WO2022173199A1 - Vaccine composition for preventing covid-19, comprising ionic complex of cationic molecular transporter and sars-cov-2 mrna - Google Patents

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

Coronavirus Infectious Disease-19 Prevention Vaccine Composition Comprising Cationic Molecular Transporter and Ion Complex of SARS-COV-2 mRNA

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). 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.

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.

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.

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.

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.

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.

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) .

[Formula 1]

In the above formula,

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.

[Formula 1]

In the above formula,

R ₁ is

, where n is an integer from 1 to 8.

According to one embodiment of the present invention, 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 (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.

The mRNA compound may form an ionic complex by electrostatic interaction with the cationic molecular transporter and/or cationic liposome.

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.

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.

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.

The mRNA sequence may include the following elements in a 5' to 3' direction.

a) 5'-CAP structure;

b) a 5'-UTR element;

c) a coding region encoding an antigenic peptide or protein;

d) a 3'-UTR element; and/or

e) Poly A tail.

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. 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.

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.

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

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. 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.

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).

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).

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.

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.

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.

According to one embodiment of the present invention, the mRNA sequence comprises a modification of the 5' untranslated region of the RNA molecule.

According to one embodiment of the present invention, the mRNA sequence comprises a modification of the 3' untranslated region of the RNA molecule.

According to one embodiment of the present invention, said modification comprises modification of poly A tail.

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.

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.

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.

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.

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).

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.

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.

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.

According to one embodiment of the present invention, the composition is lyophilized.

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.

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

[Formula 1]

In the above formula,

R1 is

, where n is an integer from 1 to 8.

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).

[Formula 1]

In the above formula,

R1 is

, where n is an integer from 1 to 8.

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%.

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).

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.

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.

According to an embodiment of the present invention, cationic liposomes include DOTAP, DOPE and cholesterol.

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.

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.

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.

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 .

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).

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).

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 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.

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. .

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

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.

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).

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.

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.

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.

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.

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.

Preparation Example 1: Preparation of mRNA compound

1. Sequence selection and vector construction of mRNA vaccine construction

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. DNA Template Preparation for mRNA Production

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

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. mRNA production through IVT (In vitro transcription)

IVT reaction

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.

Ingredients	volume (ul)	Concentration (mmol)
Nuclease-free water	1.8
ATP solution	2	150
CTP solution	2	150
UTP solution	2	150
GTP solution (1:5 dilution of GTP solution)	2	30
ARCA cap	2	120
10X reaction buffer	2
DNA template	5
Enzyme Mix	2
total volume	20

Lithium chloride precipitation

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. .

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. Confirmation of mRNA expression in HEK293 cells

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.

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).

Preparation Example 2: Preparation of an ionic complex between an mRNA compound and a cationic molecular transporter

1. Preparation of cationic molecular transporter SG6

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

[Formula 1]

In the above formula,

R ₁ is

, where n is an integer from 1 to 8.

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.

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(+) ratio	moles
	SG6	R6	PEI
0.2	815	815	81.5
0.5	326	326	32.6
One	163	163	16.3
2	81.5	81.5	8.15
5	32.6	32.6	3.26

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 .

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 ).

Example 1: Cytotoxicity evaluation of SG6 cationic molecular transporter

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.

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.

Example 2: Assessment of cytotoxicity of ion complexes

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.

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.

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).

Example 3: Measurement of mRNA encapsulation rate and loading rate

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 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

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 ).

Preparation Example 3: Preparation of ionic complex between SARS-CoV-2 RBD mRNA and cationic liposome

One. Preparation of cationic liposomes

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. Preparation of cationic liposome and SARS-CoV-2 RBD mRNA ion complex (mRNA-lipoplex)

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.

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.

Formulations	Z-Average Size (nm)	Polydispersity index	Zeta potential (mV)
Cationic liposome	165.3 ± 2.4	0.23 ± 0.03	38.5 ± 0.8
mRNA-Lipoplex	342.5 ± 16.8	0.38 ± 0.06	31.2 ± 1.7
mRNA-SG6-lipoplex	233.1 ±12.0	0.32 ± 0.09	34.2 ± 3.0
mRNA-SG6-lipoplex (lyophilized)	277.5 ± 16.7	0.25 ± 0.08	14.2 ± 1.2

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 ).

Example 4: In vitro cytotoxicity evaluation of mRNA-lipoplex

To evaluate whether mRNA-lipoplex was cytotoxic in vitro, cell viability was confirmed using Alamar Blue assay.

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.

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).

Example 5: Analysis of mRNA Encapsulation Rate of mRNA-lipoplex

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.

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

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).

Example 6: Ribonuclease (RNase) protective effect of mRNA-lipoplex

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.

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.

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).

Example 7: Evaluation of RBD protein expression efficiency using HEK293T

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.

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.

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).

Example 8: Confirmation of neutralizing antibody-forming ability of mRNA-lipoplex

The immunogenicity of the liposome ion complex as a vaccine was confirmed by inoculating the prepared mRNA-lipoplex into BALB/c mice.

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.

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).

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)

1. 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.

   [Formula 1]

   

   In the above formula,

   R ₁ is

   

   , where n is an integer from 1 to 8.
2. The vaccine composition of claim 1, wherein the ionic complex further comprises a cationic liposome.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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).
9. 8. The vaccine composition of claim 7, wherein the cationic liposome comprises DOTAP, DOPE and cholesterol.
10. The vaccine composition of claim 1 , wherein the mRNA compound comprises at least one modification that confers stability to the RNA molecule.
11. 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.
12. The vaccine composition of claim 1 , wherein the mRNA sequence comprises a modification of the 5' untranslated region of the RNA molecule.
13. The vaccine composition of claim 1 , wherein the mRNA sequence comprises a modification of the 3' untranslated region of the RNA molecule.
14. 14. The vaccine composition of claim 13, wherein the modification comprises inclusion of a poly A tail.
15. 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.
16. The vaccine composition of claim 1 , wherein the composition is lyophilized.
17. A method for preparing a vaccine composition according to any one of claims 1 to 13, comprising the steps of:

    (a) preparing a cationic molecular transporter represented by Formula 1; and

    [Formula 1]

    

    (In the above formula,

    R ₁ is

    

    , where n is an integer from 1 to 8.)

    (b) preparing an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein and mixing with the result of step (a).
18. 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.
19. The method of claim 17, further comprising the step of (c) lyophilizing the resultant of (b).

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