyophilized powder, or may be provided dissolved in an appropriate solution or buffer. If the immunostimulant is provided in a lyophilized form, it may be used by dissolving it in an appropriate solution or buffer.

[0096] In a specific embodiment of the present invention, the inventors confirmed in vivo mRNA expression according to the mixing order of liposomes, mRNA, and immunostimulants for mRNA-liposome complexes prepared by controlling the mixing order of liposomes, mRNA, and immunostimulants.

[0097] In the present invention, the mRNA, liposome, and immunostimulant may be mixed by introducing them in the order of immunostimulant → mRNA → liposome.

[0098] By mixing mRNA, liposomes, and an immunostimulant in the above order, the in vivo mRNA delivery and expression rate of the manufactured mRNA-liposome complex can be increased.

[0099] Since the mRNA and liposome of the present invention are effective ingredients of the above-described mRNA delivery composition, description of overlapping content is omitted to avoid excessive complexity of the present specification.

[0100] The mRNA delivery composition comprising a cationic liposome according to the present invention has excellent storage stability and exhibits a high intracellular delivery rate and expression rate in vivo, thereby improving the stability and efficiency of mRNA vaccines for cancer treatment / prevention or mRNA vaccines for preventing viral or bacterial infections.

[0101] Figure 1 shows the results of confirming mRNA expression in mice using liposome-mRNA complexes prepared by varying the amount of mRNA used.

[0102] Figure 2 shows the results of confirming mRNA expression in mice using liposome-mRNA complexes prepared with liposomes having different ratios of DOTAP:DOPE.

[0103] Figure 3 shows the results of confirming mRNA expression in mice using liposome-mRNA complexes prepared with liposomes having different ratios of DOTAP:DOPE:cholesterol.

[0104] Figure 4 shows the results of analyzing the size, dispersion, and zeta potential of samples of mRNA-liposome complexes prepared by varying the mixing order of mRNA and liposomes.

[0105] Figure 5 shows the results of in vivo confirmation of differences in mRNA expression according to the mixing order of mRNA and liposomes.

[0106] Figure 6 shows the results of confirming the cellular immune response (Figure 6a), antibody immune response (Figure 6b), and neutralizing antibody titer (Figure 6c) after immunizing mice with mRNA-liposome complexes prepared by varying the mixing order of mRNA and liposomes.

[0107] Figure 7 shows the results of confirming mRNA expression in mice using liposome-mRNA complexes prepared by varying the amount of dLOS used.

[0108] Figure 8 shows the results of confirming mRNA expression when mRNA-liposome-dLOS complexes prepared by varying the mixing order of mRNA, liposome, and dLOS were injected into mice (LP: liposome; R: mRNA; dL: dLOS).

[0109] Figure 9 shows the results of measuring the size (Figure 9a), dispersity (Figure 9b), and zeta potential (Figure 9c) by storage period to measure the stability of mRNA-liposome complexes manufactured with different N / P ratios.

[0110] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0111]

[0112] Manufacturing Example 1. Manufacturing of mRNA-liposome complex (mRNA-liposome)

[0113] Liposome production (film method)

[0114] DOTAP (Merck & Cie / CH2900014), DOPE (Avanti Polar Lipid), and / or cholesterol (Avanti Polar Lipid) were each mixed with chloroform and completely dissolved at 37°C for 10 minutes to prepare a liquid solution.

[0115] The above liquid solutions were mixed in a round-bottom flask at a certain weight ratio to create a lipid mixture, and the mixture was evaporated in a rotary evaporator (Buchi / B491_R200) ​​at 60°C for 30 minutes to evaporate the chloroform and create a lipid membrane film on the flask wall.

[0116] The flask in which the lipid membrane film was prepared was filled with 20 mM HEPES buffer (pH 7.4) containing 4% (w / v) sucrose, and the lipid membrane was dissolved at 60°C to form liposomes with a liposome concentration of 7.5 mg / mL. The formed liposomes were measured for particle size, zeta potential, and dispersity using a dynamic light scattering analyzer. The remaining prepared liposomes were stored at 4°C until testing.

[0117]

[0118] Preparation of mRNA-liposome complexes

[0119] The liposomes prepared above (LP-DOTAP / DOPE / Chol (40:40:20, w / w / w) or LP-DOTAP / DOPE (50:50, w / w)) and mRNA for Renilla Luciferase (SEQ ID NO: 1) were mixed in 20 mM HEPES buffer (pH 7.4) containing 4% sucrose to prepare mRNA-liposome complexes.

[0120] At this time, the 7.5 mg / mL liposome solution (LP-DOTAP / DOPE / Chol (40:40:20, w / w / w)) was used as a sample that was refrigerated immediately after preparation or for up to 2 weeks after preparation. The 1 mg / mL Renilla luciferase mRNA (SEQ ID NO: 1) solution was stored at -70°C and thawed on ice immediately before use. The 20 mM HEPES buffer (pH 7.4) containing 4% sucrose was prepared immediately before the experiment or prepared the day before the experiment and then refrigerated.

[0121] The N / P ratio, which is the mixing ratio of liposomes and mRNA, was calculated using the following formula.

[0122]

[0123]

[0124] Example 1. Confirmation of mRNA expression according to the mRNA content of the mRNA-liposome complex.

[0125] In manufacturing the liposome-mRNA complex in the above Manufacturing Example 1, the amount of mRNA was varied to 5 ㎍, 10 ㎍, and 20 ㎍, and for each case, the amount of liposomes of DOTAP:DOPE:Chol (40:40:20, w / w / w) was mixed to 18.75 ㎍, 37.5 ㎍, and 75 ㎍ to manufacture a complex (N / P ratio = 0.7), and then the expression rate was confirmed in vivo.

[0126] Specifically, each liposome-mRNA complex was injected intramuscularly (IM) into the deltoid muscle of the femur in a volume of 100 μl into shaved mice (7-week-old C57BL / 6N female, Orient Bio, Central Laboratory Animal).

[0127] Six hours after administration, mice were anesthetized with avertin (250 mg / kg) and injected with luciferase substrate stock solution (ViviRen TM 200 μL of substrate prepared by adding 2.4 mL of 1X PBS to 0.15 μg / μL of in vivo renilla luciferase substrate stock solution was injected intravenously (IV injection).

[0128] Immediately after administration, the mouse was positioned in the IVIS equipment (Ami-HTX, USA) and the exposure time was set to 60 seconds to photograph the mouse (xenogen IVIS-200). The expression level of luciferase at the injection site (region of interest, ROI) was quantified using Aura Imaging Software (Spectral Instruments Imaging, USA).

[0129] As a result, as can be confirmed in Fig. 1, there was no difference in expression rates between 5 ㎍ and 10 ㎍ mRNA, but when 20 ㎍ mRNA was included, the expression rates increased by 135% and 170%, respectively, compared to when 5 ㎍ and 10 ㎍ mRNA were included. When mRNA increased above a certain level, the in vivo expression rate increased.

[0130]

[0131] Manufacturing Example 2. Manufacturing of mRNA-liposome complex with controlled mixing ratio of cationic lipid:neutral lipid:cholesterol (DOTAP:DOPE:cholesterol) in liposomes

[0132] 2-1. Adjusting the mixing ratio of DOTAP:DOPE

[0133] Liposomes were prepared at the ratio (w / w) of DOTAP:DOPE shown in Table 1 below, and then mixed with mRNA to prepare liposome-mRNA complexes. Liposomes containing 30 μg DOTAP per 20 μg mRNA (Renilla luciferase mRNA, SEQ ID NO: 1) were used so that the NP ratio (molar ratio) of the liposome-mRNA complexes was 0.7:1. At this time, as a liposome control, LNPs (C12-200:DSPC:Chol:DMG-PEG2000 = 50:10:38.5:1.5) in which C12-200, DSPC, Cholesterol, and DMG-PEG2000 were mixed in a ratio of 50:10:38.5:1.5 using NanoAssemblr Ignite (Precision NanoSystems) using the protocol recommended by the manufacturer were used.

[0134] Experimental group 1234567DOTAP203040507090100DOPE8070605030100

[0135]

[0136] 2-2. Adjusting the mixing ratio of DOTAP:DOPE:cholesterol

[0137] Liposomes were prepared at the ratios (w / w) of DOTAP:DOPE:cholesterol shown in Table 2 below, and then mixed with mRNA to prepare liposome-mRNA complexes. Liposomes containing 30 μg DOTAP per 20 μg mRNA (Renilla luciferase mRNA, SEQ ID NO: 1) were used so that the NP ratio of the liposome-mRNA complex was 0.7:1. Invivofectamine (Thermo Fisher Scientific) was used as a liposome control.

[0138] Experimental group 123456789DOTAP504540353020406080DOPE50454035306040200Chol01020304020202020

[0139]

[0140] Example 2. Confirmation of mRNA expression according to the mixing ratio of cationic lipid:neutral lipid:cholesterol (DOTAP:DOPE:cholesterol) in liposomes.

[0141] 2-1. In vivo expression rate according to the mixing ratio of DOTAP:DOPE

[0142] For each liposome-mRNA complex prepared in Manufacturing Example 2-1 above, in vivo expression was confirmed using the same method as in Example 1 above.

[0143] As a result, as can be seen in Fig. 2, liposome-mRNA complexes prepared with liposomes at DOTAP:DOPE ratios of 40:60, 50:50, and 70:30 showed high mRNA expression rates in mice.

[0144]

[0145] 2-2. In vivo expression rate according to the mixing ratio of DOTAP:DOPE:cholesterol

[0146] For each liposome-mRNA complex prepared in Manufacturing Example 2-2, in vivo expression was confirmed using the same method as in Example 1.

[0147] As a result, as can be seen in Fig. 3, when the ratio of DOTAP:DOPE was fixed at 1:1 and the ratio of cholesterol was adjusted, liposome-mRNA complexes using liposomes prepared with a DOTAP:DOPE:cholesterol ratio of 40:40:20 to 35:35:30 showed a high mRNA expression rate in mice, and when the ratio of cholesterol was fixed, liposome-mRNA complexes using liposomes prepared with a DOTAP:DOPE:cholesterol ratio of 20:60:20 to 40:40:20 showed a high mRNA expression rate in mice.

[0148]

[0149] Example 3. Characteristics of liposomes according to the mixing ratio of cationic lipid:neutral lipid:cholesterol (DOTAP:DOPE:cholesterol)

[0150] The liposome and mRNA-liposome complexes prepared in the above Manufacturing Example 2-2 were diluted 1 / 10 with 20 mM HEPES buffer (pH 7.4) containing 4% sucrose. Dynamic Light Scattering (DLS) analysis was performed using Zetasizer Nano ZSP (Malvern Pnanlytical) to measure the size, polydispersity index (PDI), and zeta potential of the complexes.

[0151] As a result, as can be seen in Table 3 (liposome) and Table 4 (liposome-mRNA complex), the size of the liposome was 100-200 nm, and the polydispersity was less than 0.4. When DOTAP and DOPE were in the same ratio, the particle size and polydispersity increased as the cholesterol content increased, and when the cholesterol content was the same at 20, the particle size and polydispersity tended to increase when the DOPE content was higher than that of DOATP (Table 3). The liposome-mRNA complexes were generally 180-240 nm, and the polydispersity was less than 0.3, and these values ​​increased compared to liposomes without mRNA mixing. When mRNA was mixed, no change in particle size according to the cholesterol content ratio was observed.

[0152] LP-DOTAP / DOPE / Chol (Weight ratio, 2 mg / mL) Analysis items DOTAP DOPE hol Particle size (d, nm) Dispersity 50 500 116.9 0.16 145 45 10 124.0 0.16 140 40 20 128.3 0.16 235 35 30 130.8 0.16 030 30 40 17 2.3 0.34 6 20 60 20 19 2.3 0.22 5 60 20 20 13 1.8 0.16 38 00 20 111.5 0.15 6

[0153] LP-DOTAP / DOPE / Chol + mRNA Analysis Items DOTAP DOPE hol Particle size (d, nm) Dispersity Zeta potential (mV) 50 500 197.8 0.208-63.5 45 45 10 195.8 0.188-63.9 40 40 20 22 2.20 24 4-61.2 35 35 30 184.0 0.176-60.0 30 30 40 22 3.0 0.258-56.0 20 60 20 23 6.7 0.210-48.3 60 20 20 19 0.30 179-66.28 00 20 187.6 0.207-68.0

[0154]

[0155] Manufacturing Example 3. Manufacturing of mRNA-liposome complexes with controlled mixing order of liposomes and mRNA.

[0156] A 1 mg / mL Renilla luciferase mRNA solution stored at -70°C in the above Preparation Example 1, and a refrigerated liposome solution (LP-DOTAP / DOPE / Chol (40:40:20, w / w / w)) and 20 mM HEPES buffer (pH 7.4) containing 4% sucrose were used.

[0157] The following manufacturing method is an example of manufacturing 400 μL of liposome-mRNA complex. The amount was increased at a certain ratio as needed, and the solution was mixed using known methods such as pipetting and stirring.

[0158]

[0159] 3-1. mRNA → Liposome sequence

[0160] Using a 200P tip specifically for RNA, 340 μL of 20 mM HEPES buffer (pH 7.4) containing 4% sucrose was dispensed into a microtube. Using a 200P tip, 40 μL of a completely thawed 1 mg / mL Renilla luciferase mRNA solution (40 μg mRNA) was taken and placed into the microtube containing the buffer, followed by pipetting approximately 10 times. Using a 200P tip, 20 μL (150 μg liposome) of a 7.5 mg / mL liposome solution (LP-DOTAP / DOPE / Chol (40:40:20, w / w / w)) was taken and placed in a microtube containing the buffer and mRNA solution, followed by pipetting approximately 30 times. Finally, the sample containing the buffer, mRNA solution, and liposome solution was further mixed by pipetting approximately 10 times using a 1000P tip.

[0161]

[0162] 3-2. Liposome → mRNA sequence

[0163] Using a 200P tip specifically for RNA, 340 μL of 20 mM HEPES buffer (pH 7.4) containing 4% sucrose was dispensed into a microtube. Using a 200P tip, 20 μL (150 μg liposomes) of a 7.5 mg / mL liposome solution (LP-DOTAP / DOPE / Chol (40:40:20, w / w / w)) was taken and placed into the microtube containing the buffer, followed by pipetting approximately 10 times. Using a 200P tip, 40 μL (40 μg mRNA) of a completely thawed 1 mg / mL Renilla luciferase mRNA solution was taken and placed into the microtube containing the buffer and liposome solution, followed by pipetting approximately 30 times. Finally, the sample containing the above buffer, mRNA solution, and liposome solution was further mixed by pipetting about 10 times with a 1000P tip.

[0164]

[0165] Example 4. Characteristics analysis according to the mixing order of liposomes and mRNA

[0166] DLS analysis was performed on the liposome-mRNA complex prepared in the above Manufacturing Example 3 to derive the average and standard deviation of the size, polydispersity index (PDI), and zeta potential of the complex.

[0167] As a result, as can be confirmed in Fig. 4, there was no significant difference in the physical properties of each liposome-mRNA complex.

[0168]

[0169] Example 5. Confirmation of mRNA expression according to the mixing order of liposomes and mRNA.

[0170] For liposome-mRNA complexes manufactured differently according to the mixing order in the above Manufacturing Example 3, in vivo expression was analyzed using the same method as in Example 1.

[0171] As a result, as can be confirmed in Fig. 5, mRNA expression was significantly higher in the complex manufactured by mixing in the order of mRNA → Liposome.

[0172]

[0173] Example 6. Confirmation of immunogenicity according to the mixing order of liposomes and mRNA.

[0174] The following experiment was conducted on liposome-mRNA complexes prepared in a different mixing order in the same manner as in Manufacturing Example 3, except that SARS-CoV-2 S mRNA (SEQ ID NO: 3) was used instead of mRNA for Renilla Luciferase.

[0175] First, liposome-mRNA complexes prepared by different mixing orders were administered intramuscularly to the left hind thigh of 6-week-old female mice (B6C3F1 / slc, central laboratory animals) at a dose of 0.1 HD (human dose) twice at 3-week intervals.

[0176]

[0177] 6-1. Cytokines

[0178] Splenocyte restimulation

[0179] Two weeks after the last administration, mice were sacrificed by cervical dislocation, spleens were removed, and the pooled spleens were transferred to a 24-well plate containing 1% penicillin-streptomycin solution in PBS (hereinafter, PBS w / antibiotics). The media used are shown in Tables 5 and 6 below.

[0180] Complete mediaAmount addedFinal concentrationRPMI 1640 medium439.9 ml-FBS(non-heat inactivated)50 ml10 %(v / v)Penicillin streptomycin solution(100X)(antibiotics)5 mlPenicillin(100 U / ㎕)Streptomycin(100㎍ / ㎕)HEPES buffer solution(1 M)5 ml10mM0.5 M β-mercaptoethanol50 ㎕50 uMRecombinant mouse IL-212.5 ㎕0.5 ng / ㎕)

[0181] Basal mediaAmount to be addedFinal concentrationRPMI 1640 medium439.9 ml-FBS(non-heat inactivated)50 ml10 %(v / v)Penicillin streptomycin solution(100X)(antibiotics)5 mlPenicillin(100 U / ㎕)Streptomycin(100㎍ / ㎕)HEPES buffer solution(1 M)5 ml10mM

[0182]

[0183] In a clean bench, spleen tissue was picked up with forceps, washed in PBS w / antibiotics, and transferred to a 60 mm dish containing 3 mL of basal media. The tissue was mashed using a 40 μm cell strainer to isolate splenocytes. The isolated splenocytes were transferred to a 15 mL tube, centrifuged at 4 °C and 3,000 rpm for 5 minutes, the supernatant was removed, and the cells were suspended in 3 mL of RBC lysis buffer, left to stand at room temperature for 3 minutes, and then centrifuged at 4 °C and 3,000 rpm for 5 minutes. The supernatant was removed, and the cells were suspended in 3 mL of PBS w / antibiotics, centrifuged at 4 °C and 3,000 rpm for 5 minutes, the supernatant was removed, and the cells were suspended in 10 mL of complete media. The above cell suspension was cultured in complete medium at 2 X 10 7 After diluting to cells / mL, it was dispensed into a 96-well cell culture plate at 100 μl / well.

[0184] Separately, PepMix SARS-CoV-2-S1 peptide pool (JPT) and SARS-CoV-2-S2 peptide pool (JPT) were dissolved in 50 μL of DMSO in each vial, and then mixed with complete medium to a final concentration of 2.5 μg / mL to prepare a SARS CoV-2 spike peptide stimulant.

[0185] The 96-well containing the cell suspension was added with 40 μg / well of the above stimulant and 60 μl / well of complete medium, and the reaction was performed for 72 hours under conditions of 37°C and 5% CO2.

[0186] Check IFN-γ concentration

[0187] The culture medium of the stimulated splenocytes was diluted 1 / 5 with a reagent diluent (1% BSA), dispensed at 100 μl / well onto a microplate coated with anti-mouse IFN-γ capture antibody (Jackson), covered with a sealing film, and left to stand at room temperature for 2 hours. The solution in each well was removed with an ELISA washer (Tecan / Hydroflexelisa) and washed three times with a washing buffer.

[0188] Streptavidin-HRP in the IFN-γ ELISA kit (Mouse IFN-γ Duoset ELISA, R&D systems) was diluted 1 / 40 using a reagent diluent, dispensed 100 μl / well onto an immunoplate, covered with a sealing film, and left to stand at room temperature for 20 minutes. The solution in each well was removed with an ELISA washer, and washed three times using a washing buffer.

[0189] The anti-mouse IFN-γ detection antibody in the kit was diluted to 200 ng / mL using a reagent diluent, dispensed 100 μL / well onto the immunoplate, covered with a sealing film, and left to stand at room temperature for 1 hour. The solution in each well was removed using an ELISA washer (Tecan / Hydroflexelisa), and washed three times using a washing buffer.

[0190] 100 μl of TMB substrate (KPL sureblue TMB microwell peroxidase substrate, Seracare) solution was dispensed onto each immunoplate and reacted in a dark place at room temperature for 15 minutes. Then, 100 μl of 1N H2SO4 solution was dispensed onto each immunoplate to stop the reaction and the absorbance was measured at 450 nm using an ELISA reader.

[0191] As a result, as can be confirmed in Fig. 6a, the IFN-γ concentration was highest in the complex prepared by mixing in the order of mRNA → Liposome.

[0192]

[0193] 6-2. Antibody titers (IgG titers)

[0194] Two weeks after the last administration, the mice were anesthetized with an intraperitoneal injection of 250 mg / kg avertin working solution, and whole blood was collected by cardiac puncture. The collected whole blood was transferred to a microtube, allowed to stand at room temperature for 3 hours, and then centrifuged at 4°C and 15,000 rpm for 10 minutes. The supernatant was transferred to a new microtube to secure serum, which was then stored at -20°C until analysis.

[0195] Next, RBD (SARS-CoV-2 receptor binding domain) antigen (Mybiosource, USA) was diluted to 1 μg / mL using 1X PBS, dispensed 100 μl / well into the immunoplate, covered with a sealing film, and left to stand overnight at 4 °C. The solution in each well was removed with an ELISA washer (Tecan / Hydroflexelisa) and washed five times using a washing buffer (500 μl of tween20 added to 1 L of 1X PBS prepared by diluting 20X PBS with purified water). 200 μl / well of reagent diluent (1% BSA, prepared by dissolving 1 g of BSA in 100 mL of PBS) was dispensed into the immunoplate, covered with a sealing film, and left to stand in a 37 °C reactor for 1 hour. The solution in each well was removed with an ELISA washer and washed five times with washing buffer. The reagent diluent was dispensed into the immunoplate at 100 μl / well.

[0196] The serum obtained above was diluted 1:50 using a reagent diluent (1% BSA), dispensed as 100 μl into row 1 of B to G of the immunoplate, pipetted several times within the well to mix the sample, and then 100 μl from row 1 was taken and placed into row 2, thereby performing a 1 / 2 serial dilution up to row 12 on the ELISA plate. At this time, in order to evaluate the suitability of the test, hyper serum was diluted 1:200 using a reagent diluent, dispensed as 100 μl into row 1 of each immunoplate H, and serially diluted 1 / 2 in the same manner as above.

[0197] The immunoplate was covered with a sealing film and incubated in a 37°C reactor for 2 hours. The solution in each well was removed with an ELISA washer and washed five times with washing buffer. Goat anti-mouse IgG antibody (Jackson Laboratory) was diluted 1:5,000 using a reagent diluent, dispensed 100 μl per immunoplate, covered with a sealing film, and incubated in a 37°C reactor for 1 hour. The solution in each well was removed with an ELISA washer and washed five times with washing buffer.

[0198] The TMB substrate solution equilibrated to room temperature was dispensed into 100 μl of each immunoplate and reacted in a dark place at room temperature for 5 minutes. The reaction was stopped by dispensing 100 μl of 1 N H2SO4 solution into each immunoplate, and the absorbance was measured at 450 nm using an ELISA reader (Biotek / Epoch).

[0199] As a result, as can be seen in Figure 6b, the antibody immune response was the best in the complex prepared by mixing in the order of mRNA → Liposome.

[0200]

[0201] 6-3. Analysis of surrogate neutralization (%)

[0202] 60 μL of each of the negative control (DMEM medium) or the immunized mouse serum sample obtained in Example 6-2 and stored at -20 °C was mixed with 60 μL of 1:1000 diluted-HRP conjugated RBD in a 1.5 mL microtube, and reacted at 37 °C for 30 minutes. 100 μL was dispensed onto a microtiter test strip plate, covered with a sealing film, and reacted at 37 °C for 15 minutes. The solution in each well was removed with an ELISA washer and washed four times with 1X washing solution. 100 μL of TMB solution was dispensed / well, covered with a sealing film, and reacted for 15 minutes in the dark at room temperature. 50 μL of stop solution was dispensed / well to stop the reaction, and then the optical density was measured at 405 nm using an ELISA reader.

[0203] As a result, as can be confirmed in Fig. 6c, the complex prepared by mixing in the order of mRNA → Liposome showed the highest level of neutralizing antibody titer induction ability.

[0204]

[0205] Sintering

[0206] There were differences in in vivo expression and immunogenicity induction ability depending on the mixing method when manufacturing mRNA and liposome complexes, and the best effect was found when mixing in the order of mRNA → Liposome.

[0207]

[0208] Manufacturing Example 4. Manufacturing of mRNA-liposome complexes with controlled mixing order of liposomes, mRNA, and immune enhancers.

[0209] 4-1. mRNA-liposome complex containing liposomes, mRNA, and an immunostimulant

[0210] In the above manufacturing example 1, an mRNA-liposome complex was manufactured by additionally mixing the immunostimulant dLOS (detoxified Lipooligosaccharide) (TLR4 agonist; Ijin Co., Ltd., Korea).

[0211]

[0212] 4-2. Controlling the mixing order of liposomes, mRNA, and immune boosters

[0213] The 1 mg / mL Renilla luciferase mRNA solution stored at -70°C in the above Preparation Example 1, the refrigerated liposome solution (LP-DOTAP / DOPE / Chol (40:40:20, w / w / w)) and 20 mM HEPES buffer (pH 7.4) containing 4% sucrose were used. Additionally, the immunostimulant dLOS was used. The samples were mixed in the following six ways, which are the number of cases in which the mixing order of the three solutions (mRNA, liposomes, and dLOS) can be combined:

[0214] a: Liposome → mRNA → dLOS

[0215] b: Liposome → dLOS → mRNA

[0216] c: mRNA → Liposome → dLOS

[0217] d: mRNA → dLOS → Liposome

[0218] e: dLOS → Liposome → mRNA

[0219] f: dLOS → mRNA → Liposome

[0220] Specifically, a 200P tip specifically for RNA was used to take a quantitative amount of 20 mM HEPES buffer (pH 7.4) containing 4% sucrose and dispensed into a microtube. The first quantitative amount of solution was taken using the 200P tip, placed into the microtube where the buffer was dispensed, and pipetted approximately 10 times. The second quantitative amount of solution was taken using the 200P tip, placed into the microtube where the buffer was dispensed, and pipetted approximately 10 times. The third quantitative amount of solution was taken using the 200P tip, placed into the microtube where the buffer was dispensed, and pipetted approximately 10 times. Finally, the solution was mixed by pipetting 30 times using the 1000P tip.

[0221]

[0222] Example 7: Confirmation of mRNA expression according to the content of the immune enhancer.

[0223] In the above Manufacturing Example 4-1, the immune enhancer dLOS was added in different amounts to confirm the in vivo expression rate of the liposome-mRNA complex. At this time, dLOS was added in amounts of 0.25 ㎍, 0.5 ㎍, 0.75 ㎍, and 1 ㎍, and the in vivo expression confirmation was performed in the same manner as in Example 1.

[0224] As a result, as shown in Fig. 7, the expression level was found to increase when the amount of dLOS added was 0.25 ㎍ or more and 1 ㎍ or less.

[0225]

[0226] Example 8: Confirmation of mRNA expression according to the mixing order of liposomes, mRNA, and immunostimulants.

[0227] For each liposome-mRNA complex prepared in Manufacturing Example 4-2 above, in vivo expression was confirmed using the same method as in Example 1 above.

[0228] As a result, as shown in Fig. 8, a higher expression level was confirmed in mice administered a composition mixed in the order of dLOS → mRNA → Liposome.

[0229]

[0230] Example 9: Confirmation of changes in stability (physicochemical properties) of mRNA-liposome complexes over time.

[0231] [EGFP]

[0232] The changes in physicochemical properties of a complex of EGFP mRNA (SEQ ID NO: 2) and liposomes (DOTAP:DOPE:Chol 40:40:20, w / w / w) over time after preparation were confirmed through DLS measurement.

[0233] Specifically, the EGFP mRNA-liposome complex prepared above was stored in a lyophilized formulation for 9 weeks, and the size, zeta potential, and PDI were measured weekly.

[0234] As a result, as can be confirmed in Fig. 9, it was found that the NP ratio was stable for 9 weeks at 1.39:1 or less.

[0235]

[0236] [SARS-CoV-2 S]

[0237] The physicochemical properties of a complex of SARS-CoV-2 S mRNA (SEQ ID NO: 3) and liposomes (DOTAP:DOPE:Chol 40:40:20, w / w / w) in liquid and lyophilized forms were examined over time after manufacture using DLS measurements.

[0238] Specifically, the SARS-CoV-2 S mRNA-liposome complex prepared above was stored in a refrigerator for 16 weeks, and the size, zeta potential, and PDI were measured at regular intervals (0, 2, 4, 8, 12, and 16 weeks).

[0239] As a result, as can be seen in Table 7 (liquid formulation) and Table 8 (lyophilized formulation), both the liquid formulation and the lyophilized formulation were found to be stable for 16 weeks.

[0240] Category 0 Week 2 Week 4 Week 8 Week 12 Week 16 Week Particle Size (nm) 216.0 225.8 227.1 222.9 199.9 196.5 Dispersity 0.187 0.194 0.175 0.2 190.187 0.159 Zeta Potential (mV) -62.7 - 56.4 - 52.3 - 55.0 - 72.3 - 68.7

[0241] Category 0 Week 2 Week 4 Week 8 Week 12 Week 16 Week Particle Size (nm) 339.13 16.13 22.4 329.22 80.5 27 2.0 Dispersity 0.28 40.21 50.24 50.26 40.24 30.22 8 Zeta Potential (mV) -45.4 - 43.7 - 42.8 - 43.8 - 60.7 - 60.2

[0242] The present invention relates to a composition for intracellular delivery of mRNA, and more particularly, to a composition for intracellular delivery of mRNA comprising a cationic lipid-based liposome and a method for preparing the same.

Claims

1. A composition for mRNA delivery comprising cationic lipid-based liposomes.

2. A composition for mRNA delivery in claim 1, wherein the liposome additionally contains a neutral lipid.

3. A composition for mRNA delivery in the second paragraph, wherein the liposome additionally contains cholesterol.

4. In the first paragraph, the cationic lipid is dimethyldioctadecylammonium bromide (DDA), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 3β-[N-(N',N'-dimethylaminoethane) carbamoyl cholesterol (DC-Chol), 1,2-dioleoyloxy-3-dimethylammoniumpropane (DODAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 Etyle PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 Ethyl PC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 Ethyl PC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholin (18:0 Ethyl PC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 Ethyl PC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 Ethyl PC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholin (12:0 Ethyl PC),N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dimyristoyl-3-dimethylammoniumpropane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0DAP), 1,2-distearoyl-3-dimethylammonium-propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP), 1,2-dipalmitoyl-3-trimethylammoniumpropane (16:0 TA), A composition for mRNA delivery, wherein at least one is selected from the group consisting of 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP) and N4-cholesteryl-spermine (GL67).

5. In the second paragraph, the neutral lipid is 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), A composition for mRNA delivery, comprising at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), phosphatidylserine (PS), phosphoethanolamine (PE), phosphatidylglycerol (PG), phosphoric acid (PA), and phosphatidylcholine (PC).

6. A composition for mRNA delivery in the second paragraph, wherein the weight ratio of the cationic lipid and the neutral lipid is 1:9 to 9.5:0.

5.

7. A composition for mRNA delivery in claim 3, wherein the weight ratio of the cationic lipid and cholesterol is 6:1 to 1:

3.

8. A composition for mRNA delivery in claim 7, wherein the weight ratio of the cationic lipid, neutral lipid, and cholesterol is 1 to 9.5:9 to 0.5:0.05 to 3.

9. A composition for delivering mRNA in claim 1, wherein the mRNA encodes a peptide or protein that can act as an immunogen.

10. A composition for mRNA delivery in claim 9, wherein the N:P ratio of the liposome and mRNA is 0.23:1 to 1.39:

1.

11. A composition for mRNA delivery, wherein the composition further comprises an immunostimulant in claim 1.

12. A composition for mRNA delivery in claim 11, wherein the immune-enhancing agent is at least one selected from the group consisting of PAMP, saponin, CpG DNA, lipoprotein, flagella, poly I:C, squalene, tricaprin, 3D-MPL, and detoxied lipooligosaccharide (dLOS).

13. A pharmaceutical composition for the prevention or treatment of a disease selected from the group consisting of cancer, tumor, autoimmune disease, genetic disease, inflammatory disease, viral infection and bacterial infection, containing the mRNA delivery composition of any one of claims 1 to 12 as an active ingredient.

14. A vaccine containing the mRNA delivery composition of any one of claims 1 to 12 as an active ingredient.

15. A functional cosmetic composition containing the mRNA delivery composition of any one of claims 1 to 12 as an active ingredient.

16. A method for producing a composition for mRNA delivery, comprising the step of mixing mRNA and liposomes.

17. A method for producing a composition for mRNA delivery in claim 16, wherein the method comprises mixing mRNA and liposomes in that order.

18. A method for producing a composition for mRNA delivery, wherein the method further comprises a step of mixing an immunostimulant in the 16th paragraph.

19. A method for producing a composition for mRNA delivery, wherein the method comprises mixing an immune enhancer, mRNA, and liposome in that order.

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