WO2022173199A1 - Vaccine composition for preventing covid-19, comprising ionic complex of cationic molecular transporter and sars-cov-2 mrna - Google Patents
Vaccine composition for preventing covid-19, comprising ionic complex of cationic molecular transporter and sars-cov-2 mrna Download PDFInfo
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- WO2022173199A1 WO2022173199A1 PCT/KR2022/001940 KR2022001940W WO2022173199A1 WO 2022173199 A1 WO2022173199 A1 WO 2022173199A1 KR 2022001940 W KR2022001940 W KR 2022001940W WO 2022173199 A1 WO2022173199 A1 WO 2022173199A1
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- mrna
- glycero
- vaccine composition
- propane
- ethylphosphocholine
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
Definitions
- the present invention relates to a vaccine composition that efficiently delivers a nucleic acid into the body, including a cationic molecular transporter and an ionic complex of the nucleic acid.
- Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is causing a worldwide pandemic, is an RNA virus that has a spherical spike with a characteristic crown shape with an external spike protein. It is composed of (S), membrane (M), envelope (E), and nucleocapsid (N).
- SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2
- M membrane
- E envelope
- N nucleocapsid
- WHO World Health Organization
- mRNA messenger RNA
- mRNA vaccine There are three major advantages of such an mRNA vaccine.
- mRNA vaccines also have disadvantages that need to be supplemented.
- mRNA has a large molecular weight, high negative charge, and is vulnerable to RNA-degrading enzymes in the body.
- the lipid nanoparticle technology used by Pfizer/BioNtech and Moderna is being applied to vaccines, but this also has the following problems.
- Pfizer/BioNtech and Moderna vaccines cannot be said to be efficient because they use excessive amounts of mRNA of 30 ⁇ g and 100 ⁇ g, respectively, as a single inoculation dose. If mRNA usage is reduced by increasing delivery efficiency, more vaccines can be supplied with the same amount of production, and the cost of vaccines will also be reduced.
- lipid nanoparticles are unstable and must be stored at low or cryogenic temperatures.
- vaccines from Pfizer/BioNtech and Moderna must be distributed and/or stored at -70°C and -20°C or lower, so compared to most existing vaccines handled at 4°C, it is required to establish a cryogenic distribution system and storage facilities for handling institutions. Although it is the most powerful vaccine, it may have difficulties in rapidly distributing it. Therefore, there is an urgent need to develop a new delivery technology that can overcome the disadvantages of delivery efficiency and thermal stability of the above-mentioned lipid nanoparticles.
- Liposomes are in the form of microvesicles in which amphiphilic lipid molecules such as phospholipids, which are components of living cell membranes, form a double layer or multiple double layers, and have a hydrophilic space inside and a lipid membrane structure outside. Therefore, liposome preparations as various drug transporters are being developed by combining a water-soluble drug into the hydrophilic space, and a fat-soluble drug and an electric charge, as an external lipid membrane, with the advantage of excellent biocompatibility.
- cationic liposome and anionic nucleic acid are mixed in a certain ratio and the nucleic acid-liposome complex formed through electrostatic interaction facilitates endocytosis, which facilitates intracellular passage and moves through the endosome to the cytoplasm. This can be easy.
- It is an object of the present invention to provide a vaccine composition comprising (a) an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein, and (b) an ion complex comprising a cationic molecular transporter of the following formula (1) .
- R 1 is , where n is an integer from 1 to 8.
- Another object of the present invention is to prepare a cationic molecular transporter represented by the formula (1); And (b) to provide a method for preparing a vaccine composition comprising the step of preparing an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein and mixing it with the result of step (a).
- the present invention for solving the above problems is (a) an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein, and (b) a vaccine comprising an ion complex comprising a cationic molecular transporter of the following formula (1)
- a composition is provided.
- R 1 is , where n is an integer from 1 to 8.
- the ionic complex further comprises a cationic liposome.
- the liposomes of the present invention can be prepared by various techniques currently known in the art.
- Multi-lamellar vesicles are prepared by conventional techniques, for example by dissolving lipids in a suitable solvent and depositing selected lipids on the inner wall of a suitable container or vessel, and then It can be prepared by evaporating the solvent or spray drying to leave a thin film on the inside of the container. The aqueous phase can then be added to the vessel in a vortexing motion that causes the formation of MLV.
- Uni-lamellar vesicles UUVs
- UUVs can then be formed by homogenization, sonication or extrusion of multi-lamellar vesicles.
- the mRNA compound may form an ionic complex by electrostatic interaction with the cationic molecular transporter and/or cationic liposome.
- the mass ratio of the mRNA compound and the cationic liposome is 1:1 to 1:6.
- the mass ratio of the mRNA compound and the cationic liposome was 1:3, and when the mass ratio (mRNA: cationic liposome) is less than 1:1, the amount of the liposome may be insufficient to transport the mRNA, and the mass ratio When is greater than 1:6, an excess of liposomes may interfere, so that it may be difficult to transport a sufficient amount of mRNA into a cell.
- the antigenic peptide or protein is derived from a SARS-Cov-2 spike protein, a nucleocapsid protein, or a fragment or variant thereof.
- the mRNA sequence comprises an RNA sequence corresponding to the RBD sequence of the SARS-Cov-2 spike protein according to SEQ ID NO: 1.
- the mRNA sequence may include the following elements in a 5' to 3' direction.
- the 5'-UTR element may comprise or consist of an RNA sequence corresponding to the sequence according to SEQ ID NO: 2, wherein the coding region encoding the antigenic peptide or protein comprises an RNA sequence corresponding to the sequence according to SEQ ID NO: 1 wherein the 3'-UTR element comprises or consists of an RNA sequence corresponding to the sequence according to SEQ ID NO: 3, wherein the poly A tail is an RNA corresponding to the sequence according to SEQ ID NO: 4 may comprise or consist of a sequence.
- the mRNA sequences of the present invention may include non-nucleotide linkages or incorporation of modified nucleotides into the sequence.
- it may be a modification to one or both of the 3' and 5' ends of an mRNA molecule encoding an antigenic peptide or protein.
- modifications may include addition of bases to the mRNA sequence (e.g., containing a poly A tail or a longer poly A tail), complexing the mRNA with a substance (e.g., a protein or complementary nucleic acid molecule), resulting in a 3' UTR or Modification of the 5' UTR, and elements that change the structure of the mRNA molecule (eg, form a secondary structure).
- the modification may be present on a base, and may be selected from the group consisting of pseudouridine or N1-methylpseudouridine.
- the poly A tail of the present invention may be for stabilizing native mRNA.
- a long poly A tail can be added to an mRNA molecule to make the mRNA more stable.
- Poly A tails can be added using techniques known in the art.
- the length of the poly A tail was 90 to 200 adenines. Since the length of the poly A tail can affect the half-life of the mRNA molecule, the length of the poly A tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thus to control the time course of intracellular protein expression. can
- the mRNA of the present invention can be selectively bound with a reporter gene that facilitates, for example, the determination of mRNA delivery to a target cell or tissue.
- Suitable reporter genes can include, for example, green fluorescent protein mRNA (GFP mRNA), luciferase mRNA (Luciferase mRNA), firefly luciferase mRNA, or any combination thereof.
- the composition of the present invention may include a stabilizing reagent.
- the composition may include one or more agent reagents that directly or indirectly bind to and stabilize mRNA, thereby enhancing residence time in the target cell.
- agent reagents are preferably capable of improving the half-life of mRNA in the target cell.
- Stabilization reagents include one or more proteins, peptides, aptamers, translation accessory proteins, mRNA binding proteins, and/or translation initiation factors.
- the cationic liposome is dimethyldioctadecylammonium bromide (DDA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 3 ⁇ -[N-(N') ,N'-dimethylaminoethane carbamoyl cholesterol (3 ⁇ -[N-(N'N'-dimethylaminoethane) carbamoyl cholesterol, DC-Chol), 1,2-dioleoyloxy-3-dimethylammonium propane (DODAP) , 1,2-di-O-octadecenyl-3-triethylammonium propane (1,2-di-O-octadecenyl-3-trimethylammonium propane, DOTMA), 1,2-dimyristoreoyl-sn -Glycero-3-ethylphosphocholine (1,2-dimyristoyl-sn-glycero-3-e
- DDA dimethyldi
- the cationic liposome is (a) 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine, DMPC ), 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 1,2-dioleoyl-sn-glycero -3-Phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoyl- sn-glycero-3-phosphocholine, DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl
- the cationic liposome is to include DOTAP, DOPE and cholesterol.
- the molar ratio of DOTAP, DOPE and cholesterol may be 1:0.2-0.8:0.2-0.8. If the content of DOPE and cholesterol is lower than 0.2, intracellular mRNA delivery efficiency may be reduced, and if it is higher than 0.8, the capture rate of liposome mRNA may be reduced, so it is preferable to prepare in the above ratio.
- the mRNA compound may include at least one modification that imparts stability to the RNA molecule.
- Modifications that confer stability to an RNA molecule typically include in vivo degradation (eg, by exo- or endo-nucleases), ex vivo degradation (eg, by manufacturing processes prior to vaccine administration, for example). (e.g., during the manufacture of the administered vaccine solution) represents a modification that increases resistance.
- Stabilization of RNA can be achieved, for example, by providing a 5'-CAP structure, a poly A tail, or any other UTR modification.
- stabilization of RNA can be achieved by chemical modification or modification of the G/C content of the nucleic acid. A variety of other methods known in the art can be employed to achieve a modification that imparts stability.
- the ratio of the anion of the mRNA compound to the cation of the cationic molecular receptor is 0.1 to 1.
- N/P ratio when the N/P ratio is less than 1 (that is, when the ratio of cations is greater than the ratio of anions), as the ratio of N/P decreases, the electrostatic interaction increases, thereby facilitating the formation of an ionic complex.
- it is important to select an appropriate ratio because there is a possibility of causing toxicity to cells in the presence of an excess of cations.
- the N/P ratio is greater than 1 (ie, the ratio of anions is greater than the ratio of cations)
- the mRNA sequence comprises a modification of the 5' untranslated region of the RNA molecule.
- the mRNA sequence comprises a modification of the 3' untranslated region of the RNA molecule.
- said modification comprises modification of poly A tail.
- a substance for facilitating transport of the mRNA compound to the intracellular section of the target cell is further included.
- the target cells are hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, osteocytes, stem cells, mesenchymal cells, nerve cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.
- composition of the present invention may be combined with a substance for promoting transport of mRNA compounds, for example, substances that interfere with or improve the permeability of the vascular brain barrier to enhance transport of exogenous mRNA to target cells.
- composition of the present invention may include a pharmaceutically acceptable carrier, which is commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin. , calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc. It is not limited.
- composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.
- a lubricant e.g., a talc, a kaolin, a kaolin, a kaolin, a kaolin, a kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol
- composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.
- a suitable dosage of the composition of the present invention may be variously prescribed depending on factors such as formulation method, administration mode, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. have.
- composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person skilled in the art to which the present invention pertains, or It can be prepared by pouring into a multi-dose container.
- the formulation may be in the form of a solution, suspension, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.
- the composition is lyophilized.
- the lyophilized composition according to the present invention may be reconstituted prior to administration or may be reconstituted in vivo.
- the lyophilized composition may be formulated into an appropriate dosage form, for example, an intradermal dosage form such as a disc, rod or membrane, and administered such that the dosage form is rehydrated over time in vivo by a subject's body fluids.
- an appropriate dosage form for example, an intradermal dosage form such as a disc, rod or membrane
- the dosage form is rehydrated over time in vivo by a subject's body fluids.
- the freeze-dried formulation treated with the cationic molecular transporter SG6 of the present invention it exhibits a 7-fold higher neutralizing antibody-forming ability than the control, and inducing the neutralizing ability of the RBD protein without a decrease in activity. It was confirmed that it showed an excellent effect on mRNA delivery, protein expression, and stability of the vaccine composition.
- an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein, and (b) a cationic molecular transporter of Formula 1 It provides a method comprising administering a composition comprising an ionic complex comprising
- R1 is , where n is an integer from 1 to 8.
- step (a) preparing a cationic molecular transporter represented by the formula (1); And (b) provides a method for preparing a vaccine composition comprising the step of preparing an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein and mixing it with the result of step (a).
- R1 is , where n is an integer from 1 to 8.
- step (b) is a step of preparing an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein, reacting with cationic liposome, and mixing with the result of step (a).
- step (a) when the mass ratio of the mRNA compound and the cationic liposome was 1:3, the mRNA encapsulation efficiency was about 50%, but when the present invention cationic molecular transporter SG6 was treated, the encapsulation efficiency was 30% or more increased, and was measured to be greater than 80%.
- a method for producing a vaccine composition comprising the step of (c) freeze-drying the resultant of (b) after step (b).
- the ion complex provides a method in which the antigenic peptide or protein is derived from a SARS-Cov-2 spike protein, a nucleocapsid protein, or a fragment or variant thereof.
- the mRNA sequence comprises an RNA sequence corresponding to the RBD sequence of the SARS-Cov-2 spike protein according to SEQ ID NO: 1.
- cationic liposomes include DOTAP, DOPE and cholesterol.
- the ratio of the anion of the mRNA compound to the cation of the cationic molecular receptor is 0.1:1 to 1:1.
- the mass ratio of the mRNA compound to the cationic liposome is 1:1 to 1:6.
- a vaccine composition comprising an mRNA encoding an antigenic peptide or protein and a cationic molecular transporter (SG6) that forms an ion complex therewith.
- SG6 cationic molecular transporter
- the vaccine composition according to the present invention has no cytotoxicity, high mRNA delivery efficiency, and provides a vaccine composition that is easy to store because there is no decrease in mRNA activity in liquid as well as freeze-dried formulations.
- 1 is a diagram showing the composition of the mRNA of the coronavirus infection-19 vaccine to be developed in the present invention.
- FIG. 2 is a diagram showing the DNA sequence of each configuration for producing the mRNA of FIG. 1 .
- FIG. 3A shows that the plasmid DNA into which each component of FIG. 2 is inserted is extracted from E. coli, linearized with a Bbs1 restriction enzyme, and then electrophoresed using 1% agarose gel (SDS). -polyacrylamide gel electrophoresis) method,
- (b) is a diagram showing the result of electrophoresis after recovering the linearized DNA using ethanol precipitation method after (a).
- FIG. 4 is a view showing the results of confirming the mRNA produced through the IVT (in vitro transcription) reaction by electrophoresis using a 1% denaturing agarose gel (denaturing agarose gel).
- FIG. 5 is a diagram showing the results of confirming by Western blot using an RBD antibody whether mRNA produced through IVT operates normally in cells to express RBD protein.
- Figure 6 (a) is a diagram showing the electrophoresis test results confirming that the present invention cationic molecular transporter 'SG6' properly forms an ionic complex with mRNA
- Figure 6 (b) is used as a comparative example
- FIG. 7 is a diagram showing the cytotoxicity evaluation results through alamar blue assay in HEK293 cells of the cationic molecular transporter 'SG6' of the present invention and the cationic molecular transporter R6 peptide used as a comparative example and PEI. .
- FIG. 9 is a view showing a cryo-Transmission Electron Microscope (Cryo-Transmission Electron Microscope, Glacios microcscope, Thermo Fisher Scientific) photograph of the cationic liposome prepared by the Example method of the present invention.
- FIG. 10 is a view showing the electrophoresis test results to confirm that the cationic liposome prepared by the Example method of the present invention and SARS-CoV-2 RBD mRNA properly form an ion complex (mRNA lipoplex).
- FIG. 11 is a diagram showing the results of Alamar Blue analysis compared with lipofectamine whether cationic liposomes prepared by the Example method of the present invention have cytotoxicity in the HEK293T cell line.
- FIG. 12 is a result showing the mRNA loading of the cationic liposome prepared by the Example method of the present invention.
- (a) is a diagram showing the quantification of the encapsulation rate by ribogreen analysis
- (b) is a diagram showing that mRNA is maintained up to 24 hours.
- FIG. 13 is a diagram showing the results of confirming through an electrophoresis test whether mRNA encapsulated in cationic liposomes prepared by the method of the present invention is protected from ribonucleic acid degrading enzyme.
- FIG. 14 is a diagram showing the results of comparative analysis of RBD mRNA protein expression rates of the lipofectamine-RBD mRNA ion complex and the cationic liposome-RBD mRNA ion complex prepared by the Example method of the present invention by a Western blot method.
- 15 is a view showing the results of confirming the neutralizing antibody-forming ability of the cationic liposome-RBD mRNA ion complex prepared by the Example method of the present invention through an animal experiment.
- a vector including a corresponding DNA template was constructed.
- the DNA sequence of each configuration is shown in FIG. 2 .
- sequences derived from human hemoglobin and human IgE genes were used with reference to the literature (Richner et al., 2017, cell, 168, 1114-1125).
- the receptor binding domain (RBD) of SARS-CoV-2 spike protein was adopted, and the gene sequence of the RBD region (amino acids 319-541) of the spike protein of Wuhan strain (NCBI Genbank (No.: QHD43416.1) These sequences were chemically synthesized after codon optimization to ensure stable expression of mRNA in the human body, and pGS-IVT-RBD containing the T7 promoter and 102 adenine poly A tails A vector (Genscript) was constructed.
- a DNA template for the production of mRNA in the IVT reaction was prepared as follows. After propagating the E. coli DH5a bacteria into which the pGS-IVT-RBD vector was inserted in LB medium (Luria-Bertani Broth) supplemented with 100ug/ml ampicillin, the vector was isolated and purified using a plasmid preparation kit. did. For linearization, 2ug of pGS-IVT-RBD vector was quantified, and the end of the poly A tail was cut using a Bbs1-HF (NEB) restriction enzyme (37°C, 12 hours), and the reaction product was subjected to 1% agarose gel electrophoresis. was confirmed as
- mRNA was produced by an in vitro transcription method using a Megascript kit (Ambion, USA). 500ng of the DNA template sample obtained above was used. ARCA cap (Trilink, USA) was separately configured and 120 mmol was used for the reaction. The reaction composition was configured as shown in Table 1 below, and the reaction was carried out at 37°C for 12 hours. After completion of the reaction, 2 units of Turbo DNase (Ambion, USA) were treated at 37° C. for 15 minutes to remove the DNA template.
- Lithium chloride precipitation method was used to separate and purify the produced mRNA. Lithium chloride reagent and 30 ul of nuclease-free water were each added to the reaction mixture, and then precipitated at 20° C. for 30 minutes. The precipitated mRNA pellet was washed once with 70% ethanol after centrifugation, and the pellet was dried in a clean bench for 30 minutes, dissolved in nuclease-free water, and confirmed by electrophoresis using 1% denatured agarose gel. .
- mRNA produced through IVT was confirmed using a commercially available cationic delivery system. 0.5ug and 1ug mRNA were mixed with 0.75ul and 1.5ul of lipofectamine, messenger MAX TM (Invitrogen, USA), respectively, and then treated and transfected in 1x10 5 HEK293 cells in a 24-well plate for 48 hours. The supernatant was recovered and RBD expression was confirmed by Western blot.
- As the primary antibody a 1000-fold diluted SARS-CoV-2 (2019-nCoV) mouse anti-spike protein monoclonal antibody (Sino biological, China) was used.
- Cationic compound SG6 having 6 guanidine groups (+6: 1 positive charge per guanidine group) with sorbitol according to Formula 1 as a skeleton of the present invention was prepared in the method described in Example 8 of Korean Patent No. 10-0699279 prepared according to
- R 1 is , where n is an integer from 1 to 8.
- a cationic molecular transporter (Sorbitol-G6; SG6, molecular weight 1112.7 g/mol) based on sorbitol, a biocompatible material and cationic guanidinium, is used for efficient mRNA delivery.
- a cationic molecular transporter (Sorbitol-G6; SG6, molecular weight 1112.7 g/mol) based on sorbitol, a biocompatible material and cationic guanidinium
- an R6 peptide consisting of PEI (polyethyleneimine-HCl, molecular weight 4668 g/mol, Sigma) and 6 arginine, a cationic amino acid, was used.
- PEI polyethyleneimine-HCl, molecular weight 4668 g/mol, Sigma
- 6 arginine a cationic amino acid
- each material is dissolved in nuclease-free water based on 500 ng mRNA, and then the ionic complex is formed according to Table 2 for 30 minutes, followed by electrophoresis using a 1% denatured agarose gel was done .
- the cytotoxicity of SG6 and R6 itself was hardly observed, whereas in the case of PEI, the CC50 value was 50 uM (FIG. 7).
- saponin which is generally used as a positive control for cytotoxicity tests, it was similar to the CC50 value (20-30uM) known in the literature.
- lipofectamine2000 TM (Invitrogen, USA) and Messenger MAX TM (Invitrogen, USA) were used to form an ion complex with the mRNA produced according to the manufacturer's manual. 0.2ul of lipofectamine2000 TM and 0.15ul of Messenger MAX TM were used per 100ug of mRNA. After treatment with Alamar blue (Invitrogen, USA) reagent for 2 hours, absorbance was measured at 570 nm and 600 nm, and the untreated cells were set to 100% viability.
- Alamar blue Invitrogen, USA
- Preparation of ion complexes of SG6, R6 and PEI formed 1:2 and 1:5 complexes, respectively, based on 100 ng of mRNA according to the N/P ratio in Table 2. After forming the ion complex for 30 minutes, the complex was treated in 2x10 4 HEK293 cells in a 96-well plate for 48 hours, and then cell viability was measured with Alamar Blue reagent.
- RNA The encapsulation and loading rates of mRNA were measured using a cationic molecular transporter and commercially available liposomes.
- SG6 was used as a cationic molecular transporter
- Cationic DOTAP/DC-CHOL Liposomes Adjuvants (FormuMax Scientific Inc.) were used as commercially available liposomes.
- Ribogreen analysis method using ribogrin a fluorescent dye that binds to nucleic acids, was used as an analysis method. was performed.
- RBD mRNA and eGFP mRNA reacted with liposomes at room temperature in the presence and absence of SG6 to form an ionic complex, and fluorescence values were measured by adding ribogrin.
- the encapsulation rate and the loading rate were respectively calculated by the following formulas.
- Encapsulation rate (total mRNA amount-residual mRNA amount)/(total mRNA amount)X100
- Carrying rate (total mRNA amount-residual mRNA amount)/(liposome amount used)X100
- both RBD mRNA and eGFP mRNA were analyzed to be at least 80% bound to the liposome, and it was observed that at least 20% or more of the mRNA was loaded into the liposome ( FIG. 9 ).
- cationic lipids DOTAP chloride salt, 1,2-dioleoyl-3-trimethylammonium-propane
- DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
- cholesterol Choesterol
- the rehydrated suspension is downsized through a stirrer (Vortex) and a water tank type ultrasonic disperser (Sonicator), and then, for homogenization of the liposome, a polycarbonate membrane having a 0.1um pore size is repeatedly passed through a polycarbonate membrane several times to liposome. was prepared.
- the prepared liposomes were stored at 4°C until use after structural analysis (FIG. 9) and measurement of size and surface charge (Zeta potential) through a cryogenic transmission electron microscope.
- 1ug mRNA was reacted with cationic liposomes at room temperature in various mass ratios (1:1, 1:3, 1:5, 1:7), and the formed ionic complex was analyzed by 1% denaturing agarose gel electrophoresis. .
- the liposome containing SG6 was further reacted at room temperature by treating SG6 after the reaction of mRNA and the liposome at room temperature.
- mRNA lipolex liposome-mRNA
- mRNA-SG6-lipoplex SG6-treated mRNA lipoplex
- mRNA and liposome formed a stable ion complex based on a mass ratio of 1:5, confirming the possibility of mRNA loading and gene delivery ( FIG. 10 ).
- HEK293T cells a human-derived cell line, were plated in a 96-well plate at a number of 6 X 10 3 cells/well and cultured with lipofectamine or mRNA-lipoplex prepared in different mass ratios for 48 hours. Then, the cells were measured for absorbance and fluorescence by adding Alamar Blue reagent, and the degree of proliferation was measured to evaluate cytotoxicity.
- the ribogreen assay used for the detection and quantification of nucleic acids was performed to quantify the mRNA encapsulated in and out of the liposome.
- the encapsulation rate represents the content ratio of mRNA bound to the liposome with respect to the total mRNA used, and it can be evaluated that the higher the encapsulation rate, the better the mRNA-lipoplex is formed and the amount of exposed free mRNA is small.
- Example 11 To confirm this, the ion complex formed by the method of Example 11 was transferred to a 96-well plate, reacted with ribogreen, a nucleic acid binding reagent, and then the fluorescence value was measured to evaluate the encapsulation efficiency (EE%) by the following equation.
- Encapsulation Efficiency (Total mRNA Amount - Residual mRNA Amount)/(Total mRNA Amount) X 100
- the mRNA complexed with the liposome is protected from various ribonucleic acid degrading enzymes distributed in the body, and can be delivered into the cell with higher efficiency than the free RNA.
- ribonucleic acid degrading enzyme was treated to the ion complex formed in the mRNA and liposome mass ratio of 1:5 to decompose the exposed RNA, and then the ribonucleic acid degrading enzyme was inactivated with Proteinase K. Thereafter, the liposome membrane was disrupted with Triton X-100 to release the encapsulated mRNA, and the presence of mRNA was confirmed by 1% denaturing agarose gel electrophoresis.
- HEK293T a human-derived cell line
- HEK293T a human-derived cell line
- the immunogenicity of the liposome ion complex as a vaccine was confirmed by inoculating the prepared mRNA-lipoplex into BALB/c mice.
- mRNA-lipoplex prepared an inoculation sample so that the mRNA and liposome mass ratio was 1:5 based on the dose of mRNA 2 and 10ug, respectively, and at this time, some experimental groups treated SG6 in the ion complex to produce mRNA most effectively in the above example.
- Inoculation samples were prepared in the same manner as the expressed conditions.
- each sample was prepared in a freeze-dried formulation to confirm the ease of storage and long-term stability of the vaccine, and the differences in immunogenicity between formulations were compared.
- the inoculation method of the experimental group was all the same as intramuscular inoculation, and prime (1st)-boost (2nd) inoculation was performed at 3-week intervals (day 0, day 21), respectively, and serum was administered 2 weeks after the second inoculation. After separation, the ability to form neutralizing antibodies was measured by SARS-CoV-2 surrogate virus neutralization test (sVNT).
- the SG6-treated mRNA-lipoplex had at least 7-fold increased ability to form neutralizing antibodies compared to the control group (Vehicle control). It is thought to be able to help delivery and protein expression ( FIG. 15 ).
- both liquid as well as freeze-dried formulations induce RBD protein neutralization ability without decreasing activity, so just as sorbitol, the basic backbone of SG6, is widely used as a freeze-drying adjuvant, SG6 is also mRNA-liposome ion complex in the freeze-drying process.
- freeze-dried mRNA-lipoplex treated with SG6 can induce a strong humoral immune response in animal experiments, and it is also thought to be suitable for a vaccine composition for prevention because it has excellent storage easiness without decreasing activity.
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Abstract
The present invention relates to a vaccine composition comprising an ionic complex, which comprise: an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein; and a cationic molecular transporter. Provided is a vaccine in which a cationic molecular transporter based on sorbitol, which is a sugar compound that is harmless to humans, and cationic guanidinium, is applied as a carrier component, and thus is stable even during freeze-drying while having excellent mRNA delivery efficiency.
Description
본 발명은 양이온성 분자 수송체 및 핵산의 이온 복합체를 포함하여 핵산을 체내에 효율적으로 전달하는 백신 조성물에 관한 것이다.The present invention relates to a vaccine composition that efficiently delivers a nucleic acid into the body, including a cationic molecular transporter and an ionic complex of the nucleic acid.
전세계적인 팬데믹을 일으키고 있는 중증급성호흡기증후군 코로나바이러스 2(Severe acute respiratory syndrome coronavirus 2; SARS-CoV-2)는 RNA 바이러스로, 외부 스파이크(spike) 단백질이 특징적인 크라운 형태를 가지고 있는 구형으로 spike(S), membrane(M), envelope(E), nucleocapsid(N)으로 구성되어 있다. 2020년 3월 11일 세계보건기구(WHO)에서 SARS-CoV-2에 의한 질병을 코로나바이러스감염증-19(coronavirus disease 2019, COVID-19)로 명명하였고, 팬데믹을 선언한 코로나바이러스감염증-19는 현재까지 이어지고 있으며 2021년 기준 확진자 1.03억명, 사망자 224만명을 넘은 상황이다.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is causing a worldwide pandemic, is an RNA virus that has a spherical spike with a characteristic crown shape with an external spike protein. It is composed of (S), membrane (M), envelope (E), and nucleocapsid (N). On March 11, 2020, the World Health Organization (WHO) named the disease caused by SARS-CoV-2 as coronavirus disease 2019 (COVID-19) and declared a pandemic. continues to this day, and as of 2021, the number of confirmed cases has exceeded 103 million and the number of deaths has exceeded 2.24 million.
현재 불활성화 백신, 바이러스 벡터 백신, mRNA 백신, 재조합 단백질 백신 등의 백신 종류를 포함한 총 11가지 품목이 전 세계적으로 접종이 이뤄지고 있다. 이 중 가장 먼저 미국 FDA의 허가를 받은 화이자/바이오엔텍의 mRNA(BNT162b2) 백신 및 모더나의 mRNA(mRNA-1273) 백신은 항원 단백질을 합성할 수 있는 유전정보를 담은 전령 리보핵산(messenger RNA, mRNA)을 세포 내로 전달하여 항원 단백질로 발현되게 한 다음 면역반응을 유도하는 방식으로 작동한다.Currently, a total of 11 types of vaccines, including inactivated vaccines, viral vector vaccines, mRNA vaccines, and recombinant protein vaccines, are being vaccinated worldwide. Among them, the mRNA (BNT162b2) vaccine of Pfizer/BioNtech and Moderna's mRNA (mRNA-1273) vaccine, which were first approved by the US FDA, are messenger RNA (mRNA) containing genetic information for synthesizing antigenic proteins. ) into cells to be expressed as antigenic proteins, and then works by inducing an immune response.
이러한 mRNA 백신의 장점은 크게 3가지가 있다. 첫째, 생산적 측면에서 과정이 비교적 간단하여 단기간에 제조 가능하여 팬데믹과 같은 긴급 상황에서 또는 변이에 대해 신속하고 유연한 대응이 가능하다. 둘째, 기능적 측면에서 세포 내 전달이 효과적으로 이뤄진다면 체액성면역(Systemic immunity)과 세포성면역(Cellular immunity)을 모두 유도 가능하여 타 종류의 백신에 비하여 보다 효과적으로 질병 예방을 기대할 수 있다. 셋째, 안전성 측면에서 부작용이 적으며, DNA와 달리 단일 가닥인 RNA는 핵으로 들어가서 인체 유전자의 변형을 일으키지 않는 큰 장점이 있다.There are three major advantages of such an mRNA vaccine. First, in terms of productivity, the process is relatively simple and can be manufactured in a short period of time, enabling rapid and flexible response in emergency situations such as pandemics or mutations. Second, if intracellular delivery is effectively performed from a functional point of view, both humoral immunity and cellular immunity can be induced, so that disease prevention can be expected more effectively compared to other types of vaccines. Third, there are few side effects in terms of safety, and unlike DNA, single-stranded RNA has a great advantage in that it does not enter the nucleus and cause modification of human genes.
그러나 이러한 mRNA 백신은 보완해야 할 단점도 있다. mRNA는 분자량이 크고 높은 음전하를 띠는 데다가 체내의 RNA 분해효소에 취약하다. 현재는 화이자/바이오엔텍과 모더나가 사용한 지질 나노입자(Lipid nanoparticle) 기술이 백신에 적용되고 있지만, 이 역시 다음과 같은 문제점이 있다. 첫째, 전달 효율 측면에서 높은 편은 아니다. 화이자/바이오엔텍과 모더나 백신은 각각 1회 접종 용량으로 30ug 및 100ug이라는 과량의 mRNA를 사용하여 효율적이라 할 수 없다. 만약 전달 효율을 높여 mRNA의 사용량을 줄인다면 같은 생산량으로 더 많은 백신을 보급할 수 있음은 물론 백신의 단가 또한 절감하는 효과가 있을 것이다. 둘째, 지질 나노입자의 열 안정성이 낮다. 정제된 mRNA는 자체는 상당히 안정적인 물질이지만 지질 나노입자가 불안정하여 반드시 저온 또는 초저온으로 보관해야만 한다. 실제로 화이자/바이오엔텍와 모더나의 백신은 -70℃와 -20℃ 이하에서 유통 및/또는 보관해야 하므로, 4℃에서 취급되는 기존 대부분의 백신에 비하여 초저온 유통체계와 취급 기관의 보관 시설 구축이 요구되어 가장 강력한 백신임에도 신속한 보급에 어려움을 겪을 수 있다. 따라서, 위에서 언급한 지질 나노입자가 가지는 전달효율과 열안정성의 단점을 극복할 수 있는 새로운 전달 기술의 개발이 절실하다.However, these mRNA vaccines also have disadvantages that need to be supplemented. mRNA has a large molecular weight, high negative charge, and is vulnerable to RNA-degrading enzymes in the body. Currently, the lipid nanoparticle technology used by Pfizer/BioNtech and Moderna is being applied to vaccines, but this also has the following problems. First, it is not on the high side in terms of transfer efficiency. Pfizer/BioNtech and Moderna vaccines cannot be said to be efficient because they use excessive amounts of mRNA of 30 μg and 100 μg, respectively, as a single inoculation dose. If mRNA usage is reduced by increasing delivery efficiency, more vaccines can be supplied with the same amount of production, and the cost of vaccines will also be reduced. Second, the thermal stability of lipid nanoparticles is low. Purified mRNA itself is a fairly stable material, but lipid nanoparticles are unstable and must be stored at low or cryogenic temperatures. In fact, vaccines from Pfizer/BioNtech and Moderna must be distributed and/or stored at -70°C and -20°C or lower, so compared to most existing vaccines handled at 4°C, it is required to establish a cryogenic distribution system and storage facilities for handling institutions. Although it is the most powerful vaccine, it may have difficulties in rapidly distributing it. Therefore, there is an urgent need to develop a new delivery technology that can overcome the disadvantages of delivery efficiency and thermal stability of the above-mentioned lipid nanoparticles.
이에, 효과적으로 약물을 전달하기 위해 약물 전달체로서 지질 나노입자에 대한 연구가 활발히 진행되게 되었고, 생체 구성성분 중 하나인 지질 이중층으로 이루어진 리포좀 전달체가 적극적으로 활용되게 되었다. 리포좀은 생체 세포막의 구성성분인 인지질과 같은 양친매성 지질분자가 이중층 혹은 다중이중층을 이루며 내부에는 친수성 공간, 외부에는 지질막 구조를 가진 미세소포 형태이다. 따라서, 우수한 생체 적합성을 장점으로 친수성 공간인 내부로는 수용성 약물을, 외부 지질막으로는 지용성 약물과 전하성 물질을 결합하여 다양한 약물 수송체로서의 리포좀 제제가 개발되고 있다. 그 중 양이온성 리포좀과 음이온성 핵산이 일정 비율로 혼합되어 정전기적 상호작용을 통해 형성된 핵산-리포좀 복합체는 엔도사이토시스(endocytosis)가 용이해 세포 내 통과가 원활하고 엔도좀을 거쳐 세포질로의 이동이 수월할 수 있다.Accordingly, in order to effectively deliver drugs, research on lipid nanoparticles as a drug carrier has been actively conducted, and a liposome carrier composed of a lipid bilayer, which is one of the biocomponents, has been actively utilized. Liposomes are in the form of microvesicles in which amphiphilic lipid molecules such as phospholipids, which are components of living cell membranes, form a double layer or multiple double layers, and have a hydrophilic space inside and a lipid membrane structure outside. Therefore, liposome preparations as various drug transporters are being developed by combining a water-soluble drug into the hydrophilic space, and a fat-soluble drug and an electric charge, as an external lipid membrane, with the advantage of excellent biocompatibility. Among them, cationic liposome and anionic nucleic acid are mixed in a certain ratio and the nucleic acid-liposome complex formed through electrostatic interaction facilitates endocytosis, which facilitates intracellular passage and moves through the endosome to the cytoplasm. This can be easy.
본 발명자들은 지질 나노입자가 갖는 전달 효율과 열안정성의 문제점을 극복하기 위해 노력한 결과, 인체에 무해한 당화합물인 솔비톨과(Sorbitol) 양이온성의 구아니디니움(Guanidinium)을 기반으로 하는 양이온성 분자 수송체(Sorbitol-G6, 'SG6')를 전달체 성분으로 적용하는 경우, 우수한 mRNA 전달 효율을 가지면서도 안정적으로 백신을 제공할 수 있음을 확인하여 본 발명을 완성하였다.As a result of the present inventors' efforts to overcome the problems of delivery efficiency and thermal stability of lipid nanoparticles, sorbitol, a sugar compound harmless to the human body, and cationic molecule transport based on cationic guanidinium When a sieve (Sorbitol-G6, 'SG6') is applied as a carrier component, it was confirmed that a vaccine can be stably provided while having excellent mRNA delivery efficiency, thereby completing the present invention.
본 발명의 목적은 (a) 항원 펩티드 또는 단백질을 암호화하는 mRNA 서열을 포함하는 mRNA 화합물, 및 (b) 하기 화학식 1의 양이온성 분자 수송체를 포함하는 이온 복합체를 포함하는 백신 조성물을 제공하는 것이다. It is an object of the present invention to provide a vaccine composition comprising (a) an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein, and (b) an ion complex comprising a cationic molecular transporter of the following formula (1) .
[화학식 1][Formula 1]
상기 식에서,In the above formula,
R1은 
로, 여기에서 n은 1 내지 8의 정수이다. R 1 is , where n is an integer from 1 to 8.
본 발명의 다른 목적은 (a) 화학식 1로 표시되는 양이온성 분자 수송체를 제조하는 단계; 및 (b) 항원 펩티드 또는 단백질을 암호화하는 mRNA 서열을 포함하는 mRNA 화합물을 제조하여 상기 (a) 단계의 결과물과 혼합하는 단계를 포함하는 백신 조성물의 제조방법을 제공하는 것이다.Another object of the present invention is to prepare a cationic molecular transporter represented by the formula (1); And (b) to provide a method for preparing a vaccine composition comprising the step of preparing an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein and mixing it with the result of step (a).
상술한 과제를 해결하기 위한 본 발명은 (a) 항원 펩티드 또는 단백질을 암호화하는 mRNA 서열을 포함하는 mRNA 화합물, 및 (b) 하기 화학식 1의 양이온성 분자 수송체를 포함하는 이온 복합체를 포함하는 백신 조성물을 제공한다. The present invention for solving the above problems is (a) an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein, and (b) a vaccine comprising an ion complex comprising a cationic molecular transporter of the following formula (1) A composition is provided.
[화학식 1][Formula 1]
상기 식에서, In the above formula,
R1은 
로, 여기에서 n은 1 내지 8의 정수이다. R 1 is , where n is an integer from 1 to 8.
본 발명의 일 구현예에 따르면, 상기 이온 복합체는 양이온성 리포좀을 더 포함한다. According to one embodiment of the present invention, the ionic complex further comprises a cationic liposome.
본 발명의 리포좀은 당해 기술분야에 현재 공지된 다양한 기술에 의해 제조될 수 있다. 다중-라멜라 소포체(Multi-lamellar vesicle, MLV)는 기존의 기술로, 예를 들어 적절한 용매에서 지질을 용해시켜 적합한 컨테이너(container) 또는 용기(vessel)의 안쪽 벽에 선택된 지질을 퇴적시킴으로써, 그리고 이후 용기의 안쪽에 얇은 필름을 남기도록 용매를 증발시키거나 분무 건조시켜 제조될 수 있다. 이후 MLV의 형성을 야기하는 와동 운동(vortexing motion)을 하면서 용기에 수성상이 첨가될 수 있다. 이후 다중-라멜라 소포체의 균질화, 초음파처리 또는 압출에 의해 단일-라멜라 소포체(Uni-lamellar vesicle, ULV)가 형성될 수 있다. The liposomes of the present invention can be prepared by various techniques currently known in the art. Multi-lamellar vesicles (MLV) are prepared by conventional techniques, for example by dissolving lipids in a suitable solvent and depositing selected lipids on the inner wall of a suitable container or vessel, and then It can be prepared by evaporating the solvent or spray drying to leave a thin film on the inside of the container. The aqueous phase can then be added to the vessel in a vortexing motion that causes the formation of MLV. Uni-lamellar vesicles (ULVs) can then be formed by homogenization, sonication or extrusion of multi-lamellar vesicles.
상기 mRNA 화합물은 상기 양이온성 분자 수송체 및/또는 양이온성 리포좀과 정전기적 상호작용에 의해 이온 복합체를 형성하는 것일 수 있다. The mRNA compound may form an ionic complex by electrostatic interaction with the cationic molecular transporter and/or cationic liposome.
본 발명의 일 구현예에 따르면, 상기 mRNA 화합물과 상기 양이온성 리포좀의 질량비는 1:1 내지 1:6이다. 본 발명의 일 실시예에서, mRNA 화합물과 상기 양이온성 리포좀의 질량비가 1:3이었으며, 질량비(mRNA : 양이온성 리포좀)가 1:1 미만인 경우 리포좀의 양은 mRNA를 수송하는데 불충분할 수 있으며, 질량비가 1:6 초과인 경우 과량의 리포좀이 간섭을 일으켜 충분한 양의 mRNA 가 세포내로 수송되기 어려울 수 있다. According to one embodiment of the present invention, the mass ratio of the mRNA compound and the cationic liposome is 1:1 to 1:6. In one embodiment of the present invention, the mass ratio of the mRNA compound and the cationic liposome was 1:3, and when the mass ratio (mRNA: cationic liposome) is less than 1:1, the amount of the liposome may be insufficient to transport the mRNA, and the mass ratio When is greater than 1:6, an excess of liposomes may interfere, so that it may be difficult to transport a sufficient amount of mRNA into a cell.
본 발명의 일 구현예에 따르면, 상기 항원 펩티드 또는 단백질은 SARS-Cov-2의 스파이크 단백질, 뉴클레오캡시드 단백질 또는 이의 단편 또는 변이체로부터 유래되는 것이다. According to one embodiment of the present invention, the antigenic peptide or protein is derived from a SARS-Cov-2 spike protein, a nucleocapsid protein, or a fragment or variant thereof.
본 발명의 일 구현예에 따르면, 상기 mRNA 서열은 서열번호 1에 따른 SARS-Cov-2 스파이크 단백질의 RBD 서열에 상응하는 RNA 서열을 포함하는 것이다. According to one embodiment of the present invention, the mRNA sequence comprises an RNA sequence corresponding to the RBD sequence of the SARS-Cov-2 spike protein according to SEQ ID NO: 1.
상기 mRNA 서열은 5'에서 3' 방향으로 다음 요소를 포함하는 것일 수 있다. The mRNA sequence may include the following elements in a 5' to 3' direction.
a) 5'-CAP 구조; a) 5'-CAP structure;
b) 5'-UTR 요소;b) a 5'-UTR element;
c) 항원 펩티드 또는 단백질을 암호화하는 암호화 영역;c) a coding region encoding an antigenic peptide or protein;
d) 3'-UTR 요소; 및/또는 d) a 3'-UTR element; and/or
e) 폴리 A 꼬리.e) Poly A tail.
상기 5'-UTR 요소는 서열번호 2에 따른 서열에 상응하는 RNA 서열을 포함하거나 이로 구성될 수 있으며, 상기 항원 펩티드 또는 단백질을 암호화하는 암호화 영역은 서열번호 1에 따른 서열에 상응하는 RNA 서열을 포함하거나 이로 구성될 수 있으며, 상기 3'-UTR 요소는 서열번호 3에 따른 서열에 상응하는 RNA 서열을 포함하거나 이로 구성될 수 있으며, 상기 폴리 A 꼬리는 서열번호 4에 따른 서열에 사응하는 RNA 서열을 포함하거나 이로 구성될 수 있다.The 5'-UTR element may comprise or consist of an RNA sequence corresponding to the sequence according to SEQ ID NO: 2, wherein the coding region encoding the antigenic peptide or protein comprises an RNA sequence corresponding to the sequence according to SEQ ID NO: 1 wherein the 3'-UTR element comprises or consists of an RNA sequence corresponding to the sequence according to SEQ ID NO: 3, wherein the poly A tail is an RNA corresponding to the sequence according to SEQ ID NO: 4 may comprise or consist of a sequence.
본 발명의 mRNA 서열은 서열 내로 비-뉴클레오티드의 연결 또는 변형된 뉴클레오티드의 편입을 포함할 수 있다. 예를 들어, 항원 펩티드 또는 단백질을 암호화하는 mRNA 분자의 3' 및 5' 말단 중 하나 또는 둘 모두에 대한 변형일 수 있다. 이러한 변형은 mRNA 서열에 염기의 첨가(예를 들어, 폴리 A 꼬리 또는 더 긴 폴리 A 꼬리의 함유), 물질(예를 들어, 단백질 또는 상보적인 핵산 분자)과 mRNA를 복합시켜, 3' UTR 또는 5' UTR의 변형, 및 mRNA 분자의 구조를 변화(예를 들어, 2차 구조를 형성)시키는 요소를 포함한다. 또한, 변형은 염기 상에 존재할 수 있으며, 슈도우리딘(psuedouridine) 또는 N1-메틸슈도우리딘으로 이루어진 그룹으로부터 선택될 수 있다. The mRNA sequences of the present invention may include non-nucleotide linkages or incorporation of modified nucleotides into the sequence. For example, it may be a modification to one or both of the 3' and 5' ends of an mRNA molecule encoding an antigenic peptide or protein. Such modifications may include addition of bases to the mRNA sequence (e.g., containing a poly A tail or a longer poly A tail), complexing the mRNA with a substance (e.g., a protein or complementary nucleic acid molecule), resulting in a 3' UTR or Modification of the 5' UTR, and elements that change the structure of the mRNA molecule (eg, form a secondary structure). In addition, the modification may be present on a base, and may be selected from the group consisting of pseudouridine or N1-methylpseudouridine.
본 발명의 폴리 A 꼬리는 천연 mRNA을 안정화하기 위한 것일 수 있다. 예를 들어, 긴 폴리 A 꼬리는 mRNA 분자에 첨가되어 mRNA를 더 안정하게 만들 수 있다. 폴리 A 꼬리는 당해 기술분야에서 공지된 기술을 이용하여 첨가될 수 있다.The poly A tail of the present invention may be for stabilizing native mRNA. For example, a long poly A tail can be added to an mRNA molecule to make the mRNA more stable. Poly A tails can be added using techniques known in the art.
본 발명의 일 구현예에서, 폴리 A 꼬리의 길이는 90 내지 200개 아데닌을 사용하였다. 폴리 A 꼬리의 길이가 mRNA 분자의 반감기에 영향을 줄 수 있기 때문에, 뉴클레아제에 대한 mRNA의 저항의 수준을 변형하고 따라서 세포내 단백질 발현의 시간 경과를 제어하기 위해 폴리 A 꼬리의 길이가 조정될 수 있다. In one embodiment of the present invention, the length of the poly A tail was 90 to 200 adenines. Since the length of the poly A tail can affect the half-life of the mRNA molecule, the length of the poly A tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thus to control the time course of intracellular protein expression. can
본 발명의 mRNA는 예를 들어, 표적 세포 또는 조직으로의 mRNA 전달의 결정을 촉진시키는 리포터 유전자(reporter gene)와 선택적으로 결합될 수 있다. 적합한 리포터 유전자는 예를 들어, 녹색 형광 단백질 mRNA(GFP mRNA), 루시퍼라아제 mRNA (Luciferase mRNA), 반딧불이 루시퍼라아제 mRNA, 또는 이들의 임의의 조합을 포함할 수 있다. The mRNA of the present invention can be selectively bound with a reporter gene that facilitates, for example, the determination of mRNA delivery to a target cell or tissue. Suitable reporter genes can include, for example, green fluorescent protein mRNA (GFP mRNA), luciferase mRNA (Luciferase mRNA), firefly luciferase mRNA, or any combination thereof.
본 발명의 상기 조성물은 안정화 시약을 포함할 수 있다. 조성물은 mRNA에 직접적으로 또는 간접적으로 결합하고, 안정화하여, 표적 세포내 체류 시간을 증진시키는 하나 이상의 제제 시약을 포함할 수 있다. 이러한 시약은 바람직하게 표적 세포 내 mRNA의 반감기를 개선할 수 있다. 안정화 시약은 하나 이상의 단백질, 펩티드, 압타머, 번역 부속 단백질, mRNA 결합 단백질, 및/또는 번역 개시 인자를 포함한다.The composition of the present invention may include a stabilizing reagent. The composition may include one or more agent reagents that directly or indirectly bind to and stabilize mRNA, thereby enhancing residence time in the target cell. Such reagents are preferably capable of improving the half-life of mRNA in the target cell. Stabilization reagents include one or more proteins, peptides, aptamers, translation accessory proteins, mRNA binding proteins, and/or translation initiation factors.
본 발명의 일 구현예에 따르면, 상기 양이온성 리포좀은 디메틸디옥타데실암모늄 브로마이드(DDA), 1,2-디올레오일-3-트리메틸암모늄프로페인(DOTAP), 3β-[N-(N',N'-디메틸아미노에테인 카바모일 콜레스테롤(3β-[N-(N'N'-dimethylaminoethane) carbamoyl cholesterol, DC-Chol), 1,2-디올레오일옥시-3-디메틸암모늄프로페인(DODAP), 1,2-디-O-옥타데세닐-3-트리에틸암모늄 프로페인(1,2-di-O-octadecenyl-3-trimethylammonium propane, DOTMA), 1,2-디미리스토레오일-sn-글리세로-3-에틸포스포콜린(1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 14:1 Ethyle PC), 1-팔미토일-2-올레오일-sn-글리세로-3-에틸포스포콜린(1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, 16:0-18:1 Ethyl PC), 1,2-디올레오일-sn-글리세로-3-에틸포스포콜린(1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 18:1 Ethyl PC), 1,2-디스테아로일-sn-글리세로-3-에틸포스포콜린(1,2-distearoyl-sn-glycero-3-ethylphosphocholin, 18:0 Ethyl PC), 1,2-디팔미토일-sn-글리세로-3-에틸포스포콜린(1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 16:0 Ethyl PC), 1,2-디미리스토일-sn-글리세로-3-에틸포스포콜린(1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 14:0 Ethyl PC), 1,2-디라우로일-sn-글리세로-3-에틸포스포콜린(1,2-dilauroyl-sn-glycero-3-ethylphosphocholin, 12:0 Ethyl PC), N1-[2-((1S)-1-[(3-아미노프로필)아미노]-4-[디(3-아미노-프로필)아미노]부틸카복사미도)에틸]-3,4-디[올레일옥시]-벤자마이드(N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide, MVL5), 1,2-디미리스토일-3-디메틸암모늄-프로페인(1,2-dimyristoyl-3-dimethylammonium-propane, 14:0 DAP), 1,2-디팔미토일-3-디메틸암모늄-프로페인(1,2-dipalmitoyl-3-dimethylammonium-propane, 16:0 DAP), 1,2-디스테아로일-3-디메틸암모늄-프로페인(1,2-distearoyl-3-dimethylammonium-propane, 18:0 DAP), N-(4-카복시벤질)-N,N-디메틸-2,3-비스(올레오일옥시)프로판-1-아미늄(N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium, DOBAQ), 1,2-스테아로일-3-트리메틸암모늄-프로페인(1,2-stearoyl-3-trimethylammonium-propane, 18:0 TAP), 1,2-디팔미토일-3-트리메틸암모늄-프로페인(1,2-dipalmitoyl-3-trimethylammonium-propane, 16:0 TA), 1,2-디미리스토일-3-트리메틸암모늄-프로페인(1,2-dimyristoyl-3-trimethylammonium-propane, 14:0 TAP) 및 N4-콜레스테릴-스퍼민(N4-Cholesteryl-Spermine, GL67)으로 구성된 군으로부터 선택되는 양이온성 지질인 것이다. According to one embodiment of the present invention, the cationic liposome is dimethyldioctadecylammonium bromide (DDA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 3β-[N-(N') ,N'-dimethylaminoethane carbamoyl cholesterol (3β-[N-(N'N'-dimethylaminoethane) carbamoyl cholesterol, DC-Chol), 1,2-dioleoyloxy-3-dimethylammonium propane (DODAP) , 1,2-di-O-octadecenyl-3-triethylammonium propane (1,2-di-O-octadecenyl-3-trimethylammonium propane, DOTMA), 1,2-dimyristoreoyl-sn -Glycero-3-ethylphosphocholine (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 14:1 Ethyle PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethyl Phosphocholine (1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, 16:0-18:1 Ethyl PC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 18:1 Ethyl PC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (1,2-distearoyl-sn) -glycero-3-ethylphosphocholine, 18:0 Ethyl PC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 16: 0 Ethyl PC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 14:0 Ethyl PC), 1,2 -dilauroyl-sn-glycero-3-ethylphosphocholine (1,2-dilauroyl-sn-glycero-3-ethylphosphocholin, 12:0 Ethyl PC), N1-[2-((1S)-1 -[(3-aminopro Phil)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (N1-[2-((1S)- 1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide, MVL5), 1,2-dimyristo yl-3-dimethylammonium-propane (1,2-dimyristoyl-3-dimethylammonium-propane, 14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (1,2-dipalmitoyl-propane) 3-dimethylammonium-propane, 16:0 DAP), 1,2-distearoyl-3-dimethylammonium-propane (1,2-distearoyl-3-dimethylammonium-propane, 18:0 DAP), N-( 4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium(N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy) )propan-1-aminium, DOBAQ), 1,2-stearoyl-3-trimethylammonium-propane (1,2-stearoyl-3-trimethylammonium-propane, 18:0 TAP), 1,2-dipalmi Toyl-3-trimethylammonium-propane (1,2-dipalmitoyl-3-trimethylammonium-propane, 16:0 TA), 1,2-dimyristoyl-3-trimethylammonium-propane (1,2-dimyristoyl) It is a cationic lipid selected from the group consisting of -3-trimethylammonium-propane, 14:0 TAP) and N4-Cholesteryl-Spermine (GL67).
본 발명의 일 구현예에 따르면, 상기 양이온성 리포좀은 (a) 1,2-디미리스토일-sn-글리세로-3-포스파티딜콜린(1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine, DMPC), 1,2-디올레오일-sn-글리세로-3-포스포콜린(1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 1,2-디올레오일-sn-글리세로-3-포스포에탄올아민(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-디팔미토일-sn-글리세로-3-포스포콜린(1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), 1,2-디스테아로일-sn-글리세로-3-포스포콜린(1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-디리노레오일-sn-글리세로-3-포스포콜린(1,2-dilinoleoyl-sn-glycero-3-phosphocholine, DLPC), 포스파티딜세린(PS), 포스포에탄올라민(PE), 포스파티딜글리세롤(PG), 포스포릭액시드(PA) 및 포스파티딜콜린(PC)으로 구성된 군으로부터 선택되는 중성 지질; 및 (b) 콜레스테롤(Cholesterol)을 추가로 포함하는 것이다. According to one embodiment of the present invention, the cationic liposome is (a) 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine, DMPC ), 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 1,2-dioleoyl-sn-glycero -3-Phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoyl- sn-glycero-3-phosphocholine, DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), 1, 2-Dilinoleoyl-sn-glycero-3-phosphocholine (1,2-dilinoleoyl-sn-glycero-3-phosphocholine, DLPC), phosphatidylserine (PS), phosphoethanolamine (PE), phosphatidyl a neutral lipid selected from the group consisting of glycerol (PG), phosphoric acid (PA) and phosphatidylcholine (PC); and (b) cholesterol (Cholesterol).
본 발명의 일 구현예에 따르면, 상기 양이온성 리포좀은 DOTAP, DOPE 및 콜레스테롤을 포함하는 것이다. DOTAP, DOPE 및 콜레스테롤의 몰 비는 1 : 0.2 ~ 0.8 : 0.2 ~ 0.8일 수 있다. DOPE와 콜레스테롤의 함량이 0.2 보다 낮은 경우 세포 내 mRNA 전달 효율이 감소될 수 있고, 0.8 보다 높은 경우 리포좀의 mRNA의 포획률이 감소될 수 있으므로, 상기 비율로 조제함이 바람직하다.According to one embodiment of the present invention, the cationic liposome is to include DOTAP, DOPE and cholesterol. The molar ratio of DOTAP, DOPE and cholesterol may be 1:0.2-0.8:0.2-0.8. If the content of DOPE and cholesterol is lower than 0.2, intracellular mRNA delivery efficiency may be reduced, and if it is higher than 0.8, the capture rate of liposome mRNA may be reduced, so it is preferable to prepare in the above ratio.
본 발명의 일 구현예에 따르면, 상기 mRNA 화합물은 RNA 분자에 안정도를 부여하는 적어도 한 가지 변형을 포함할 수 있다. RNA 분자에 안정도를 부여하는 변형은 전형적으로, 생체 내 분해(예를 들어, 엑소- 또는 엔도-뉴클레아제에 의한 분해), 생체 외 분해(예를 들어, 백신 투여 전 제조 과정에 의해, 예를 들어 투여되는 백신 용액의 제조 과정에서)에 대한 내성을 증가시키는 변형을 나타낸다. RNA의 안정화는 예를 들어 5'-CAP 구조, 폴리 A 꼬리, 또는 임의의 기타 UTR 변형의 제공에 의해 달성될 수 있다. 또한, RNA의 안정화는 화학적 변형 또는 핵산의 G/C 함량의 변형에 의해 달성될 수 있다. 안정도를 부여하는 변형을 달성하기 위해 당해 분야에 공지된 다양한 다른 방법을 도입할 수 있다. According to one embodiment of the present invention, the mRNA compound may include at least one modification that imparts stability to the RNA molecule. Modifications that confer stability to an RNA molecule typically include in vivo degradation (eg, by exo- or endo-nucleases), ex vivo degradation (eg, by manufacturing processes prior to vaccine administration, for example). (e.g., during the manufacture of the administered vaccine solution) represents a modification that increases resistance. Stabilization of RNA can be achieved, for example, by providing a 5'-CAP structure, a poly A tail, or any other UTR modification. In addition, stabilization of RNA can be achieved by chemical modification or modification of the G/C content of the nucleic acid. A variety of other methods known in the art can be employed to achieve a modification that imparts stability.
본 발명의 일 구현예에 따르면, 상기 mRNA 화합물의 음이온/상기 양이온성 분자 수용체의 양이온의 비율(N/P ratio)이 0.1 내지 1인 것이다. 본 발명 실시예에서 N/P 비율이 1 미만인 경우(즉, 양이온의 비율이 음이온의 비율보다 큰 경우) N/P 비율이 감소함에 따라 정전기적 상호작용이 증가하여 이온복합체 형성이 용이하다. 그러나, 과량의 양이온 존재시 세포에 독성을 유발할 가능성이 있으므로 적절한 비율의 선택이 중요하다. 한편 N/P 비율이 1 초과인 경우(즉, 음이온의 비율이 양이온의 비율보다 큰 경우) 정전기적 상호작용의 불균형으로 완전한 이온 복합체가 형성되기 어렵다.According to one embodiment of the present invention, the ratio of the anion of the mRNA compound to the cation of the cationic molecular receptor (N/P ratio) is 0.1 to 1. In an embodiment of the present invention, when the N/P ratio is less than 1 (that is, when the ratio of cations is greater than the ratio of anions), as the ratio of N/P decreases, the electrostatic interaction increases, thereby facilitating the formation of an ionic complex. However, it is important to select an appropriate ratio because there is a possibility of causing toxicity to cells in the presence of an excess of cations. On the other hand, when the N/P ratio is greater than 1 (ie, the ratio of anions is greater than the ratio of cations), it is difficult to form a complete ionic complex due to an imbalance in the electrostatic interaction.
본 발명의 일 구현예에 따르면, 상기 mRNA 서열은 상기 RNA 분자의 5'비번역 영역의 변형을 포함한다. According to one embodiment of the present invention, the mRNA sequence comprises a modification of the 5' untranslated region of the RNA molecule.
본 발명의 일 구현예에 따르면, 상기 mRNA 서열은 상기 RNA 분자의 3' 비번역 영역의 변형을 포함한다. According to one embodiment of the present invention, the mRNA sequence comprises a modification of the 3' untranslated region of the RNA molecule.
본 발명의 일 구현예에 따르면, 상기 변형은 폴리 A 꼬리의 변형을 포함한다. According to one embodiment of the present invention, said modification comprises modification of poly A tail.
본 발명의 일 구현예에 따르면, 표적 세포의 세포 내 구간으로 mRNA 화합물의 수송을 촉진시키기 위한 물질을 추가로 포함하는 것이다. According to one embodiment of the present invention, a substance for facilitating transport of the mRNA compound to the intracellular section of the target cell is further included.
상기 표적세포는 간세포, 상피세포, 조혈세포, 상피세포, 내피세포, 폐세포, 골세포, 줄기세포, 간엽세포, 신경세포, 심장세포, 지방세포, 혈관 평활근 세포, 심근세포, 골격근 세포, 베타 세포, 뇌하수체 세포, 활액 내층 세포, 난소세포, 고환세포, 섬유아세포, B 세포, T 세포, 망상적혈구, 백혈구, 과립구 및 종양 세포로 구성된 그룹에서 선택되는 것이다. The target cells are hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, osteocytes, stem cells, mesenchymal cells, nerve cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.
본 발명의 조성물은 mRNA 화합물의 수송을 촉진시키기 위한 물질, 예를 들어, 혈관 뇌 장벽의 투과성을 방해하거나 개선하여 표적 세포로 외인성 mRNA의 수송을 증진시키는 물질과 결합될 수 있다.The composition of the present invention may be combined with a substance for promoting transport of mRNA compounds, for example, substances that interfere with or improve the permeability of the vascular brain barrier to enhance transport of exogenous mRNA to target cells.
본 발명의 조성물은 약제학적으로 허용되는 담체를 포함할 수 있으며, 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. The composition of the present invention may include a pharmaceutically acceptable carrier, which is commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin. , calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc. It is not limited.
본 발명의 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences(19th ed., 1995)에 상세히 기재되어 있다.The composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 조성물은 경구 또는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 정맥 내 주입, 피하 주입, 근육 주입, 복강 주입, 경피 투여 등으로 투여할 수 있다.The composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.
본 발명의 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다.A suitable dosage of the composition of the present invention may be variously prescribed depending on factors such as formulation method, administration mode, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. have.
본 발명의 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person skilled in the art to which the present invention pertains, or It can be prepared by pouring into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.
본 발명의 일 구현예에 따르면, 상기 조성물은 동결건조되는 것이다. According to one embodiment of the present invention, the composition is lyophilized.
본 발명에 따른 동결건조된 조성물은 투여에 앞서 복원될 수 있고 또는 생체 내에서 복원될 수 있다. 예를 들어, 동결건조된 조성물은 적절한 제형, 예를 들어, 원판, 막대 또는 막과 같은 피내 제형으로 조제될 수 있고 제형이 개체의 체액에 의해 생체 내에서 시간이 지남에 따라 재수화되도록 투여될 수 있다. 본 발명의 일 실시예에서, 본 발명 양이온성 분자 수송체 SG6가 처리된 동결건조 제형의 경우, 대조군에 비해 7배 높은 중화항체 형성능을 나타내고, 활성의 감소 없이 RBD 단백질의 중화능을 유도하는 것을 확인하여, mRNA 전달과 단백질 발현 및 백신 조성물의 안정성에 우수한 효과를 나타냄을 확인하였다.The lyophilized composition according to the present invention may be reconstituted prior to administration or may be reconstituted in vivo. For example, the lyophilized composition may be formulated into an appropriate dosage form, for example, an intradermal dosage form such as a disc, rod or membrane, and administered such that the dosage form is rehydrated over time in vivo by a subject's body fluids. can In one embodiment of the present invention, in the case of the freeze-dried formulation treated with the cationic molecular transporter SG6 of the present invention, it exhibits a 7-fold higher neutralizing antibody-forming ability than the control, and inducing the neutralizing ability of the RBD protein without a decrease in activity. It was confirmed that it showed an excellent effect on mRNA delivery, protein expression, and stability of the vaccine composition.
본 발명의 다른 양태에 따르면, 질병을 예방, 치료하는 방법에 있어서, (a) 항원 펩티드 또는 단백질을 암호화하는 mRNA 서열을 포함하는 mRNA 화합물, 및 (b) 하기 화학식 1의 양이온성 분자 수송체를 포함하는 이온 복합체를 포함하는 조성물을 투여하는 단계를 포함하는 방법을 제공하며, 상기 조성물의 투여 후 상기 mRNA는 상기 항원 펩티드 또는 단백질을 생산하기 위해 표적 세포에서 발현되는 것인, 방법을 제공한다.According to another aspect of the present invention, in a method for preventing or treating a disease, (a) an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein, and (b) a cationic molecular transporter of Formula 1 It provides a method comprising administering a composition comprising an ionic complex comprising
[화학식 1][Formula 1]
상기 식에서, In the above formula,
R1은 
로, 여기에서 n은 1 내지 8의 정수이다. R1 is , where n is an integer from 1 to 8.
본 발명의 다른 양태에 따르면, (a) 화학식 1로 표시되는 양이온성 분자 수송체를 제조하는 단계; 및 (b) 항원 펩티드 또는 단백질을 암호화하는 mRNA 서열을 포함하는 mRNA 화합물을 제조하여 상기 (a) 단계의 결과물과 혼합하는 단계를 포함하는 백신 조성물의 제조방법을 제공한다. According to another aspect of the present invention, (a) preparing a cationic molecular transporter represented by the formula (1); And (b) provides a method for preparing a vaccine composition comprising the step of preparing an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein and mixing it with the result of step (a).
[화학식 1][Formula 1]
상기 식에서, In the above formula,
R1은 
로, 여기에서 n은 1 내지 8의 정수이다. R1 is , where n is an integer from 1 to 8.
본 발명의 일 구현예에 따르면, 상기 (b) 단계는 항원 펩티드 또는 단백질을 암호화하는 mRNA 서열을 포함하는 mRNA 화합물을 제조하여 양이온성 리포좀과 반응시킨 후 상기 (a) 단계의 결과물과 혼합하는 단계일 수 있다. 본 발명의 일 실시예에서 mRNA 화합물과 상기 양이온성 리포좀의 질량비가 1 : 3인 경우 mRNA의 캡슐화 효율이 약 50% 였으나, 본 발명 양이온성 분자 수송체 SG6를 처리하는 경우 캡슐화 효율이 30% 이상 증가되어, 80% 이상으로 측정되었다. According to one embodiment of the present invention, step (b) is a step of preparing an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein, reacting with cationic liposome, and mixing with the result of step (a). can be In an embodiment of the present invention, when the mass ratio of the mRNA compound and the cationic liposome was 1:3, the mRNA encapsulation efficiency was about 50%, but when the present invention cationic molecular transporter SG6 was treated, the encapsulation efficiency was 30% or more increased, and was measured to be greater than 80%.
본 발명의 일 구현예에 따르면, 상기 (b) 단계 이후 (c) 상기 (b)의 결과물을 동결건조하는 단계를 포함하는 백신 조성물의 제조방법을 제공한다. According to one embodiment of the present invention, there is provided a method for producing a vaccine composition comprising the step of (c) freeze-drying the resultant of (b) after step (b).
본 발명의 일 구현예에 따르면, 상기 이온 복합체는 항원 펩티드 또는 단백질은 SARS-Cov-2의 스파이크 단백질, 뉴클레오캡시드 단백질 또는 이의 단편 또는 변이체로부터 유래되는 방법을 제공한다.According to one embodiment of the present invention, the ion complex provides a method in which the antigenic peptide or protein is derived from a SARS-Cov-2 spike protein, a nucleocapsid protein, or a fragment or variant thereof.
본 발명의 일 구현예에 따르면, 상기 mRNA 서열은 서열번호 1에 따른 SARS-Cov-2 스파이크 단백질의 RBD 서열에 상응하는 RNA 서열을 포함하는 것이다. According to one embodiment of the present invention, the mRNA sequence comprises an RNA sequence corresponding to the RBD sequence of the SARS-Cov-2 spike protein according to SEQ ID NO: 1.
본 발명의 일 구현예에 따르면 양이온성 리포좀은 DOTAP, DOPE 및 콜레스테롤을 포함하는 것이다. According to an embodiment of the present invention, cationic liposomes include DOTAP, DOPE and cholesterol.
본 발명의 일 구현예에 따르면, 상기 mRNA 화합물의 음이온 및 상기 양이온성 분자 수용체의 양이온의 비율이 0.1:1 내지 1:1인 것이다. According to one embodiment of the present invention, the ratio of the anion of the mRNA compound to the cation of the cationic molecular receptor is 0.1:1 to 1:1.
본 발명의 일 구현예에 따르면, 상기 mRNA 화합물과 상기 양이온성 리포좀의 질량비가 1:1 내지 1:6인 것이다.According to one embodiment of the present invention, the mass ratio of the mRNA compound to the cationic liposome is 1:1 to 1:6.
상기와 같은 본 발명에 따르면, 항원 펩티드 또는 단백질을 암호화하는 mRNA 및 이와 이온 복합체를 형성하는 양이온성 분자 수송체(SG6)를 포함하는 백신 조성물을 제공한다. According to the present invention as described above, there is provided a vaccine composition comprising an mRNA encoding an antigenic peptide or protein and a cationic molecular transporter (SG6) that forms an ion complex therewith.
본 발명에 따른 백신 조성물은 세포 독성이 없으면서도, mRNA 전달 효율이 높으며, 액상은 물론 동결 건조 제형에서 mRNA 활성 감소가 없어 보관이 용이한 백신 조성물을 제공한다.The vaccine composition according to the present invention has no cytotoxicity, high mRNA delivery efficiency, and provides a vaccine composition that is easy to store because there is no decrease in mRNA activity in liquid as well as freeze-dried formulations.
도 1은 본 발명에서 개발하고자 하는 코로나바이러스감염증-19 백신의 mRNA를 구성을 나타낸 도이다. 1 is a diagram showing the composition of the mRNA of the coronavirus infection-19 vaccine to be developed in the present invention.
도 2는 도 1의 mRNA를 생산하기 위한 각 구성의 DNA 서열을 나타낸 도이다. FIG. 2 is a diagram showing the DNA sequence of each configuration for producing the mRNA of FIG. 1 .
도 3의 (a)는 도 2의 각 구성요소가 삽입되어 있는 플라스미드 DNA를 대장균에서 추출하여 Bbs1 제한효소로 선형화(linearization)한 후 1% 아가로스 겔(agarose gel)을 이용하여 전기영동(SDS-polyacrylamide gel electrophoresis) 법으로 확인한 도이며, (b)는 (a) 이후 에탄올 침전법을 이용하여 선형화한 DNA를 회수하여 전기영동법으로 확인한 결과를 나타낸 도이다. 3A shows that the plasmid DNA into which each component of FIG. 2 is inserted is extracted from E. coli, linearized with a Bbs1 restriction enzyme, and then electrophoresed using 1% agarose gel (SDS). -polyacrylamide gel electrophoresis) method, (b) is a diagram showing the result of electrophoresis after recovering the linearized DNA using ethanol precipitation method after (a).
도 4는 IVT(In vitro transcription) 반응을 통하여 생산한 mRNA를 1% 변성 아가로스 겔(denaturing agarose gel)을 이용한 전기영동법으로 확인한 결과를 나타낸 도이다. 4 is a view showing the results of confirming the mRNA produced through the IVT (in vitro transcription) reaction by electrophoresis using a 1% denaturing agarose gel (denaturing agarose gel).
도 5는 IVT를 통해 생산한 mRNA가 실제 세포에서 정상 작동하여 RBD 단백질을 발현하는지 RBD 항체를 이용하여 웨스턴 블롯(Western blot)으로 확인한 결과를 나타낸 도이다. 5 is a diagram showing the results of confirming by Western blot using an RBD antibody whether mRNA produced through IVT operates normally in cells to express RBD protein.
도 6(a)는 본 발명 양이온성 분자 수송체 'SG6'가 mRNA와 이온 복합체(ionic complex)를 적절하게 형성하는지 확인한 전기영동 시험결과를 나타낸 도이고, 도 6(b)는 비교예로 사용한 양이온성 분자 수송체 R6 펩타이드(RRRRRR)와 PEI(polyethyleneimine HCl)가 mRNA와 이온 복합체(ionic complex)를 적절하게 형성하는지 확인한 전기영동 시험결과를 나타낸 도이다. Figure 6 (a) is a diagram showing the electrophoresis test results confirming that the present invention cationic molecular transporter 'SG6' properly forms an ionic complex with mRNA, Figure 6 (b) is used as a comparative example A diagram showing the results of an electrophoresis test confirming that the cationic molecular transporter R6 peptide (RRRRRR) and PEI (polyethyleneimine HCl) properly form an ionic complex with mRNA.
도 7은 본 발명 양이온성 분자 수송체 'SG6' 및 비교예로 사용한 양이온성 분자 수송체 R6 펩타이드와 PEI의 HEK293 세포에서 알라마 블루 분석(alamar blue assay)을 통한 세포독성 평가 결과를 나타낸 도이다. 7 is a diagram showing the cytotoxicity evaluation results through alamar blue assay in HEK293 cells of the cationic molecular transporter 'SG6' of the present invention and the cationic molecular transporter R6 peptide used as a comparative example and PEI. .
도 8은 본 발명 양이온성 분자 수송체 'SG6' 및 비교예로 사용한 리포좀 형태의 양이온성 분자 수송체 lipofectamine 2000™, MessengerMax™, R6, PEI(polyethyleneimine HCl) 물질 자체의 세포독성 및 이들 각각과 mRNA와의 이온 복합체의 세포독성 평가 결과를 나타낸 도이다. 8 is a cationic molecular transporter of the present invention 'SG6' and a liposome-type cationic molecular transporter used as a comparative example, lipofectamine 2000™, MessengerMax™, R6, and the cytotoxicity of the PEI (polyethyleneimine HCl) material itself and each of them and mRNA It is a diagram showing the cytotoxicity evaluation result of the ion complex with
도 9는 본 발명의 실시예 방법으로 제조된 양이온성 리포좀의 극저온 투과 전자 현미경(Cryo-Transmission Electron Microscope, Glacios microcscope, Thermo Fisher Scientific) 사진을 나타낸 도이다. 9 is a view showing a cryo-Transmission Electron Microscope (Cryo-Transmission Electron Microscope, Glacios microcscope, Thermo Fisher Scientific) photograph of the cationic liposome prepared by the Example method of the present invention.
도 10은 본 발명의 실시예 방법으로 제조된 양이온성 리포좀과 SARS-CoV-2 RBD mRNA가 적절하게 이온 복합체(mRNA lipoplex)를 형성하는지 확인하기 위하여 전기영동 시험결과를 나타낸 도이다.10 is a view showing the electrophoresis test results to confirm that the cationic liposome prepared by the Example method of the present invention and SARS-CoV-2 RBD mRNA properly form an ion complex (mRNA lipoplex).
도 11는 본 발명의 실시예 방법으로 제조된 양이온성 리포좀이 HEK293T 세포주에서 세포독성을 가지는지 여부를 리포펙타민과 비교한 알라마 블루 분석 결과를 나타낸 도이다.11 is a diagram showing the results of Alamar Blue analysis compared with lipofectamine whether cationic liposomes prepared by the Example method of the present invention have cytotoxicity in the HEK293T cell line.
도 12은 본 발명의 실시예 방법으로 제조된 양이온성 리포좀의 mRNA 로딩을 나타낸 결과이다. (a)는 리보그린 분석법으로 봉입률을 정량화한 도이며 (b)는 mRNA가 24시간까지 유지됨을 나타낸 도이다.12 is a result showing the mRNA loading of the cationic liposome prepared by the Example method of the present invention. (a) is a diagram showing the quantification of the encapsulation rate by ribogreen analysis, and (b) is a diagram showing that mRNA is maintained up to 24 hours.
도 13는 본 발명의 실시예 방법으로 제조된 양이온성 리포좀에 봉입된 mRNA가 리보핵산 분해효소로부터 보호되는지 여부를 전기영동 시험을 통하여 확인한 결과를 나타낸 도이다.13 is a diagram showing the results of confirming through an electrophoresis test whether mRNA encapsulated in cationic liposomes prepared by the method of the present invention is protected from ribonucleic acid degrading enzyme.
도 14는 리포펙타민-RBD mRNA 이온 복합체 및 본 발명의 실시예 방법으로 제조된 양이온성 리포좀-RBD mRNA 이온 복합체의 RBD mRNA 단백질 발현율을 웨스턴 블롯 방법으로 비교 분석한 결과를 나타낸 도이다.14 is a diagram showing the results of comparative analysis of RBD mRNA protein expression rates of the lipofectamine-RBD mRNA ion complex and the cationic liposome-RBD mRNA ion complex prepared by the Example method of the present invention by a Western blot method.
도 15는 본 발명의 실시예 방법으로 제조된 양이온성 리포좀-RBD mRNA 이온 복합체의 중화항체 형성능을 동물실험을 통해 확인한 결과를 나타낸 도이다.15 is a view showing the results of confirming the neutralizing antibody-forming ability of the cationic liposome-RBD mRNA ion complex prepared by the Example method of the present invention through an animal experiment.
이하, 본 발명의 내용을 하기의 실시예 및 실험예를 통해 더욱 상세히 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예 및 실험예에만 한정되는 것은 아니고, 이와 등가의 기술적 사상의 변형까지를 포함한다.Hereinafter, the content of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, and includes modifications of the technical idea equivalent thereto.
제조예 1: mRNA 화합물의 제조Preparation Example 1: Preparation of mRNA compound
1. mRNA 백신 구성의 서열 선정 및 벡터 구축
1. Sequence selection and vector construction of mRNA vaccine construction
도 1 구성의 mRNA를 생산하기 위하여 그에 상응하는 DNA 템플레이트를 포함하는 벡터를 구축하였다. 각 구성의 DNA 서열은 도 2에 나타냈다. 5'UTR, 시그널 펩타이드(signal peptide), 3’UTR 서열은 문헌(Richner et al., 2017, cell, 168, 1114-1125)을 참고하여 인간 헤모글로빈과 인간 IgE 유전자 유래 서열을 사용하였다. 백신 항원으로는 SARS-CoV-2 스파이크 단백질의 RBD(receptor binding domain)를 채택하였으며, 우한 균주(Wuhan strain)의 스파이크 단백질의 RBD 영역(아미노산 319~541)의 유전자 서열(NCBI Genbank(No.: QHD43416.1)을 사용하였다. 이 서열들은 인체에서 mRNA가 안정적으로 발현되도록 코돈 최적화(codon optimization)를 거친 후 화학 합성하였으며, T7 프로모터와 102개 아데닌의 폴리 A 꼬리가 포함된 pGS-IVT-RBD 벡터(Genscript)를 구축하였다.In order to produce the mRNA of the configuration of Fig. 1, a vector including a corresponding DNA template was constructed. The DNA sequence of each configuration is shown in FIG. 2 . For 5'UTR, signal peptide, and 3'UTR sequences, sequences derived from human hemoglobin and human IgE genes were used with reference to the literature (Richner et al., 2017, cell, 168, 1114-1125). As the vaccine antigen, the receptor binding domain (RBD) of SARS-CoV-2 spike protein was adopted, and the gene sequence of the RBD region (amino acids 319-541) of the spike protein of Wuhan strain (NCBI Genbank (No.: QHD43416.1) These sequences were chemically synthesized after codon optimization to ensure stable expression of mRNA in the human body, and pGS-IVT-RBD containing the T7 promoter and 102 adenine poly A tails A vector (Genscript) was constructed.
2. mRNA 생산을 위한 DNA 템플레이트 준비
2. DNA Template Preparation for mRNA Production
IVT 반응 시 mRNA를 생산하기 위한 DNA 템플레이트는 다음과 같이 준비하였다. pGS-IVT-RBD 벡터가 삽입된 E. coli DH5a 균을 100ug/ml 앰피실린이 첨가된 LB 배지(Luria-Bertani broth)에서 증식한 후 플라스미드 준비 키트(plasmid preparation kit)를 이용하여 벡터를 분리정제 하였다. 선형화는 2ug의 pGS-IVT-RBD 벡터를 정량한 후 Bbs1-HF(NEB) 제한효소를 이용하여 폴리 A 꼬리의 말단을 절단하였으며(37℃, 12시간), 반응물은 1% 아가로스 겔 전기영동으로 확인하였다. A DNA template for the production of mRNA in the IVT reaction was prepared as follows. After propagating the E. coli DH5a bacteria into which the pGS-IVT-RBD vector was inserted in LB medium (Luria-Bertani Broth) supplemented with 100ug/ml ampicillin, the vector was isolated and purified using a plasmid preparation kit. did. For linearization, 2ug of pGS-IVT-RBD vector was quantified, and the end of the poly A tail was cut using a Bbs1-HF (NEB) restriction enzyme (37°C, 12 hours), and the reaction product was subjected to 1% agarose gel electrophoresis. was confirmed as
그 결과, 약 4kb의 선형화 된 pGS-IVT-RBD 벡터가 높은 순도로 얻어짐을 확인하였다(도 3a). 그 후 제한효소 반응물에서 순수한 선형화된 DNA를 얻기 위하여 에탄올 침전법으로 불순물을 제거하여 mRNA 생산에 사용할 순도 95% 이상의 DNA 템플레이트를 확보하였다(도 3b).As a result, it was confirmed that a linearized pGS-IVT-RBD vector of about 4 kb was obtained with high purity (Fig. 3a). Thereafter, in order to obtain pure linearized DNA from the restriction enzyme reaction, impurities were removed by ethanol precipitation to obtain a DNA template with a purity of 95% or more for use in mRNA production (FIG. 3b).
3. IVT(In vitro transcription)를 통한 mRNA 생산
3. mRNA production through IVT (In vitro transcription)
IVT 반응IVT reaction
mRNA는 Megascript kit(Ambion, USA)을 이용하여 시험관 내 전사(in vitro transcription) 방법으로 생산하였다. 위에서 확보한 DNA 템플레이트 시료 500ng을 사용하였다. ARCA cap(Trilink, USA)은 별도로 구성하여 120mmol을 반응에 사용하였다. 반응 조성물은 아래 표 1과 같이 구성하였으며, 37℃에서 12시간 동안 반응을 진행하였다. 반응이 종료된 후 Turbo DNase(Ambion, USA) 2 유닛(units)을 37℃에서 15분 동안 처리하여 DNA 템플레이트를 제거하였다.mRNA was produced by an in vitro transcription method using a Megascript kit (Ambion, USA). 500ng of the DNA template sample obtained above was used. ARCA cap (Trilink, USA) was separately configured and 120 mmol was used for the reaction. The reaction composition was configured as shown in Table 1 below, and the reaction was carried out at 37°C for 12 hours. After completion of the reaction, 2 units of Turbo DNase (Ambion, USA) were treated at 37° C. for 15 minutes to remove the DNA template.
| 구성 성분Ingredients | 부피(ul)volume (ul) | 농도(mmol)Concentration (mmol) |
| Nuclease-free waterNuclease-free water | 1.81.8 | |
|
ATP solution |
22 | 150150 |
|
CTP solution |
22 | 150150 |
|
UTP solution |
22 | 150150 |
|
GTP solution (1:5 dilution of GTP solution)GTP solution (1:5 dilution of GTP solution) |
22 | 3030 |
|
ARCA cap |
22 | 120120 |
|
10X reaction buffer |
22 | |
|
DNA template |
55 | |
|
Enzyme Mix |
22 | |
|
총 부피 |
2020 |
염화리튬(Lithium chloride) 침전Lithium chloride precipitation
생산된 mRNA를 분리 정제하기 위하여 염화리튬 침전법을 이용하였다. 염화리튬 시약 및 뉴클레아제 무함유 물(nuclease-free water) 각각 30ul를 반응물에 첨가 후 20℃에서 30분 동안 침전시켰다. 침전된 mRNA 펠렛은 원심분리 후 70% 에탄올을 이용하여 1회 세척하였으며 펠렛은 클린벤치 내에서 30분간 건조시킨 후 뉴클레아제 무함유 물에 녹여서 1% 변성 아가로스 겔을 이용한 전기영동법으로 확인하였다. Lithium chloride precipitation method was used to separate and purify the produced mRNA. Lithium chloride reagent and 30 ul of nuclease-free water were each added to the reaction mixture, and then precipitated at 20° C. for 30 minutes. The precipitated mRNA pellet was washed once with 70% ethanol after centrifugation, and the pellet was dried in a clean bench for 30 minutes, dissolved in nuclease-free water, and confirmed by electrophoresis using 1% denatured agarose gel. .
그 결과, 약 1kb 크기의 순도 95% 이상되는 mRNA가 생산됨을 확인하였으며(도 4), 정량결과 1회 반응 시 약 50ug 이상의 수율로 확인되었다.As a result, it was confirmed that mRNA having a size of about 1 kb and having a purity of 95% or more was produced (FIG. 4), and as a result of quantification, it was confirmed that a yield of about 50 μg or more was obtained in one reaction.
4. HEK293 세포에서의 mRNA 발현 확인
4. Confirmation of mRNA expression in HEK293 cells
IVT를 통해 생산된 mRNA의 정상적 발현 여부를 상업적으로 판매되는 양이온성 전달체를 이용하여 확인하였다. 0.5ug, 1ug의 mRNA를 각각 0.75ul, 1.5ul의 리포펙타민, messenger MAXTM(Invitrogen, USA)와 혼합 후 24 웰 플레이트에서 1x105 HEK293 세포에 48시간 처리하여 트랜스펙션(transfection)한 후 상층액을 회수하여 웨스턴 블롯(Western blot)으로 RBD 발현을 확인하였다. 1차 항체로 SARS-CoV-2(2019-nCoV) mouse anti-spike protein monoclonal antibody(Sino biological, China)를 1000배 희석하여 사용하였다. Normal expression of mRNA produced through IVT was confirmed using a commercially available cationic delivery system. 0.5ug and 1ug mRNA were mixed with 0.75ul and 1.5ul of lipofectamine, messenger MAX TM (Invitrogen, USA), respectively, and then treated and transfected in 1x10 5 HEK293 cells in a 24-well plate for 48 hours. The supernatant was recovered and RBD expression was confirmed by Western blot. As the primary antibody, a 1000-fold diluted SARS-CoV-2 (2019-nCoV) mouse anti-spike protein monoclonal antibody (Sino biological, China) was used.
그 결과, 약 37kDa RBD 단백질 발현을 확인함으로 mRNA가 정상 작동되는 것을 정성적으로 확인하였다(도 5).As a result, it was qualitatively confirmed that the mRNA was normally operated by confirming the expression of about 37 kDa RBD protein (FIG. 5).
제조예 2 : mRNA 화합물과 양이온성 분자 수송체 간의 이온 복합체의 제조Preparation Example 2: Preparation of an ionic complex between an mRNA compound and a cationic molecular transporter
1. 양이온성 분자 수송체 SG6의 제조
1. Preparation of cationic molecular transporter SG6
본 발명의 화학식 1에 따른 솔비톨을 골격으로 하고 6개의 구아니딘기(+6 : 구아니딘기 1개 당 1개의 양전하)를 갖는 양이온성 화합물 SG6을 대한민국 특허 제10-0699279호의 실시예 8에 기재된 방식에 따라 제조하였다.Cationic compound SG6 having 6 guanidine groups (+6: 1 positive charge per guanidine group) with sorbitol according to Formula 1 as a skeleton of the present invention was prepared in the method described in Example 8 of Korean Patent No. 10-0699279 prepared according to
[화학식 1][Formula 1]
상기 식에서, In the above formula,
R1은 
로, 여기에서 n은 1 내지 8의 정수이다. R 1 is , where n is an integer from 1 to 8.
본 발명에서는 mRNA 효율적인 전달을 위하여 생체적합성 물질인 솔비톨(Sorbitol)과 양이온성의 구아니디니움(Guanidinium)을 기반으로 하는 양이온성 분자수송체(Sorbitol-G6; SG6, 분자량 1112.7g/mol)를 이용하여, 정전기적 상호작용에 의한 이온 복합체(ionic complex)를 제조하였다. In the present invention, a cationic molecular transporter (Sorbitol-G6; SG6, molecular weight 1112.7 g/mol) based on sorbitol, a biocompatible material and cationic guanidinium, is used for efficient mRNA delivery. Thus, an ionic complex by electrostatic interaction was prepared.
비교예로서 PEI(polyethyleneimine-HCl, 분자량 4668 g/mol, Sigma)과 양이온성 아미노산인 알지닌 6개로 구성된 R6 펩타이드를 사용하였다. 각 물질별 1개 분자당 이온수는 mRNA는 980개의 음이온, SG6와 R6는 각각 6개의 양이온, PEI는 60개의 양이온을 띠고 있다. 이를 기반으로 이온 복합체의 제조비율을 설정하였으며, 음이온/양이온 비율(Negative/positive charge ratio, N/P ratio)에 따라 mRNA 1몰당 각 양이온성 전달체의 몰수를 나타내었다(표 2).As a comparative example, an R6 peptide consisting of PEI (polyethyleneimine-HCl, molecular weight 4668 g/mol, Sigma) and 6 arginine, a cationic amino acid, was used. As for the number of ions per molecule for each substance, mRNA has 980 anions, SG6 and R6 each have 6 cations, and PEI has 60 cations. Based on this, the production ratio of the ion complex was set, and the number of moles of each cationic transporter per 1 mole of mRNA was shown according to the negative/positive charge ratio (N/P ratio) (Table 2).
| N(-)/P(+) ratioN(-)/P(+) ratio | 몰수(moles)moles | ||
| SG6SG6 | R6R6 | PEIPEI | |
| 0.20.2 | 815815 | 815815 | 81.581.5 |
| 0.50.5 | 326326 | 326326 | 32.632.6 |
| 1One | 163163 | 163163 | 16.316.3 |
| 22 | 81.581.5 | 81.581.5 | 8.158.15 |
| 55 | 32.632.6 | 32.632.6 | 3.263.26 |
이온 복합체가 적절하게 형성되는지 알아보기 위하여 500ng mRNA를 기준으로 각 물질을 뉴클레아제 무함유 물에 녹인 후 표 2에 따라 30분간 이온 복합체를 형성시킨 후, 1% 변성 아가로스 겔을 이용한 전기영동법을 행하였다.
In order to check whether the ionic complex is properly formed, each material is dissolved in nuclease-free water based on 500 ng mRNA, and then the ionic complex is formed according to Table 2 for 30 minutes, followed by electrophoresis using a 1% denatured agarose gel was done .
그 결과, N/P 비율이 1 이하(0.2, 0.5)일 때 양이온성의 복합체가 형성되는 것을 확인하였다(도 6).As a result, it was confirmed that the cationic complex was formed when the N/P ratio was 1 or less (0.2, 0.5) ( FIG. 6 ).
실시예 1: SG6 양이온성 분자 수송체의 세포독성 평가 Example 1: Cytotoxicity evaluation of SG6 cationic molecular transporter
양이온성 분자수송체 자체의 세포독성을 평가하기 위해 분자구조적으로 유사한 R6 펩타이드, PEI 수송체와 알라마 블루 분석(alamar blue assay)을 처리농도 200uM 까지 수행하였다. In order to evaluate the cytotoxicity of the cationic molecular transporter itself, molecularly structurally similar R6 peptide, PEI transporter and alamar blue assay were performed up to a treatment concentration of 200 uM.
그 결과, SG6와 R6 자체의 세포독성은 거의 나타나지 않는 반면, PEI의 경우 CC50 값이 50uM로 나타났다(도 7). 세포독성 시험의 양성대조군으로 일반적으로 사용되는 사포닌의 경우 문헌에서 알려진 CC50 값(20~30uM)과 유사하게 나타났다.As a result, the cytotoxicity of SG6 and R6 itself was hardly observed, whereas in the case of PEI, the CC50 value was 50 uM (FIG. 7). In the case of saponin, which is generally used as a positive control for cytotoxicity tests, it was similar to the CC50 value (20-30uM) known in the literature.
실시예 2: 이온 복합체의 세포독성 평가Example 2: Assessment of cytotoxicity of ion complexes
상업적으로 사용되는 리포좀 형태의 양이온성 분자 수송체인 lipofectamine2000TM(Invitrogen, USA)과 Messenger MAXTM(Invitrogen, USA)를 제조사의 매뉴얼에 따라 생산된 mRNA와 이온 복합체를 형성시켰다. mRNA 100ug 당 lipofectamine2000TM은 0.2ul, Messenger MAXTM는 0.15ul을 사용하였다. 알라마 블루(Alamar blue, Invitrogen, USA) 시약을 2시간 동안 처리한 후 570nm, 600nm로 흡광도를 측정하였으며, 아무것도 처리하지 않은 세포를 생존율(viability) 100%로 설정하였다. Commercially used liposome-type cationic molecular transporters lipofectamine2000 TM (Invitrogen, USA) and Messenger MAX TM (Invitrogen, USA) were used to form an ion complex with the mRNA produced according to the manufacturer's manual. 0.2ul of lipofectamine2000 TM and 0.15ul of Messenger MAX TM were used per 100ug of mRNA. After treatment with Alamar blue (Invitrogen, USA) reagent for 2 hours, absorbance was measured at 570 nm and 600 nm, and the untreated cells were set to 100% viability.
SG6, R6와 PEI의 이온 복합체 제조는 표 2의 N/P 비율에 따라 mRNA 100ng 기준으로 각각 1:2와 1:5 복합체를 형성하였다. 30분간 이온 복합체를 형성 후, 96-웰 플레이트에서 2x104 개의 HEK293 세포에 복합체를 48시간 동안 처리한 후 알라마 블루 시약으로 세포 생존율을 측정하였다. Preparation of ion complexes of SG6, R6 and PEI formed 1:2 and 1:5 complexes, respectively, based on 100 ng of mRNA according to the N/P ratio in Table 2. After forming the ion complex for 30 minutes, the complex was treated in 2x10 4 HEK293 cells in a 96-well plate for 48 hours, and then cell viability was measured with Alamar Blue reagent.
그 결과, 권장 사용 농도에서 시약 자체만으로는 사용된 양이온성 수송체 lipo2000, MMAX, SG6, R6, PEI의 경우 세포활성이 약 10% 감소되었다(도 8). 이온 복합체 형성 후, lipofectamine2000TM과 Messenger MAXTM의 경우 25~40% 이상의 세포활성 감소를 나타낸 반면, SG6, R6, PEI의 경우 세포활성 감소가 거의 나타나지 않았다(도 8).As a result, in the case of the cationic transporters lipo2000, MMAX, SG6, R6, and PEI used only with the reagent itself at the recommended concentration, cell activity was reduced by about 10% (FIG. 8). After formation of the ion complex, lipofectamine2000 TM and Messenger MAX TM showed a decrease in cellular activity of 25-40% or more, whereas SG6, R6, and PEI showed almost no decrease in cellular activity (FIG. 8).
실시예 3: mRNA 봉입율 및 담지율 측정Example 3: Measurement of mRNA encapsulation rate and loading rate
양이온성 분자 수송체 및 상업적으로 이용되는 리포좀을 이용하여 mRNA의 봉입율 및 담지율을 측정하였다. 양이온성 분자 수송체로는 SG6, 상업적으로 이용되는 리포좀으로는 Cationic DOTAP/DC-CHOL Liposomes Adjuvants(FormuMax Scientific Inc.)를 사용하였고, 분석법으로는 핵산과 결합하는 형광염료인 리보그린을 이용한 리보그린 분석법을 수행하였다.The encapsulation and loading rates of mRNA were measured using a cationic molecular transporter and commercially available liposomes. SG6 was used as a cationic molecular transporter, and Cationic DOTAP/DC-CHOL Liposomes Adjuvants (FormuMax Scientific Inc.) were used as commercially available liposomes. Ribogreen analysis method using ribogrin, a fluorescent dye that binds to nucleic acids, was used as an analysis method. was performed.
RBD mRNA와 eGFP mRNA는 SG6의 존재와 부재하에 리포좀과 상온에서 반응하여 이온 복합체를 형성하였고, 이에 리보그린을 더해 형광값을 측정하였다. 봉입률 및 담지율은 하기 식으로 각각 계산되었다.RBD mRNA and eGFP mRNA reacted with liposomes at room temperature in the presence and absence of SG6 to form an ionic complex, and fluorescence values were measured by adding ribogrin. The encapsulation rate and the loading rate were respectively calculated by the following formulas.
봉입률 = (총 mRNA 양-잔여 mRNA 양)/(총 mRNA 양)X100, Encapsulation rate = (total mRNA amount-residual mRNA amount)/(total mRNA amount)X100,
담지율 = (총 mRNA 양-잔여 mRNA 양)/(사용된 리포좀 양)X100Carrying rate = (total mRNA amount-residual mRNA amount)/(liposome amount used)X100
그 결과 RBD mRNA 및 eGFP mRNA는 모두 최소 80% 이상 리포좀에 결합한 것으로 분석되며, 최소 20% 이상의 mRNA가 리포좀에 로딩된 것으로 관찰되었다(도 9).As a result, both RBD mRNA and eGFP mRNA were analyzed to be at least 80% bound to the liposome, and it was observed that at least 20% or more of the mRNA was loaded into the liposome ( FIG. 9 ).
제조예 3: SARS-CoV-2 RBD mRNA와 양이온성 리포좀 간의 이온 복합체 제조Preparation Example 3: Preparation of ionic complex between SARS-CoV-2 RBD mRNA and cationic liposome
1.
양이온성 리포좀(Cationic liposome)의 제조
One. Preparation of cationic liposomes
클로로포름(Choloform)을 용매로 하여 양이온성 지질 DOTAP(chloride salt, 1,2-dioleoyl-3-trimethylammonium-propane), DOPE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 콜레스테롤(Cholesterol)은 둥근바닥 플라스크에 1: 0.5:0.5의 몰 비로 취해진다. 혼합된 지질용액은 회전 농축기(Rotary evaporator)를 이용하여 플라스크 내벽에 얇은 지질막(Thin-layer film) 형태가 형성될 때까지 충분히 건조하고, 20mM HEPES 용액을 추가하여 재수화시켰다. 재수화된 현탁액은 교반기(Vortex)와 수조형 초음파 분산기(Sonicator)를 통해 다운사이징(downsizing)되고, 이어 리포좀의 균질화를 위해 0.1um 기공(Pore) 크기를 가진 폴리카보네이트 막을 수차례 반복 통과시켜 리포좀을 제조하였다. 준비된 리포좀은 극저온 투과 전자 현미경을 통한 구조분석(도 9)과 크기 및 표면전하(Zeta potential)를 측정한 뒤 사용하기 전까지 4℃에서 보관하였다.Using chloroform as a solvent, cationic lipids DOTAP (chloride salt, 1,2-dioleoyl-3-trimethylammonium-propane), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), cholesterol (Cholesterol) is taken into a round bottom flask in a molar ratio of 1:0.5:0.5. The mixed lipid solution was sufficiently dried until a thin-layer film was formed on the inner wall of the flask using a rotary evaporator, and rehydrated by adding 20 mM HEPES solution. The rehydrated suspension is downsized through a stirrer (Vortex) and a water tank type ultrasonic disperser (Sonicator), and then, for homogenization of the liposome, a polycarbonate membrane having a 0.1um pore size is repeatedly passed through a polycarbonate membrane several times to liposome. was prepared. The prepared liposomes were stored at 4°C until use after structural analysis (FIG. 9) and measurement of size and surface charge (Zeta potential) through a cryogenic transmission electron microscope.
2. 양이온성 리포좀과 SARS-CoV-2 RBD mRNA의 이온 복합체(mRNA-lipoplex)의 제조
2. Preparation of cationic liposome and SARS-CoV-2 RBD mRNA ion complex (mRNA-lipoplex)
1ug의 mRNA는 다양한 질량비(1:1, 1:3, 1:5, 1:7)로 양이온성 리포좀과 상온에서 반응시켰으며, 형성된 이온 복합체는 1% 변성 아가로스 겔 전기영동법을 통해 분석하였다. 또한, SG6를 포함하는 리포좀은 mRNA와 리포좀의 상온 반응 후 SG6를 처리하여 추가로 상온에서 반응시켰다. 1ug mRNA was reacted with cationic liposomes at room temperature in various mass ratios (1:1, 1:3, 1:5, 1:7), and the formed ionic complex was analyzed by 1% denaturing agarose gel electrophoresis. . In addition, the liposome containing SG6 was further reacted at room temperature by treating SG6 after the reaction of mRNA and the liposome at room temperature.
리포좀, 리포좀-mRNA(이하, mRNA lipolex) 및 SG6 처리된 mRNA lipoplex(이하, mRNA-SG6-lipoplex)의 입자크기, 다분산 지수(PDI) 및 표면전하 특성(characterization)은 나노 입도 분석기(Zetasizer Nano ZSP, Malvern)를 사용하여, 그 결과를 표 3에 나타냈다.The particle size, polydispersity index (PDI) and surface charge characteristics (characterization) of liposomes, liposome-mRNA (hereinafter, mRNA lipolex) and SG6-treated mRNA lipoplex (hereinafter, mRNA-SG6-lipoplex) were measured using a nano particle size analyzer (Zetasizer Nano). ZSP, Malvern), and the results are shown in Table 3.
| FormulationsFormulations | Z-Average Size (nm)Z-Average Size (nm) | Polydispersity indexPolydispersity index | Zeta potential (mV)Zeta potential (mV) |
| Cationic liposomeCationic liposome | 165.3 ± 2.4165.3 ± 2.4 | 0.23 ± 0.030.23 ± 0.03 | 38.5 ± 0.838.5 ± 0.8 |
| mRNA-LipoplexmRNA-Lipoplex | 342.5 ± 16.8342.5 ± 16.8 | 0.38 ± 0.060.38 ± 0.06 | 31.2 ± 1.731.2 ± 1.7 |
| mRNA-SG6-lipoplexmRNA-SG6-lipoplex | 233.1 ±12.0233.1 ±12.0 | 0.32 ± 0.090.32 ± 0.09 | 34.2 ± 3.034.2 ± 3.0 |
| mRNA-SG6-lipoplex (lyophilized)mRNA-SG6-lipoplex (lyophilized) | 277.5 ± 16.7277.5 ± 16.7 | 0.25 ± 0.080.25 ± 0.08 | 14.2 ± 1.214.2 ± 1.2 |
mRNA와 리포좀은 질량비 1:5를 기준으로 안정적인 이온 복합체를 형성하여, mRNA 로딩 및 유전자 전달체로서의 가능성을 확인하였다(도 10).mRNA and liposome formed a stable ion complex based on a mass ratio of 1:5, confirming the possibility of mRNA loading and gene delivery ( FIG. 10 ).
실시예 4: mRNA-lipoplex의 생체 외 세포독성 평가Example 4: In vitro cytotoxicity evaluation of mRNA-lipoplex
mRNA-lipoplex의 생체 외 세포 독성 여부를 평가하기 위해 알라마 블루 분석법을 이용하여 세포 생존율을 확인하였다. To evaluate whether mRNA-lipoplex was cytotoxic in vitro, cell viability was confirmed using Alamar Blue assay.
인간 유래 세포주인 HEK293T 세포는 96-웰 플레이트에 6 X 103 세포/웰의 수로 플레이팅 되어 리포펙타민 혹은 각기 다른 질량비로 제조된 mRNA-lipoplex와 함께 48시간 동안 배양하였다. 이어 상기 세포는 알라마 블루 시약을 추가하여 흡광도 및 형광값을 측정하였으며, 증식정도를 측정하여 세포독성을 평가하였다. HEK293T cells, a human-derived cell line, were plated in a 96-well plate at a number of 6 X 10 3 cells/well and cultured with lipofectamine or mRNA-lipoplex prepared in different mass ratios for 48 hours. Then, the cells were measured for absorbance and fluorescence by adding Alamar Blue reagent, and the degree of proliferation was measured to evaluate cytotoxicity.
그 결과, 비교군으로 사용된 동량의 리포펙타민이 처리된 실험군에서는 리포펙타민의 농도가 높아질수록 세포증식이 감소하고 세포독성이 증가되는 반면 mRNA-lipoplex가 처리된 실험군에서는 각기 다른 리포좀 질량비로 형성된 그룹 모두에서 세포독성이 현저히 낮은 것을 확인하였다(도 11).As a result, in the experimental group treated with the same amount of lipofectamine used as a comparison group, cell proliferation decreased and cytotoxicity increased as the concentration of lipofectamine increased, whereas in the experimental group treated with mRNA-lipoplex, different liposome mass ratios were used. It was confirmed that the cytotoxicity was significantly low in all the formed groups (FIG. 11).
실시예 5: mRNA-lipoplex의 mRNA 봉입률 분석Example 5: Analysis of mRNA Encapsulation Rate of mRNA-lipoplex
mRNA와 리포좀이 이온결합체를 형성하였을 때, 리포좀의 내외부로 봉입된 mRNA를 정량화하고자 핵산의 검출 및 정량에 사용되는 리보그린 분석법을 수행하였다. 봉입률은 사용된 전체 mRNA에 대하여 리포좀에 결합된 mRNA의 함량비율을 나타내며, 봉입률이 높을수록 mRNA-lipoplex가 잘 형성되고 노출된 유리(free) mRNA가 적은 것으로 평가할 수 있다. When the mRNA and the liposome form an ionic compound, the ribogreen assay used for the detection and quantification of nucleic acids was performed to quantify the mRNA encapsulated in and out of the liposome. The encapsulation rate represents the content ratio of mRNA bound to the liposome with respect to the total mRNA used, and it can be evaluated that the higher the encapsulation rate, the better the mRNA-lipoplex is formed and the amount of exposed free mRNA is small.
이를 확인하기 위해 실시예 11의 방법으로 형성된 이온 복합체는 96 웰 플레이트로 옮겨져 핵산 결합 시약인 리보그린과 반응한 뒤 형광값을 측정하여 다음 식으로 캡슐화 효율(Encapsulation Efficiency, EE%)을 평가하였다.To confirm this, the ion complex formed by the method of Example 11 was transferred to a 96-well plate, reacted with ribogreen, a nucleic acid binding reagent, and then the fluorescence value was measured to evaluate the encapsulation efficiency (EE%) by the following equation.
캡슐화 효율 = (총 mRNA 양-잔여 mRNA양)/(총 mRNA 양) X 100Encapsulation Efficiency = (Total mRNA Amount - Residual mRNA Amount)/(Total mRNA Amount) X 100
그 결과, mRNA와 리포좀 질량비 1:3의 mRNA-lipoplex는 약 50%의 mRNA를 캡슐화하고, 질량비 1:5는 약 90%의 mRNA를 각각 캡슐화하였으며 상기 결과는 높은 결과적 유의성을 나타내었다. 또한, 흥미롭게도 mRNA와 리포좀 질량비 1:3의 mRNA-lipoplex는 봉입률이 약 50%인 반면 SG6가 처리된 이온 복합체는 30% 이상 증가된 80% 이상으로 측정되었다. 이는 SG6가 이온 복합체의 형성에 유의미한 영향을 끼친 것으로 생각되며, 이렇게 형성된 이온 복합체는 24시간까지 리포좀의 분해나 mRNA 용출 없이 안정성이 유지됨을 실험적 결과로 확인하였다(도 12a, 도 12b).As a result, the mRNA-lipoplex of the mRNA and liposome mass ratio of 1:3 encapsulated about 50% of the mRNA, and the mass ratio of 1:5 encapsulated about 90% of the mRNA, respectively, and the results showed high significance. Interestingly, the mRNA-lipoplex with a mass ratio of mRNA and liposome of 1:3 had an encapsulation rate of about 50%, whereas the SG6-treated ion complex increased by more than 30% to 80% or more. It is thought that SG6 had a significant effect on the formation of the ion complex, and it was confirmed as an experimental result that the ionic complex thus formed was maintained without degradation of the liposome or mRNA elution until 24 hours (Fig. 12a, Fig. 12b).
실시예 6: mRNA-lipoplex의 리보핵산 분해효소(RNase) 보호 효과Example 6: Ribonuclease (RNase) protective effect of mRNA-lipoplex
리포좀과 복합체를 형성한 mRNA는 체내에 분포하는 다양한 리보핵산 분해효소로부터 보호되어 노출된(free) RNA에 비해 높은 효율로 세포내에 전달될 수 있다. The mRNA complexed with the liposome is protected from various ribonucleic acid degrading enzymes distributed in the body, and can be delivered into the cell with higher efficiency than the free RNA.
이를 확인하기 위해 mRNA와 리포좀 질량비 1:5로 형성된 이온 복합체에 리보핵산 분해효소를 처리하여 노출된 RNA를 분해 후 Proteinase K로 리보핵산 분해효소를 비활성화시켰다. 이후 Triton X-100으로 리포좀 막을 파괴시켜 봉입된 mRNA를 방출시킨 뒤, 1% 변성 아가로스 겔 전기영동법으로 mRNA 여부를 확인하였다. To confirm this, ribonucleic acid degrading enzyme was treated to the ion complex formed in the mRNA and liposome mass ratio of 1:5 to decompose the exposed RNA, and then the ribonucleic acid degrading enzyme was inactivated with Proteinase K. Thereafter, the liposome membrane was disrupted with Triton X-100 to release the encapsulated mRNA, and the presence of mRNA was confirmed by 1% denaturing agarose gel electrophoresis.
그 결과, 리포좀이 처리되지 않은 노출된 mRNA는 리보핵산 분해효소에 의해 모두 분해된 반면 리포좀과 이온 복합체를 형성한 mRNA는 리포좀에 의해 리보핵산 분해효소로부터 보호되어 더욱 효과적으로 mRNA를 전달할 수 있음을 확인하였다(도 13).As a result, it was confirmed that the liposome-untreated exposed mRNA was all degraded by ribonuclease, whereas the mRNA that formed an ion complex with the liposome was protected from ribonuclease by the liposome, thereby enabling more effective mRNA delivery. (FIG. 13).
실시예 7: HEK293T를 이용한 RBD 단백질 발현 효율 평가Example 7: Evaluation of RBD protein expression efficiency using HEK293T
상기 실시예 6에서 확인한 리포좀 이온 복합체의 리보핵산 분해효소 보호 효과와 더불어 보호된 mRNA의 세포 내 전달 및 단백질 발현여부를 평가하기 위해 웨스턴 블롯 분석을 수행하였다. Western blot analysis was performed to evaluate the intracellular delivery and protein expression of the protected mRNA as well as the ribonucleic acid degrading enzyme protective effect of the liposome ion complex confirmed in Example 6 above.
먼저 인간유래 세포주인 HEK293T는 24 웰 플레이트에 2 X 104개 세포/웰의 수로 플레이팅되어 각기 다른 질량비로 반응시킨 mRNA-lipoplex와 48시간 동안 배양하였으며, 비교군으로 사용된 리포펙타민은 제조사의 매뉴얼에 따라 처리되었다. 배양된 세포는 원심분리 후 상등액만 취하여 4~15% 구배 겔(gradient gel)로 전기영동을 수행한 뒤 SARS-CoV-2 스파이크 항체를 이용하여 웨스턴 블롯 방법으로 분석하였다. First, HEK293T, a human-derived cell line, was plated at a number of 2 X 10 4 cells/well in a 24-well plate and cultured with mRNA-lipoplex reacted at different mass ratios for 48 hours. was processed according to the manual of After centrifugation, only the supernatant was collected, electrophoresed on a 4-15% gradient gel, and then analyzed by Western blot using SARS-CoV-2 spike antibody.
그 결과, 약 37kDa RBD 단백질 발현을 확인함으로서 mRNA가 리포좀과의 복합체를 형성하여 효과적으로 세포 내로 전달되었음을 확인하였고, 비교군으로 사용된 동량의 리포펙타민보다 현저히 높은 발현량을 통해 유전자 전달체로서의 높은 가능성을 확인하였다(도 14). 또한, 단백질 발현량을 상대적으로 비교 측정한 결과 mRNA-lipoplex 형성 시 SG6가 추가 처리된 이온 복합체에서 더 높은 단백질 발현 효율을 관찰할 수 있었기 때문에 mRNA의 세포 내 도입과 단백질 발현증가에 SG6의 처리가 보다 효과적임을 확인할 수 있었다(도 14).As a result, by confirming the expression of about 37 kDa RBD protein, it was confirmed that the mRNA was effectively delivered into the cell by forming a complex with the liposome, and a high possibility as a gene carrier through a significantly higher expression level than the same amount of lipofectamine used as a control group was confirmed (FIG. 14). In addition, as a result of comparatively measuring the protein expression level, higher protein expression efficiency was observed in the ion complex further treated with SG6 during mRNA-lipoplex formation. It was confirmed that it was more effective (FIG. 14).
실시예 8: mRNA-lipoplex의 중화항체 형성능 확인Example 8: Confirmation of neutralizing antibody-forming ability of mRNA-lipoplex
상기 제조된 mRNA-lipoplex를 BALB/c 마우스에 접종함으로써 리포좀 이온 복합체의 백신으로서의 면역원성을 확인하였다. The immunogenicity of the liposome ion complex as a vaccine was confirmed by inoculating the prepared mRNA-lipoplex into BALB/c mice.
구체적으로 mRNA-lipoplex는 각각 mRNA 2, 10ug의 용량을 기준으로 mRNA와 리포좀 질량비 1:5가 되도록 접종시료를 제조하였고, 이때 일부 실험군은 이온 복합체에 SG6를 처리하여 상기 실시예에서 가장 효과적으로 mRNA가 발현되는 조건과 동일하게 접종 시료를 제조하였다. 또한, 백신의 보관 용이성과 장기간 안정성 확인을 위해 각 시료는 동결건조 제형으로도 제조되어 제제간 면역원성의 차이를 비교하였다.Specifically, mRNA-lipoplex prepared an inoculation sample so that the mRNA and liposome mass ratio was 1:5 based on the dose of mRNA 2 and 10ug, respectively, and at this time, some experimental groups treated SG6 in the ion complex to produce mRNA most effectively in the above example. Inoculation samples were prepared in the same manner as the expressed conditions. In addition, each sample was prepared in a freeze-dried formulation to confirm the ease of storage and long-term stability of the vaccine, and the differences in immunogenicity between formulations were compared.
실험군의 접종방법은 모두 근육 내 접종으로 동일하며, 각각 3주 간격 (0일, 21일)에 맞춰 프라임(1차)-부스트(2차) 접종을 수행하여 2차 접종 실시 2주 후 혈청을 분리해 SARS-CoV-2 대리 바이러스 중화 검사(SARS-CoV-2 surrogate virus neutralization test, sVNT)로 중화항체 형성능을 측정하였다.The inoculation method of the experimental group was all the same as intramuscular inoculation, and prime (1st)-boost (2nd) inoculation was performed at 3-week intervals (day 0, day 21), respectively, and serum was administered 2 weeks after the second inoculation. After separation, the ability to form neutralizing antibodies was measured by SARS-CoV-2 surrogate virus neutralization test (sVNT).
그 결과, 용량간 차이는 적었지만 SG6가 처리된 mRNA-리포플렉스는 대조군(Vehicle control)에 비해 중화항체 형성능이 최소 7배 이상 증가하였으며, 이는 SG6가 세포실험에 이어 동물실험에서도 mRNA의 세포 내 전달과 단백질 발현에 도움을 줄 수 있는 것으로 생각된다(도 15). 또한, 액상은 물론 동결건조제형 모두 활성 감소 없이 RBD 단백질 중화능을 유도하는 것으로 보아 SG6의 기본 백본(backbone)인 솔비톨이 동결건조 보조제로 널리 사용되는 것처럼 SG6 역시 동결건조 과정에서 mRNA-리포좀 이온 복합체의 안정성에 기여한 것으로 관찰된다. 결과적으로 SG6가 처리된 동결건조형 mRNA-lipoplex는 동물실험에서 강력한 체액성 면역 반응을 유도할 수 있으며, 활성 감소 없이 보관의 용이성 또한 우수하여 예방용 백신 조성물에 적합할 것으로 생각된다.As a result, although the difference between doses was small, the SG6-treated mRNA-lipoplex had at least 7-fold increased ability to form neutralizing antibodies compared to the control group (Vehicle control). It is thought to be able to help delivery and protein expression ( FIG. 15 ). In addition, both liquid as well as freeze-dried formulations induce RBD protein neutralization ability without decreasing activity, so just as sorbitol, the basic backbone of SG6, is widely used as a freeze-drying adjuvant, SG6 is also mRNA-liposome ion complex in the freeze-drying process. was observed to contribute to the stability of As a result, the freeze-dried mRNA-lipoplex treated with SG6 can induce a strong humoral immune response in animal experiments, and it is also thought to be suitable for a vaccine composition for prevention because it has excellent storage easiness without decreasing activity.
Claims (19)
- (a) 항원 펩티드 또는 단백질을 암호화하는 mRNA 서열을 포함하는 mRNA 화합물, 및 (b) 하기 화학식 1의 양이온성 분자 수송체를 포함하는 이온 복합체를 포함하는 백신 조성물.A vaccine composition comprising (a) an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein, and (b) an ion complex comprising a cationic molecular transporter of Formula 1 below.[화학식 1][Formula 1]
상기 식에서, In the above formula,R1은로, 여기에서 n은 1 내지 8의 정수이다.R 1 is
, where n is an integer from 1 to 8. - 제1항에 있어서, 상기 이온 복합체는 양이온성 리포좀을 더 포함하는 것인, 백신 조성물.The vaccine composition of claim 1, wherein the ionic complex further comprises a cationic liposome.
- 제2항에 있어서, 상기 mRNA 화합물과 상기 양이온성 리포좀의 질량비가 1:1 내지 1:6인 것인 백신 조성물.The vaccine composition according to claim 2, wherein the mass ratio of the mRNA compound and the cationic liposome is 1:1 to 1:6.
- 제1항에 있어서, 상기 항원 펩티드 또는 단백질은 병원성 항원, 종양 항원, 알레르기 항원 또는 자가면역 자가항원 또는 이의 단편 또는 변이체로부터 유래되는 것인, 백신 조성물.The vaccine composition according to claim 1, wherein the antigenic peptide or protein is derived from a pathogenic antigen, a tumor antigen, an allergen or an autoimmune autoantigen or a fragment or variant thereof.
- 제1항에 있어서, 상기 항원 펩티드 또는 단백질은 SARS-Cov-2의 스파이크 단백질, 뉴클레오캡시드 단백질 또는 이의 단편 또는 변이체로부터 유래되는 것인, 백신 조성물.The vaccine composition of claim 1, wherein the antigenic peptide or protein is derived from a spike protein of SARS-Cov-2, a nucleocapsid protein, or a fragment or variant thereof.
- 제1항에 있어서, 상기 mRNA 서열은 서열번호 1에 따른 SARS-Cov-2 스파이크 단백질의 RBD 서열에 상응하는 RNA 서열을 포함하는 것인, 백신 조성물.The vaccine composition according to claim 1, wherein the mRNA sequence comprises an RNA sequence corresponding to the RBD sequence of the SARS-Cov-2 spike protein according to SEQ ID NO: 1.
- 제2항에 있어서, 상기 양이온성 리포좀은 디메틸디옥타데실암모늄 브로마이드(DDA), 1,2-디올레오일-3-트리메틸암모늄프로페인(DOTAP), 3β-[N-(N',N'-디메틸아미노에테인 카바모일 콜레스테롤(3β-[N-(N'N'-dimethylaminoethane) carbamoyl cholesterol, DC-Chol), 1,2-디올레오일옥시-3-디메틸암모늄프로페인(DODAP), 1,2-디-O-옥타데세닐-3-트리에틸암모늄 프로페인(1,2-di-O-octadecenyl-3-trimethylammonium propane, DOTMA), 1,2-디미리스토레오일-sn-글리세로-3-에틸포스포콜린(1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 14:1 Ethyle PC), 1-팔미토일-2-올레오일-sn-글리세로-3-에틸포스포콜린(1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, 16:0-18:1 Ethyl PC), 1,2-디올레오일-sn-글리세로-3-에틸포스포콜린(1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 18:1 Ethyl PC), 1,2-디스테아로일-sn-글리세로-3-에틸포스포콜린(1,2-distearoyl-sn-glycero-3-ethylphosphocholin, 18:0 Ethyl PC), 1,2-디팔미토일-sn-글리세로-3-에틸포스포콜린(1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 16:0 Ethyl PC), 1,2-디미리스토일-sn-글리세로-3-에틸포스포콜린(1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 14:0 Ethyl PC), 1,2-디라우로일-sn-글리세로-3-에틸포스포콜린(1,2-dilauroyl-sn-glycero-3-ethylphosphocholin, 12:0 Ethyl PC), N1-[2-((1S)-1-[(3-아미노프로필)아미노]-4-[디(3-아미노-프로필)아미노]부틸카복사미도)에틸]-3,4-디[올레일옥시]-벤자마이드(N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide, MVL5), 1,2-디미리스토일-3-디메틸암모늄-프로페인(1,2-dimyristoyl-3-dimethylammonium-propane, 14:0 DAP), 1,2-디팔미토일-3-디메틸암모늄-프로페인(1,2-dipalmitoyl-3-dimethylammonium-propane, 16:0 DAP), 1,2-디스테아로일-3-디메틸암모늄-프로페인(1,2-distearoyl-3-dimethylammonium-propane, 18:0 DAP), N-(4-카복시벤질)-N,N-디메틸-2,3-비스(올레오일옥시)프로판-1-아미늄(N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium, DOBAQ), 1,2-스테아로일-3-트리메틸암모늄-프로페인(1,2-stearoyl-3-trimethylammonium-propane, 18:0 TAP), 1,2-디팔미토일-3-트리메틸암모늄-프로페인(1,2-dipalmitoyl-3-trimethylammonium-propane, 16:0 TA), 1,2-디미리스토일-3-트리메틸암모늄-프로페인(1,2-dimyristoyl-3-trimethylammonium-propane, 14:0 TAP) 및 N4-콜레스테릴-스퍼민(N4-Cholesteryl-Spermine, GL67)으로 구성된 군으로부터 선택되는 양이온성 지질인 것인, 백신 조성물.3. The method of claim 2, wherein the cationic liposome is dimethyldioctadecylammonium bromide (DDA), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 3β-[N-(N',N') -Dimethylaminoethane carbamoyl cholesterol (3β-[N-(N'N'-dimethylaminoethane) carbamoyl cholesterol, DC-Chol), 1,2-dioleoyloxy-3-dimethylammonium propane (DODAP), 1, 2-di-O-octadecenyl-3-triethylammonium propane (1,2-di-O-octadecenyl-3-trimethylammonium propane, DOTMA), 1,2-dimyristoreoyl-sn-glycero -3-ethylphosphocholine (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 14:1 Ethyle PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, 16:0-18:1 Ethyl PC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine, 18:1 Ethyl PC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (1,2-distearoyl-sn-glycero- 3-ethylphosphocholine, 18:0 Ethyl PC), 1,2-Dipalmitoyl-sn-glycero-3-ethylphosphocholine (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 16:0 Ethyl PC ), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 14:0 Ethyl PC), 1,2-dilau Royl-sn-glycero-3-ethylphosphocholine (1,2-dilauroyl-sn-glycero-3-ethylphosphocholine, 12:0 Ethyl PC), N1-[2-((1S)-1-[( 3-aminopropyl)amino]-4 -[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (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 (1,2-dimyristoyl-3-dimethylammonium-propane, 14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (1,2-dipalmitoyl-3-dimethylammonium-propane, 16:0 DAP), 1,2-distearoyl-3-dimethylammonium-propane (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 (1,2-stearoyl-3-trimethylammonium-propane, 18:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium -propane (1,2-dipalmitoyl-3-trimethylammonium-propane, 16:0 TA), 1,2-dimyristoyl-3-trimethylammonium-propane (1,2-dimyristoyl-3-trimethylammonium-propane) , 14:0 TAP) and N4-cholesteryl-spermine (N4-Cholesteryl-Spermine, GL67) is a cationic lipid selected from the group consisting of, the vaccine composition.
- 제3항에 있어서, 상기 양이온성 리포좀은 (a) 1,2-디미리스토일-sn-글리세로-3-포스파티딜콜린(1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine, DMPC), 1,2-디올레오일-sn-글리세로-3-포스포콜린(1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 1,2-디올레오일-sn-글리세로-3-포스포에탄올아민(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-디팔미토일-sn-글리세로-3-포스포콜린(1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), 1,2-디스테아로일-sn-글리세로-3-포스포콜린(1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-디리노레오일-sn-글리세로-3-포스포콜린(1,2-dilinoleoyl-sn-glycero-3-phosphocholine, DLPC), 포스파티딜세린(PS), 포스포에탄올라민(PE), 포스파티딜글리세롤(PG), 포스포릭액시드(PA) 및 포스파티딜콜린(PC)으로 구성된 군으로부터 선택되는 중성 지질; 및 (b) 콜레스테롤(Cholesterol)을 추가로 포함하는 것인, 백신 조성물.4. The method of claim 3, wherein the cationic liposome comprises (a) 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine, DMPC), 1 ,2-Dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 1,2-dioleoyl-sn-glycero-3- Phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoyl-sn-glycero) -3-phosphocholine, DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-di Linoleoyl-sn-glycero-3-phosphocholine (1,2-dilinoleoyl-sn-glycero-3-phosphocholine, DLPC), phosphatidylserine (PS), phosphoethanolamine (PE), phosphatidylglycerol (PG) ), a neutral lipid selected from the group consisting of phosphoric acid (PA) and phosphatidylcholine (PC); and (b) cholesterol (Cholesterol).
- 제7항에 있어서 상기 양이온성 리포좀은 DOTAP, DOPE 및 콜레스테롤을 포함하는 것인, 백신 조성물.8. The vaccine composition of claim 7, wherein the cationic liposome comprises DOTAP, DOPE and cholesterol.
- 제1항에 있어서, 상기 mRNA 화합물은 RNA 분자에 안정도를 부여하는 적어도 한 가지 변형을 포함하는 것인, 백신 조성물.The vaccine composition of claim 1 , wherein the mRNA compound comprises at least one modification that confers stability to the RNA molecule.
- 제1항에 있어서, 상기 mRNA 화합물의 음이온 및 상기 양이온성 분자 수용체의 양이온의 비율(N/P)이 0.1 내지 1인 것인, 백신 조성물.According to claim 1, wherein the ratio of the anion of the mRNA compound to the cation of the cationic molecular receptor (N/P) is 0.1 to 1, the vaccine composition.
- 제1항에 있어서, 상기 mRNA 서열은 상기 RNA 분자의 5' 비번역 영역의 변형을 포함하는 것인, 백신 조성물.The vaccine composition of claim 1 , wherein the mRNA sequence comprises a modification of the 5' untranslated region of the RNA molecule.
- 제1항에 있어서, 상기 mRNA 서열은 상기 RNA 분자의 3' 비번역 영역의 변형을 포함하는 것인, 백신 조성물.The vaccine composition of claim 1 , wherein the mRNA sequence comprises a modification of the 3' untranslated region of the RNA molecule.
- 제13항에 있어서, 상기 변형은 폴리 A 꼬리의 함유를 포함하는 것인, 백신 조성물.14. The vaccine composition of claim 13, wherein the modification comprises inclusion of a poly A tail.
- 제1항에 있어서, 표적 세포의 세포 내 구간으로 mRNA 화합물의 수송을 촉진시키기 위한 물질을 추가로 포함하는 것인, 백신 조성물.The vaccine composition of claim 1, further comprising a substance for facilitating transport of the mRNA compound to the intracellular segment of the target cell.
- 제1항에 있어서, 상기 조성물은 동결건조되는 것인, 백신 조성물.The vaccine composition of claim 1 , wherein the composition is lyophilized.
- 하기 단계를 포함하는 제1항 내지 제13항 중 어느 한 항에 따른 백신 조성물의 제조방법:A method for preparing a vaccine composition according to any one of claims 1 to 13, comprising the steps of:(a) 화학식 1로 표시되는 양이온성 분자 수송체를 제조하는 단계; 및 (a) preparing a cationic molecular transporter represented by Formula 1; and[화학식 1][Formula 1]
(상기 식에서, (In the above formula,R1은로, 여기에서 n은 1 내지 8의 정수이다.) R 1 is
, where n is an integer from 1 to 8.)(b) 항원 펩티드 또는 단백질을 암호화하는 mRNA 서열을 포함하는 mRNA 화합물을 제조하여 상기 (a) 단계의 결과물과 혼합하는 단계.(b) preparing an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein and mixing with the result of step (a). - 제17항에 있어서, 상기 (b) 단계는 항원 펩티드 또는 단백질을 암호화하는 mRNA 서열을 포함하는 mRNA 화합물을 제조하여 양이온성 리포좀과 반응시킨 후 상기 (a) 단계의 결과물과 혼합하는 단계인 것인, 백신 조성물 제조방법.The method according to claim 17, wherein step (b) is a step of preparing an mRNA compound containing an mRNA sequence encoding an antigenic peptide or protein, reacting it with cationic liposome, and mixing it with the result of step (a). , a method for preparing a vaccine composition.
- 제17항에 있어서, (c) 상기 (b)의 결과물을 동결건조하는 단계를 추가로 포함하는 것인, 백신 조성물 제조방법.The method of claim 17, further comprising the step of (c) lyophilizing the resultant of (b).
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| KR10-2022-0010609 | 2022-01-25 | ||
| KR1020220010609A KR20220117133A (en) | 2021-02-15 | 2022-01-25 | Vaccine composition for the prevention of COVID-19 containing ion complex of cationic molecular carrier and SARS-CoV-2 mRNA |
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| US11898186B1 (en) | 2022-08-10 | 2024-02-13 | Genscript Usa Inc. | Compositions and methods for preparing capped mRNA |
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| KR20060112791A (en) * | 2005-04-28 | 2006-11-02 | 학교법인 포항공과대학교 | Molecular transporters based on sugar or its analogues and processes for the preparation thereof |
| KR20160001419A (en) * | 2014-06-27 | 2016-01-06 | 포항공과대학교 산학협력단 | Composition for skin permeation comprising cationic molecular transporters and anionic bioactive substance |
| CN111218458A (en) * | 2020-02-27 | 2020-06-02 | 珠海丽凡达生物技术有限公司 | mRNAs encoding SARS-CoV-2 virus antigen and vaccine and preparation method of vaccine |
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| KR20060112791A (en) * | 2005-04-28 | 2006-11-02 | 학교법인 포항공과대학교 | Molecular transporters based on sugar or its analogues and processes for the preparation thereof |
| KR20160001419A (en) * | 2014-06-27 | 2016-01-06 | 포항공과대학교 산학협력단 | Composition for skin permeation comprising cationic molecular transporters and anionic bioactive substance |
| CN111218458A (en) * | 2020-02-27 | 2020-06-02 | 珠海丽凡达生物技术有限公司 | mRNAs encoding SARS-CoV-2 virus antigen and vaccine and preparation method of vaccine |
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| US11898186B1 (en) | 2022-08-10 | 2024-02-13 | Genscript Usa Inc. | Compositions and methods for preparing capped mRNA |
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