Complex for gene delivery

Abstract

The present invention relates to a nucleic acid-based drug delivery platform. A complex according to the present invention has moderated toxicity and exhibits stable nucleic acid-binding ability due to osmotic activity. In addition, the complex promotes cell uptake of the nucleic acid supported on a polymer, and improves an endosomal release effect of the nucleic acid through degradation of nanoparticles in the cytoplasm. The complex according to the present invention has effects of maximizing gene expression of nucleic acids in a cell and contributing to maintaining the expression for a long time, and thus can be applied to various fields of immune activity, vaccine development, and the like.

Description

Gene delivery complex

[0001] The present invention relates to a complex for gene delivery, and more particularly, to a delivery platform for nucleic acid-based drugs.

[0002] Before and after the COVID-19 pandemic, the development of preventive vaccines using mRNA has rapidly progressed, demonstrating their effectiveness and utility. Since then, efforts to develop mRNA gene delivery systems for various therapeutic purposes have continued. Most mRNA gene delivery systems currently under development are based on lipid-based nanoparticles, and the development of polymer-based gene delivery systems that can be modified for various purposes is emerging as a hot topic. Examples of cationic polymers include poly-L-lysine (PLL), poly(4-hydroxy-L-proline ester), polyethyleneimine (PEI), poly[α-(4-aminobutyl)-L-glycolic acid], polyamidoamine dendrimers, and poly[N,N'-(dimethylamino)ethyl] methacrylate (PDMAEMA). These polymers compress therapeutic nucleic acids, such as DNA, into nanoparticles, protecting them from enzymatic degradation and facilitating rapid penetration into cells and endosome exit. However, these methods have limitations, including relatively low transduction efficiency, cytotoxicity, and limited particle size. Therefore, there is a need to develop a gene delivery vehicle that maintains the advantages of existing non-viral vectors while reducing toxicity and increasing transduction efficiency.

[0003] The present invention aims to provide a polymer for nucleic acid delivery.

[0004] The present invention aims to provide a nucleic acid delivery vehicle comprising the above polymer and nucleic acid.

[0005] The present invention provides a vaccine composition comprising the nucleic acid delivery vehicle.

[0006] 1. A polymer having the following chemical formula 1 as a repeating unit:

[0007] [Chemical Formula 1]

[0008]

[0009] (In the formula, each R is independently a hydrogen or fluorine-containing functional group, except when all R are hydrogen).

[0010] 2. A polymer in which the fluorine-containing functional group is substituted by 1 to 30% in the above 1.

[0011] 3. In the above 1, the fluorine-containing functional group is perfluorooctanoic acid (C8HF 15 O2), octafluoropentyl-tetrafluoroethyl ether (C7H4F 12 O), 2-(perfluorobutyl)ethyl methacrylate (C 10 H9F9O2), octafluoropentyl methacrylate (C9H8F8O2), perfluorobutanoylcyclopentanone (C9H7F7O2), octafluoropentyl ether (C8H8F8O), octafluorooctadienoic acid (C8H4F8O2), octafluoropentylacrylate (C8H6F8O2), heptafluoroisobutyl methacrylate (C8H7F7O2), heptafluorohexanediol (C6H7F7O2), hexafluorobutyl methacrylate (C8H8F6O2), hexafluoroisopropyl methacrylate (C7H6F6O2), tetrafluoropropyl methacrylate (C7H8F4O2), trifluoromethylphenylacetic acid (C9H7F3O2), trifluoromethyl acetylacetonate (C6H7F3O2), A polymer selected from the group consisting of methyltrifluoroacetoacetate (C5H5F3O3), trifluoroethyl acetate (C4H5F3O2), and methyltrifluoroacetate (C3H3F3O2).

[0012] 4. A nucleic acid delivery system comprising any one of the polymers 1 to 3 above and a nucleic acid.

[0013] 5. A nucleic acid delivery vehicle in the above 4, wherein the nucleic acid is at least one selected from the group consisting of gDNA, cDNA, pDNA, mRNA, tRNA, rRNA, siRNA, shRNA, miRNA, aptamer, antisense oligodeoxynucleotide (AS-ODN), DNA-RNA hybrid, and ribozyme.

[0014] 6. A nucleic acid carrier in the above 4, wherein the weight ratio (w / w) of the polymer is 1 to 20.

[0015] 7. A nucleic acid carrier having a size of 20 to 150 nm in the above 4.

[0016] 8. In the above 4, a nucleic acid delivery vehicle that promotes proliferation specifically of immune cells.

[0017] 9. In the above 8, the immune cell is a T cell, B cell, NK cell or hematopoietic progenitor cell, nucleic acid delivery vehicle.

[0018] 10. A vaccine composition comprising any one of the nucleic acid carriers 4 to 9 above.

[0019] 11. In the above 10, the vaccine composition is for preventing or treating one or more infections selected from the group consisting of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and influenza virus.

[0020] The polymer according to the present invention provides the effect of alleviating cytotoxicity and improving biocompatibility.

[0021] The polymer according to the present invention provides the effect of forming stable nanoparticles by binding to nucleic acids.

[0022] The nucleic acid delivery system according to the present invention provides improved effects of intracellular nucleic acid delivery and gene expression within the nucleic acid.

[0023] The vaccine composition according to the present invention provides improved effects of enhancing immunity and preventing infection.

[0024] Figures 1a and 1b are diagrams showing the manufacturing reaction scheme of PS polymer.

[0025] Figures 2a and 2b are diagrams showing the manufacturing reaction scheme of fluorinated PEI (PEI-F).

[0026] Figure 3a shows the survival rate of HK2 cells treated with PEI-F-5%, PEI-F-10%, PEI-F-15%, and PEI-F-20% (PC: untreated, NC: Triton X-100 (0.1%) treated).

[0027] Figure 3b is a diagram showing the transfection efficiency through evaluation of pDNA nucleic acid expression of PEI-F-5%, PEI-F-10%, PEI-F-15%, and PEI-F-20%.

[0028] Figure 3c shows the viability of HK2 cells treated with PEI-F-5%-pFluc, PEI-F-10%-pFluc, PEI-F-15%-pFluc, and PEI-F-20%-pFluc (PC: untreated, NC: Triton X-100 (0.1%) treated).

[0029] Figures 4a and 4b are diagrams showing the manufacturing reaction scheme of PFS polymer.

[0030] Figure 5a is a diagram showing a PS-mGLuc polyplex in which hGLuc mRNA is transfected into a PS polymer.

[0031] Figure 5b is a diagram showing a PFS-mGLuc polyplex transfected with hGLuc mRNA into a PFS polymer.

[0032] Figure 6a is a diagram showing the composition analysis of PS polymer confirmed by proton nuclear magnetic resonance (NMR) spectroscopy.

[0033] Figure 6b is a diagram showing the composition analysis of the PFS polymer using proton nuclear magnetic resonance (NMR) spectroscopy.

[0034] Figure 6c is a diagram confirming the successful polymer formation of SDA and PEI in PS polymer using Fourier transform infrared (FTIR) spectroscopy.

[0035] Figure 6d is a diagram confirming the successful polymer formation of SDA and PEI-F of PFS polymer using Fourier transform infrared (FTIR) spectroscopy.

[0036] Figure 7a shows the hydrodynamic diameters of PS-mGLuc (w / w = 2.52) polyplexes and PFS-mGLuc (w / w = 10) polyplexes confirmed by dynamic light scattering (DLS) analysis.

[0037] Figure 7b shows the surface charge of PS-mGLuc (w / w = 2.52) polyplex and PFS-mGLuc (w / w = 10) polyplex confirmed by dynamic light scattering (DLS) analysis.

[0038] Figure 8a is a diagram showing the formation of PS-mGLuc polyplexes according to the weight ratio (w / w) of PS polymers confirmed by agarose gel electrophoresis.

[0039] Figure 8b is a diagram showing the formation of PFS-mGLuc polyplexes according to the weight ratio (w / w) of PFS polymers confirmed by agarose gel electrophoresis.

[0040] Figure 8c is a diagram showing an FE-TEM image of PS-mGLuc polyplex.

[0041] Figure 8d is a diagram showing an FE-TEM image of PFS-mGLuc polyplex.

[0042] Figure 9 is a diagram showing the cytotoxicity of PS-mGLuc polyplex and PFS-mGLuc polyplex evaluated using the WST-1 assay.

[0043] Figure 10 is a diagram showing the Gaussia Luciferase mRNA expression profiles of PS-mGLuc polyplexes and PFS-mGLuc polyplexes.

[0044] Figure 11a is a graph showing mRNA labeled with Cy5.5 and quantified according to a standard protocol.

[0045] Figure 11b is a diagram confirming the intracellular uptake of PFS-mGluc / Cy5.5 nanoparticles.

[0046] Figure 12a is a diagram showing the mRNA expression profiles of PS-mGLuc polyplexes and PFS-mGLuc polyplexes pretreated with the ciliary endocytic inhibitor Filipin-III.

[0047] Figure 12b is a diagram showing the mRNA expression profiles of PS-mGLuc polyplexes and PFS-mGLuc polyplexes pretreated with the ciliary endocytic inhibitor methyl-β-cyclodextrin.

[0048] Figure 12c is a diagram showing the mRNA expression profiles of PS-mGLuc polyplexes and PFS-mGLuc polyplexes pretreated with chlorpromazine, a clathrin-mediated cell proliferation inhibitor.

[0049] Figure 13a is a diagram showing the mRNA expression profile for 144 hours after intramuscular injection of PS-mGLuc polyplexes prepared with PS polymers at weight ratios (w / w) of 1.60, 2.10, 2.52, 3.15, and 3.80 into mice.

[0050] Figure 13b is a diagram showing the mRNA expression profile for 144 hours after intramuscular injection of PFS-mGLuc polyplexes prepared at weight ratios (w / w) of 8, 10, 12, 15, and 18 PFS polymers into mice.

[0051] Figures 14a and 14b show the production of IFN-γ in serum two weeks after the initial vaccination with the PS-SmRNA vaccine and the PFS-SmRNA vaccine, as determined by ELISpot analysis. Statistical analysis was performed using an ordinary one-way ANOVA with a P value of <0.0001.

[0052] Figures 14c-14d show the production of IFN-γ in serum two weeks after additional vaccination with the PS-SmRNA vaccine and PFS-SmRNA vaccine, as determined by ELISpot analysis. Statistical analysis was performed using an ordinary one-way ANOVA with a P value of <0.0001.

[0053] Figure 15a shows the results of ELISA analysis to confirm the production of IFN-γ in serum 3 days after the initial vaccination with PS-SmRNA vaccine and PFS-SmRNA vaccine. Statistical analysis was performed using ordinary one-way ANOVA with a P value of <0.0001 (n=3).

[0054] Figure 15a shows the results of ELISA analysis to confirm the production of TNF-α in serum 3 days after vaccination with PS-SmRNA vaccine and PFS-SmRNA vaccine. Statistical analysis was performed using ordinary one-way ANOVA with a P value of <0.0001 (n=3).

[0055] Figure 16a shows the detection of spike peptide antibodies in serum after the initial vaccination with the PS-SmRNA vaccine and the PFS-SmRNA vaccine using an ELISA assay. Statistical analysis was performed using an ordinary one-way ANOVA with a P value of <0.0001.

[0056] Figure 16b shows the detection of spike peptide antibodies after additional vaccination with PS-SmRNA vaccine and PFS-SmRNA vaccine using ELISA analysis. Statistical analysis was performed using an ordinary one-way ANOVA with a P value of <0.0001.

[0057] Figure 17a shows the detection of PRNT50 in serum isolated after the initial vaccination with PS-SmRNA vaccine and PFS-SmRNA vaccine (2 weeks after vaccination) using ELISA analysis. Statistical analysis was performed using ordinary one-way ANOVA with a P value of <0.0001 (n=5).

[0058] Figure 17b shows the detection of PRNT50 in serum isolated after booster vaccination with PS-SmRNA vaccine and PFS-SmRNA vaccine (5 weeks after vaccination) using ELISA analysis. Statistical analysis was performed using ordinary one-way ANOVA with a P value of <0.0001 (n=5).

[0059] Figure 18 shows H&E staining analysis of major organs and muscles of mice vaccinated with the PS-SmRNA vaccine and PFS-SmRNA vaccine. Tissue samples were collected postmortem, embedded in paraffin, sectioned, and stained with H&E.

[0060] The success of nucleic acid-based drug delivery systems depends on their ability to effectively deliver nucleic acids into cells. Essential requirements for delivery systems include stable binding to nucleic acids such as mRNA molecules, effective intracellular delivery, and release of nucleic acids through nanoparticle degradation in the cytoplasm.

[0061] The present invention provides a configuration that effectively overcomes individual barriers in the nucleic acid delivery and expression process of a delivery vehicle to maximize gene expression of nucleic acids.

[0062] The present invention provides, from one aspect, a polymer having the following chemical formula 1 as a repeating unit.

[0063] [Chemical Formula 1]

[0064]

[0065] In the formula, each R independently represents a hydrogen or fluorine-containing functional group. The case where all R are hydrogen is excluded. In the formula, n is an integer from 1 to 500.

[0066] The polymer of the present invention is characterized by having a repeating unit of a bond of fluorinated modified polyethyleneimine (PEI-F) and sorbitol dimethacrylate (SDA), thereby enhancing the intracellular nucleic acid delivery and gene expression characteristics of the nucleic acid.

[0067] Polyethylenimine (PEI) is a cationic polymer that can bind to negatively charged biological molecules, such as nucleic acids, through strong electrostatic interactions. Based on these properties, PEI is utilized as a drug delivery system. However, its strong cationicity leads to nonspecific interactions with anionic biological molecules in the body, resulting in cytotoxicity. Furthermore, its poor biocompatibility limits its ability to deliver and express nucleic acids.

[0068] The fluorinated modified polyethyleneimine (PEI-F) of the present invention has reduced cytotoxicity of polyethyleneimine (PEI) and improved binding ability to nucleic acids.

[0069] The polyethyleneimine (PEI) used in the present invention may be linear or branched, but is not limited thereto. Furthermore, the molecular weight may range from 100 to 2,000 Da. A molecular weight less than 100 Da reduces its ability to bind to nucleic acids, while a molecular weight exceeding 2,000 Da may make it difficult to excrete from the body through the kidneys.

[0070] Fluorinated modified polyethyleneimine (PEI-F) can be used by dissolving in, but is not limited to, DMSO, methanol, absolute ethanol or acidified DW (pH less than 5).

[0071] The fluorine-containing functional group may be substituted in 1 to 30% of polyethyleneimine (PEI), preferably in 5 to 25%, and more preferably in 10 to 20%. By substituting the primary amine among the amine groups inherent in polyethyleneimine (PEI) with the fluorine-containing functional group within the above range, a low toxicity effect can be obtained without damaging the binding ability to nucleic acids. If the substitution exceeds 30%, complex formation with nucleic acids may not be successful.

[0072] Fluorine-containing functional groups include perfluorooctanoic acid (C8HF 15O2), octafluoropentyl-tetrafluoroethyl ether (C7H4F 12 O), 2-(perfluorobutyl)ethyl methacrylate (C 10 H9F9O2), octafluoropentyl methacrylate (C9H8F8O2), perfluorobutanoylcyclopentanone (C9H7F7O2), octafluoropentyl ether (C8H8F8O), octafluorooctadienoic acid (C8H4F8O2), octafluoropentylacrylate (C8H6F8O2), heptafluoroisobutyl methacrylate (C8H7F7O2), heptafluorohexanediol (C6H7F7O2), hexafluorobutyl methacrylate (C8H8F6O2), hexafluoroisopropyl methacrylate (C7H6F6O2), tetrafluoropropyl methacrylate (C7H8F4O2), trifluoromethylphenylacetic acid (C9H7F3O2), trifluoromethyl acetylacetonate (C6H7F3O2), It may be any one selected from the group consisting of methyltrifluoroacetoacetate (C5H5F3O3), trifluoroethyl acetate (C4H5F3O2), and methyltrifluoroacetate (C3H3F3O2), but is not limited thereto.

[0073] The polymer of the present invention can be formed by combining fluorinated modified polyethyleneimine (PEI) and sorbitol dimethacrylate (SDA). The combination may be, but is not limited to, a Michael addition reaction.

[0074] “Sorbitol dimethacrylate (SDA)” helps to effectively release nucleic acids from the endosomal state by inducing osmotic pressure, and in particular, it binds to lysosomes and contributes to preventing degradation during intracellular transport.

[0075] The polymer of the present invention is a polymer in which a fluorinated modified polyethyleneimine (PEI) and sorbitol dimethacrylate (SDA) are combined in a 1:1 ratio, but is not limited thereto.

[0076] The polymer of the present invention can have reduced cytotoxicity and improved biocompatibility through primary amine fluorination modification of fluorinated modified polyethyleneimine (PEI-F).

[0077] The polymer of the present invention may have improved stable binding to nucleic acids and nanoparticle formation based on the secondary amine of fluorinated modified polyethyleneimine (PEI-F).

[0078] The polymer of the present invention can enhance the endosomal escape effect of nucleic acids by lowering the endosomal pH through the proton sponge effect in which the secondary and tertiary amines of fluorinated modified polyethyleneimine (PEI-F) bind to hydrogen ions introduced into the endosome.

[0079] The polymer of the present invention effectively releases nucleic acids, such as mRNA molecules, from endosomal states through the osmotic pressure induced by sorbitol dimethacrylate (SDA). Specifically, sorbitol sugar binds to lysosomes and undergoes an intracellular transport process that does not degrade them. Sorbitol sugar induces caveolae-based endocytosis, thereby minimizing degradation of mRNA molecules.

[0080] The polymer of the present invention can induce release of nucleic acids and gene expression through ester bond cleavage within nanoparticles within the cytoplasm.

[0081] In one embodiment, the polymer has a repeating chemical structure as shown in FIG. 4B. The polymer is a fluorinated modified polyethyleneimine (PEI-F) having 5 to 20% octafluoropentyl ether (glycidyl 2,2,3,3,4,4,5,5-octafluoropentyl ether) functional groups substituted on branched polyethyleneimine (PEI) and bonded to sorbitol dimethacrylate (SDA) by a Michael addition reaction.

[0082] From another perspective, the present invention provides a nucleic acid delivery system comprising a polymer having chemical formula 1 as a repeating unit and a nucleic acid.

[0083] The polymer is produced by a combined reaction of fluorinated modified polyethyleneimine (PEI-F) and sorbitol dimethacrylate (SDA), as described above.

[0084] The nucleic acid contained in the nucleic acid delivery system may be at least one selected from the group consisting of gDNA, cDNA, pDNA, mRNA, tRNA, rRNA, siRNA, shRNA, miRNA, aptamer, antisense oligodeoxynucleotide (AS-ODN), DNA-RNA hybrid, and ribozyme. Preferably, it may be mRNA, but is not limited thereto.

[0085] The nucleic acid carrier may have a weight ratio (w / w) of the polymer to nucleic acid of 1 to 20. Specifically, if the weight ratio (w / w) of the polymer to nucleic acid exceeds 20, toxicity may increase and cell stability may decrease.

[0086] The nucleic acid carrier may be 20 to 150 nm in size, preferably 50 to 120 nm, but is not limited thereto.

[0087] In one embodiment, the TEM image results of the mRNA polyplex using the polymer according to the present invention confirmed a nanoparticle size of 55 to 100 nm, and such a small size can be expected to promote rapid uptake of the nucleic acid delivery vehicle into cells.

[0088] The nucleic acid delivery system according to the present invention has improved intracellular delivery and release effects of nucleic acids.

[0089] In one embodiment, the PFS-mRNA polyplex experimental group demonstrated a consistently successful hGLuc mRNA expression pattern, as confirmed through in vitro and in vivo experiments. In particular, animal experiments confirmed that hGLuc mRNA expression in the Moderna LNP-administered group rapidly decreased after day 3, whereas hGLuc mRNA expression in the PFS-mRNA polyplex-administered group was maintained for up to day 6. Therefore, it was confirmed that the fluorination of the PFS polymer according to the present invention enhanced mRNA delivery and gene expression within the mRNA molecule. Therefore, effects such as immune activation and vaccination efficacy can be expected.

[0090] The nucleic acid delivery system according to the present invention can specifically promote the proliferation of immune cells. The immune cells may be T cells, B cells, NK cells, or hematopoietic progenitor cells, and preferably T cells.

[0091] The present invention provides a vaccine composition comprising the nucleic acid delivery vehicle from another perspective.

[0092] The vaccine composition positively influences the immune response of the subject, providing an enhanced systemic or local immune response induced by a cellular immune response or a humoral immune response.

[0093] The vaccine composition may be for the prevention or treatment of one or more infections selected from the group consisting of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and influenza virus.

[0094] In one embodiment, when a fluorinated modified-polyethyleneimine (PEI) and sorbitol dimethacrylate (SDA) conjugated PFS polymer was used as an mRNA carrier compared to a non-fluorinated polyethyleneimine (PEI) and sorbitol dimethacrylate (SDA) conjugated PS polymer used as an mRNA carrier using a SARS-COVID-2 spike protein coding mRNA, the number of IFN-γ-producing T cells was significantly increased, and in particular, a statistically significant similarity in the proliferation rate of T cells was observed compared to the positive control group, Moderna LNP administration group, demonstrating the biocompatibility, intracellular delivery of mRNA, and enhancement of gene expression within the mRNA molecule according to the fluorinated polymer.

[0095] The vaccine composition may additionally comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, 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.

[0096] In addition to the above ingredients, the vaccine composition may additionally include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.

[0097] The vaccine composition may include other ingredients, such as stabilizers, excipients, other pharmaceutically acceptable compounds, or any other antigen or portion thereof. The vaccine may be in the form of a lyophilized preparation or suspension, all of which are common in the field of vaccine production.

[0098] The dosage form of the vaccine composition may be in the form of an enteric-coated unit, an intraperitoneal, intramuscular, or subcutaneous injection, an aerosol spray, or oral or intranasal use. It may also be administered in drinking water or as edible pellets.

[0099] The vaccine composition may be delivered as a single vaccine by expressing heterologous antigens and immune modulatory molecules such as cytokines within the same recombinant, and may be administered together with an immune adjuvant.

[0100] An adjuvant is a compound or mixture that enhances an immune response and / or promotes the rate of absorption after vaccination, including any absorption-promoting agent. Acceptable adjuvants include, but are not limited to, Freund's complete adjuvant, Freund's incomplete adjuvant, saponins, mineral gels such as aluminum hydroxide gel, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, chiholmpet hemocyanin, dinitrophenol, IMS1313, and the like.

[0101] Hereinafter, the present invention will be described in detail with examples.

[0102]

[0103] Manufacturing Example 1. PS polymer synthesis

[0104] Polyethyleneimine (hereinafter referred to as PEI) and sorbitol diacrylate (hereinafter referred to as SDA) having high osmotic pressure activity were prepared, and PEI-SDA was prepared using Michael addition reaction coupling (Fig. 1a-b).

[0105] Specifically, PEI and SDA, each with a molecular weight of 12 kDa, were dissolved in anhydrous DMSO, respectively. The SDA solution was slowly added to the PEI solution at a 1:1 ratio and the mixture was allowed to react at -80 °C for 24 h with magnetic stirring. The reactant was dialyzed against distilled water (MWCO: 3500 Da) at 4 °C for 48 h and then freeze-dried to obtain the PEI-SDA (hereinafter, PS) polymer. The PS polymer was stored at -80 °C until used in the experiment.

[0106]

[0107] Manufacturing Example 2. PFS polymer synthesis

[0108] To reduce the inherent toxicity of PEI, enhance hydrophobicity, and increase cell permeability, PEI was fluorinated and combined with SDA through a Michael addition reaction to produce PEI-F-SDA according to the following steps.

[0109] Step 1: Manufacturing fluorinated PEI-F

[0110] First, fluorinated PEI (PEI-F) was prepared by bonding fluorine-containing functional groups to PEI. Fluorination was performed by an oxirane reaction (Fig. 2a-b).

[0111] Specifically, a PEI solution was prepared by dissolving 200 mg of branched PEI (MW 1200 Da) in distilled water. Subsequently, a GOFPE solution, prepared by dissolving glycidyl 2,2,3,3,4,4,,5,5-octafluoropentyl ether (GOFPE) in ethanol, was mixed with the PEI solution. The content of GOFPE, a fluorine-containing functional group, was determined to correspond to 5, 10, 15, and 20% of the total amine groups (primary, secondary, and tertiary amines) of PEI, as shown in Table 1.

[0112] % feed ratio of fluorine compound with respect to primary amines (theoretical calculation)% equivalent of PEI occupied in 1mg fluorination productTotal PEI 2K occupied in 1mg of Fluorinated PEI (nmol)Equivalent fluorination content in fluorinated PEI % by weight (theoretical)Fluorination content (nmol)Ratio of fluorination with respect to PEI (mol) ratio)PEI-F-5%97.43(974.3 μg)487.152.57(25.7 μg)89.191:0.18PEI-F-10%86.93(869.3 μg)434.6513.07(130.7 μg)453.591:1.04PEI-F-15%69.83(698.3 μg)349.1530.17(301.7 μg)1047.061:3PEI-F-20%60.13(601.3 μg)300.6539.87(398.7 μg)1383.701:4.6

[0113] Theoretically, the molecular weight of a single repeating unit of ethyleneimine (C2H5N) is 43.04, and each repeating unit contains 1 mol of amine. Table 1 shows the calculated fluorinated compound supply ratio for the fluorination of PEI 2000 K by Copper II acetate analysis. For 1 mg PEI-F-15, 698.3 μg PEI is identified, accounting for 69.83 wt%. The remaining 30.17 wt% is 301.7 μg, and a fluorination content of 1047.06 mmol can be inferred. Therefore, since PEI 1200 K contains 27 mmol of amine, if 15% for 1 g of PEI, the addition amount of GOFPE can be derived as 1.1526 g (4 mmol).

[0114] The mixture of GOFPE solution and PEI solution was stirred at room temperature for 48 hours, then sequentially dialyzed against ethanol solution and distilled water, and the resulting products were lyophilized to obtain PEI-F-5%, PEI-F-10%, PEI-F-15%, and PEI-F-20%.

[0115] Step 2: Analysis of toxicity and transfection efficiency of PEI-F according to fluorination rate

[0116] The toxicity of fluorinated PEI polymers at various concentrations was evaluated. HK2 cells were treated with fluorinated PEI-F-5%, PEI-F-10%, PEI-F-15%, and PEI-F-20% at concentrations ranging from 0.073 to 150 μg / ml, and the cytotoxicity was analyzed using the WST-1 reagent. The results showed that toxicity tended to increase with increasing fluorination and hydrophobicity, but stable cell viability was observed in the therapeutic range of N / P 10 and 15 (Fig. 3a).

[0117] We introduced pDNA into fluorinated PEI polymer to prepare PEI-pFluc polyplexes and then examined their transfection efficiency. As a result, PEI-F-15%-pFluc and PEI-F-20%-pFluc were observed to have similarly high transfection efficiencies at N / P of 10 and 15 (Fig. 3b). Next, we analyzed the cytotoxicity of PEI-pFluc polyplexes and observed stable cell viability for PEI-F-15%-pFluc and PEI-F-20%-pFluc at N / P of 10 and 15 (Fig. 3c). Based on these results, PEI-F-15% was selected for use in subsequent experiments.

[0118] Step 3: Synthesis of PFS polymer using fluorinated PEI-F-15%

[0119] PEI-F-15% was dissolved in anhydrous DMSO, and SDA solution was slowly added at a 1:1 ratio, followed by a reaction at -80 °C for 24 h with magnetic stirring. The reaction product was dialyzed against distilled water (MWCO: 3500 Da) at 4 °C for 48 h and then lyophilized to obtain PEI-F-SDA (hereinafter referred to as PFS), which was stored at -80 °C until use in the experiment (Fig. 4a-b).

[0120]

[0121] Manufacturing Example 3. Manufacturing of mRNA-polyplexes

[0122] To confirm the nucleic acid delivery ability of the PS polymer synthesized by the method of Manufacturing Example 1 and the PFS polymer synthesized by the method of Manufacturing Example 2, mRNA-polyplexes were prepared using hGLuc mRNA (Gaussia Luciferase reporter mRNA) (Fig. 5a-b).

[0123] hGLuc mRNA was synthesized from the pUCIDT plasmid according to the kit protocol. After acidifying hGLuc mRNA with sodium acetate buffer, polyplexes were formed by conjugating PS polymer or PFS polymer to hGLuc mRNA at various weight (w / w) ratios ranging from 1 to 30. Each polyplex was incubated at room temperature for 30 minutes and then electrophoresed on a 1% agarose gel for 30 minutes to obtain PS-mGLuc polyplexes and PFS-mGLuc polyplexes.

[0124]

[0125] Example 1. Analysis of physicochemical properties of PS polymer and PFS polymer

[0126] The composition analysis of the PS polymer synthesized by the method of Manufacturing Example 1 and the PFS polymer synthesized by the method of Manufacturing Example 2 was confirmed through proton nuclear magnetic resonance (NMR) spectroscopy.

[0127] As a result of NMR analysis, the peaks at 5.8-6.8 ppm (1) originating from the acrylate group present in the SDA spectral range and the peak at 7.4 ppm originating from the OH-signal (2) disappear for the PS polymer and PFS polymer after synthesis, and the CH2-signal resonance peaks at 3.2-4.8 ppm originating from the vinyl group of SDA and the methylene group of the polymer (3) are observed. In particular, in the case of the PFS polymer, the OH peak (4) formed from the fluorine-containing functional group of PEI-F was observed in the spectral range of 6 ppm, confirming the fluorination of the primary amine group (Fig. 6a-b).

[0128] The synthesis of PS polymer and PFS polymer was verified by Fourier transform infrared (FTIR) spectroscopy.

[0129] FTIR analysis results showed that the primary amine stretches of PEI were detected at 3274 and 3351 cm-1, the acrylate group double bond of SDA was detected at 1635 cm-1, and the carbonyl stretch was detected at 1725 cm-1 for the PS polymer and PFS polymer after synthesis. In addition, the additional peak at 1744 cm-1 confirmed the presence of unconjugated fragment esters in the polymers. In particular, the successful polymer formation of SDA and PEI-F was confirmed by the detection of a carbonyl-fluorine stretch (CF stretch) at 1116 cm-1 for the PFS polymer (Fig. 6c-d). GPC analysis of the PFS polymer revealed the presence of a copolymer with a size of 4.65 kDa.

[0130]

[0131] Example 2. Biochemical Characterization of mRNA-Polyplexes

[0132] Polyplex formation of PS-mGLuc and PFS-mGLuc manufactured by the method of Manufacturing Example 3 was confirmed through dynamic light scattering (DLS) measurement, agarose gel electrophoresis, and FE-TEM analysis.

[0133] First, the DLS measurement results showed that PS-mGLuc contained small particles with a hydrodynamic size of 121.13 ± 2.31 nm and a surface charge of +33.33 ± 1.25 mV. In contrast, PFS-mGLuc showed a slightly larger particle size of 197.03 ± 6.43 nm and a surface charge of +30.3 ± 2.74 mV (Fig. 7a-b). Thus, PFS-mGLuc was confirmed to have a larger diameter and lower surface charge than PS-mGLuc. This is because the polyethyleneimine (PEI) changed from a strong cationic to a weak cationic after fluorination. This is expected to reduce the inherent toxicity of PEI.

[0134] Additionally, the agarose gel electrophoresis results showed that in the case of PS-mGLuc, polyplexes transfected with reporter mRNA began to form when the weight ratio (w / w) of the PS polymer was 0.84 or higher (Fig. 8a), and in the case of PFS-mGLuc, polyplexes began to form when the weight ratio (w / w) of the PFS polymer was 2 or higher (Fig. 8b).

[0135] FE-TEM images of mRNA-polyplexes showed that PS-mGLuc (w / w 2.52) had an average particle size of 75.6 nm (Fig. 8c), while PFS-mGLuc (w / w 10) had a particle size of 55–100 nm (Fig. 8d). Specifically, it was observed that the particle size of PFS-mGLuc increased due to aggregation, and it can be inferred that this aggregation, which was not observed in PS-mGLuc, is due to the hydrophobic nature of PFS imparted by fluorination.

[0136] Meanwhile, PFS-mGLuc was confirmed to have a nanoparticle size smaller than that determined by hydrodynamic diameter analysis, and thus, it is expected that mRNA polyplexes using the PFS polymer according to the present invention will be rapidly absorbed into cells.

[0137]

[0138] Example 3. Cytotoxicity analysis of mRNA-polyplexes (in vitro)

[0139] The biocompatibility of PS-mGLuc and PFS-mGLuc manufactured by the method of Manufacturing Example 3 was evaluated using a mouse macrophage cell line. Comparison was made with PEI1200-mGLuc as a control.

[0140] Raw 264.7 cells were exposed to various PS-mGLuc, PFS-mGLuc, and PEI1200-mGLuc with polymer weight ratios (w / w) of 1 to 20, cultured for 4 hours, replaced with fresh medium, and further cultured for 24 hours. 10 μL of WST-1 reagent was injected and exposed for 2 hours, and then cell viability was quantified using a TECAN plate reader. As a result, the PS-mGLuc and PFS-mGLuc experimental groups exhibited lower cytotoxicity compared to the control group, PEI1200-mGLuc. In particular, the PFS-mGLuc experimental group showed a stable cell viability of over 80% in all weight ratios (w / w) of 1 to 20 (Fig. 9). Compared to the PS-mGLuc experimental group, where cell viability decreased rapidly from a weight ratio of 15 (w / w) or higher, the toxicity mitigation effect due to fluorination of the PFS polymer according to the present invention was confirmed.

[0141]

[0142] Example 4. Analysis of nucleic acid delivery capacity of mRNA-polyplexes (in vitro)

[0143] The nucleic acid delivery ability of PS-mGLuc and PFS-mGLuc manufactured by the method of Manufacturing Example 3 was evaluated using a mouse macrophage cell line.

[0144] PS-mGLuc and PFS-mGLuc with a polymer weight ratio (w / w) of 0.5 - 25 were prepared, and PEI (MW = 25000) polyplexes with an N / P ratio of 10 were prepared as a positive control. Raw 264.7 cells were seeded in 24-well plates at a density of 5 × 10 per well. 4 Cells were seeded in RPMI medium at a cell density of 1 μg and cultured overnight at 37°C with 5% CO2. Then, the cells were exposed to each polyplex containing 1 μg of Gaussia Luciferase mRNA per well and cultured. GLuc expression was evaluated using the Pierce™ Gaussia Luciferase Glow assay kit in the cultured lysate. As a result, both PS-mGLuc and PFS-mGLuc showed successful expression patterns, and the GLuc expression pattern of PFS-mGLuc was improved compared to PS-mGLuc. These results confirmed the effect of fluorination of the PFS polymer according to the present invention on the improvement of nucleic acid delivery ability (Fig. 10).

[0145]

[0146] Experimental Example 5. Internalization Analysis of mRNA-Polyplexes

[0147] To visually analyze the internalization of PFS-mGluc polyplexes, confocal microscopy was performed using Cy5.5-labeled mRNA.

[0148] Figure 11a is a graph showing mRNA labeled with Cy5.5 and quantified according to a standard protocol. After 5 hours of treatment with Raw 264.7 cells containing PFS-mGluc / Cy5.5, a complex of Cy5.5-labeled mRNA and PFS-mGluc polyplex, successful internalization of PFS-mGluc / Cy5.5 nanoparticles into the cells was confirmed (Figure 11b). This result demonstrates the effective cellular uptake of PFS-mGluc polyplex.

[0149]

[0150] Experimental Example 6. Analysis of the Internalization Pathway of mRNA-Polyplexes

[0151] The relationship between the endocytosis pathway and infection efficiency of PFS-mRNA polyplexes was further investigated using filipin-III, methyl-β-cyclodextrin, and chlorpromazine. RAW 264.7 cells were seeded at 5 × 10 per well in 24-well plates. 4 Cells were seeded in RPMI medium at a density of 10 μg / well and cultured overnight at 37°C in 5% CO2. Prior to treatment with polyplexes, cells were pretreated with 1 μmol of Filipin-III and 20 mmol of methyl-β-cyclodextrin for 30 min, and 20 μmol of chlorpromazine (20 μmol) was used for clathrin-mediated cytotoxicity. Cells were then exposed to each polyplex containing 1 μg of mRNA per well and cultured. As positive controls, polyplexes containing lipofectamine, LNP, and PEI (MW = 25,000 and 40,000) at an N / P ratio of 10 were prepared and administered. After 24 h of culture, the obtained cell lysates were evaluated by bioluminescence.

[0152] As a result, both PS-mGluc and PFS-mGluc polyplexes pretreated with cilia-mediated cytotoxicity inhibitors Filipin-III and methyl-β-cyclodextrin showed a significant decrease in mRNA expression (Fig. 12a-b). These inhibitors are known to interfere with sponge formation and alter membrane organization. However, chlorpromazine, a clathrin-mediated cytostatic agent, was observed to have no effect on the infection efficiency of both polyplexes. In contrast, the control groups, lipofectamine (Lipo), PEI 25K, PEI 40K, and LNP, showed a significant decrease in mRNA expression (Fig. 12c). These results demonstrate that PS-mGluc and PFS-mGluc polyplexes are primarily internalized through cilia-mediated endocytosis and do not rely on the clathrin-mediated pathway for cellular uptake.

[0153]

[0154] Experimental Example 7. Analysis of nucleic acid delivery capacity of mRNA-polyplexes (in vivo)

[0155] Following the in vitro mRNA delivery and expression analysis of Example 4, the in vivo expression profiles of PS-mGLuc and PFS-mGluc polyplexes were analyzed using C57BL / 6 mice. Seven-week-old female BALB / c and C57BL / 6 mice were used in this experiment. The mice were purchased from Orient (Korea) and handled according to the guidelines of the host institution, with approval from the animal experiment committee of the institution.

[0156] BALB / c mice were intramuscularly injected with PS-mGLuc at weight ratios (w / w) of 1.60, 2.10, 2.52, 3.15, and 3.80 and PFS-mGLuc at weight ratios (w / w) of 8, 10, 12, 15, and 18, respectively. Blood samples were collected at various time intervals, and the obtained serum was separated to quantify Gluc expression. gLuc LNP (Moderna) and linear PEI 40000 were used as positive controls.

[0157] The Moderna LNP experimental group showed high expression from 3 hours after administration to mice, but the expression decreased rapidly after 72 hours. On the other hand, the PS-mGLuc experimental group and the PFS-mGLuc experimental group showed expression levels similar to the Moderna LNP experimental group from 12 hours after administration to mice, showed the highest expression at 96 hours, and maintained an effective expression level for up to 144 hours. In addition, depending on the weight ratio (w / w), the optimal GLuc expression level was confirmed at a weight ratio of 2.10 to 3.15 (w / w) for the PS-mGLuc experimental group and at a weight ratio of 10 to 15 (w / w) for the PFS-mGLuc experimental group (Fig. 13a-b).

[0158]

[0159] Example 8. Evaluation of vaccine delivery efficacy of mRNA-polyplexes

[0160] Based on the GLuc expression levels according to the weight ratio (w / w) in Example 7, PS-mGLuc at a weight ratio (w / w) of 2.52 and PFS-mGLuc at a weight ratio (w / w) of 10 were prepared. PS-SmRNA and PFS-SmRNA vaccines conjugated with SmRNA (mRNA encoding the SARS-COVID-2 spike protein) were manufactured, and the immune response efficacy was evaluated. Moderna LNPs containing SmRNA (molar ratio, 50:10:38.5:1.5 = ionized lipid: DSPC: cholesterol: DMG-PEG-2000) were used as a positive control, and PBS was prepared as a negative control.

[0161] Seven-week-old C57BL / 6 mice were divided into five groups (n=10) and administered two intramuscular injections with 6 μg each of PBS, SmRNA, LNP, PS-SmRNA, and PFS-SmRNA.

[0162] Measurement of IFN-γ-secreting T cell populations using ELISpot analysis

[0163] Vaccine efficacy was assessed by administering PS-SmRNA (2.52 w / w) and PFS-SmRNA (10 w / w) to C57 / BL6 mice, followed by initial and booster doses at 2-week intervals. Mice were then euthanized, and spleen cells were isolated and stimulated with a crude mixture of SARS-CoV-2 spike glycoprotein to induce IFN-γ production.

[0164] The analysis results showed that the PFS-SmRNA administration group showed superior immune efficacy compared to the PS-SmRNA administration group, and in particular, there was no significant difference compared to the positive control group, the Moderna LNP administration group (Fig. 14a-b). After the additional vaccination, the PFS-SmRNA administration group showed a significant difference from the Moderna LNP administration group, but a significant increase in the IFN-γ-producing T cell population was observed in the PFS-SmRNA administration group (Fig. 14c-d). Therefore, the successful immunization of the vaccine formulation using the PFS polymer according to the present invention was confirmed.

[0165] Measurement of IFN-γ and TNF-α using ELISA analysis

[0166] Key cytokines, such as IFN-γ and TNF-α, play a crucial role in the immune response to vaccination and serve as important biomarkers for assessing vaccine immunogenicity. We investigated the detection of IFN-γ and TNF-α cytokines in the serum of mice administered PS-SmRNA and PFS-SmRNA after vaccination.

[0167] Three days after vaccination, mouse serum was separated from the blood and the serum samples were diluted 50-fold using a sample diluent. To measure IFN-γ (interferon-gamma) and TNF-α (tumor necrosis factor-alpha), ELISA was performed according to the manufacturer's protocol using the Invitrogen Mouse IFN-γ Uncoated ELISA Kit (Cat #A411505) and Invitrogen Mouse TNF-α Uncoated ELISA Kit (Cat #88-7324-88). Absorbance was measured at 450 nm with wavelength compensation at 570 nm, and standard curves corresponding to IFN-γ and TNF-α were constructed using the provided standards, and cytokine concentrations in the serum samples were calculated based on these standard curves.

[0168] As a result, from the third day after vaccination, both the PS-SmRNA administration group and the PFS-SmRNA administration group reached the peak in IFN-γ and TNF-α production, and in particular, the PFS-SmRNA administration group induced significantly higher cytokine production than the PS-SmRNA administration group, confirming that it had a superior protective immune response ability (Fig. 15a-b).

[0169] Spike peptide antibody measurement

[0170] To assess antibody formation after mRNA expression, spike peptide antibodies were measured via ELISA. Specifically, the receptor binding domain (RBD) antigen (ab275986-recombinant human coronavirus SARS-CoV-2 spike glycoprotein RBD-His tag) was coated onto the wells of an ELISA plate at a concentration of 100 ng / 50 μL using ELISA coating buffer and then incubated overnight at 4°C. After incubation, the coating buffer was discarded, and the wells were washed twice with washing buffer (PBS + 0.05% Tween-20, i.e., PBST). Then, a blocking step was performed by adding PBS + 1% bovine serum albumin (BSA) buffer to each well (100 μL / well) and incubating for 2 hours at 37°C, followed by washing twice with washing buffer. Serum samples were diluted 1:40 through two-fold serial dilution using PBS + 1% BSA buffer, and the diluted samples (100 μL / well) were added to the wells, incubated at 37°C for 1 h, and washed three times with wash buffer. Then, HRP-labeled secondary antibodies diluted 1:5000 were added to the wells (50 μL / well), incubated at 37°C for 1 h, and washed five times with wash buffer. Finally, 3,3′′tetramethylbenzidine solution was added to the wells (50 μL / well), incubated at room temperature for 10 min, and the reaction was stopped by adding 2 M H2SO4 (50 μL / well). The absorbance was then measured at 450 nm using a Tecan microplate reader.

[0171] As a result, after the initial vaccination, spike peptide antibodies were detected in the PFS-SmRNA group at two weeks post-vaccination at a level that was not significantly different from that in the Moderna LNP group (Fig. 16a). After the booster vaccination, the PFS-SmRNA group at five weeks post-vaccination maintained significant and sustained antibody levels, albeit at a lower level than the Moderna LNP group (Fig. 16b). The presence of spike peptide-specific antibodies indicates successful in vivo delivery of spike mRNA and expression of the spike antigen, confirming the effective mRNA delivery capability of the PFS polymer according to the present invention.

[0172] Measurement of neutralizing antibody titers

[0173] To quantify neutralizing antibody titers against SARS-COVID-2 in mouse serum samples, the PRNT50 assay was performed. The PRNT50 assay measures the ability of antibodies to neutralize viral infection by quantifying the reduction in viral plaques formed in cell culture.

[0174] To analyze vaccination efficacy, PRNT50 was performed with heat-inactivated serum against the Wuhan and variant COVID-19 virus strains. This experiment was performed in a biosafety level 3 facility. Mouse serum samples were serially diluted from 1 / 4 to 2-fold. The diluted serum was mixed with 50 PFU of the Wuhan or variant COVID-19 virus strains and incubated for 1 hour at 37°C with 5% CO2. Vero cells were inoculated into 12-well plates the previous day. The serum-virus mixture was then inoculated onto Vero cells and incubated for 1 hour at 37°C. After removing the serum-virus mixture and adding 1% methylcellulose overlay medium, the medium was incubated at 37°C with 5% CO2 for 3 days. After virus plaque formation, the cells were fixed and stained with crystal violet. The number of plaques in each well was counted and the neutralizing antibody titer was calculated as the reciprocal of the serum dilution that reduced the number of plaques by 50% compared to the virus-only control wells.

[0175] Analysis results showed that the Moderna LNP-administered group showed a greater reduction in viral plaques than the PS-SmRNA and PFS-SmRNA-administered groups in serum collected two weeks after the initial vaccination (Fig. 17a). However, after the booster dose, the PFS-SmRNA-administered group showed a greater reduction in viral plaques compared to the two-week post-inoculation baseline (Fig. 17b). These results demonstrate that PFS-SmRNA polyplexes can effectively prime the immune system and generate potent and long-lasting neutralizing antibodies when administered as a priming-boost regimen. The enhanced neutralizing capacity of PFS-SmRNA demonstrates its ability to successfully induce an immune response.

[0176] These results confirm the excellent suitability of the PFS polymer according to the present invention as a potential mRNA vaccine delivery formulation for the prevention and treatment of infections such as COVID-19.

[0177] Histopathological analysis

[0178] H&E staining analysis of major internal organs and muscles after euthanasia of mice revealed an abundance of extramedullary hematopoietic cells in the spleen of both the Moderna LNP- and PFS-SmRNA-treated groups. This phenomenon may be due to transient hematopoietic stress caused by the vaccine-induced immune response and compensatory mechanisms activated in response to the increased immune activity following vaccination. This process may stimulate extramedullary hematopoiesis in the spleen to ensure adequate blood cell production during periods of increased immune demand. No other abnormalities were observed in other major internal organs, including the muscles at the vaccination site (Fig. 18).

Claims

1. A polymer having the following chemical formula 1 as a repeating unit: [Chemical Formula 1] (In the formula, each R is independently a hydrogen or fluorine-containing functional group, except when all R are hydrogen.) 2. A polymer according to claim 1, wherein the fluorine-containing functional group is substituted in 5 to 20% of the polymer.

3. In claim 1, the fluorine-containing functional group is perfluorooctanoic acid (C 8 HF 15 O 2 ), octafluoropentyl-tetrafluoroethyl ether (C 7 H 4 F 12 O), 2-(perfluorobutyl)ethyl methacrylate (C 10 H 9 F 9 O 2 ), octafluoropentyl methacrylate (C 9 H 8 F 8 O 2 ), perfluorobutanoylcyclopentanone (C 9 H 7 F 7 O 2 ), octafluoropentyl ether (C 8 H 8 F 8 O), octafluorooctadienoic acid (C 8 H4F 8 O 2 ), octafluoropentylacrylate (C 8 H 6 F 8 O 2 ), heptafluoroisobutyl methacrylate (C 8 H 7 F 7 O 2 ), heptafluorohexanediol (C 6 H 7 F 7 O 2 ), hexafluorobutyl methacrylate (C 8 H 8 F 6 O 2 ), hexafluoroisopropyl methacrylate (C 7 H 6 F 6 O 2 ), tetrafluoropropyl methacrylate (C 7 H 8 F 4 O 2 ), trifluoromethylphenylacetic acid (C 9 H 7 F 3 O 2 ), trifluoromethylacetylacetonate (C 6 H 7 F 3 O 2 ), methyl trifluoroacetoacetate (C 5 H 5 F 3 O 3 ), trifluoroethyl acetate (C 4 H 5 F 3 O 2 ) and methyl trifluoroacetate (C 3 H 3 F 3 O 2 ) is selected from the group consisting of a polymer.

4. A nucleic acid delivery vehicle comprising a polymer and a nucleic acid according to any one of claims 1 to 3.

5. A nucleic acid delivery vehicle according to claim 4, wherein the nucleic acid is at least one selected from the group consisting of gDNA, cDNA, pDNA, mRNA, tRNA, rRNA, siRNA, shRNA, miRNA, aptamer, antisense oligodeoxynucleotide (AS-ODN), DNA-RNA hybrid, and ribozyme.

6. A nucleic acid delivery vehicle according to claim 4, wherein the weight ratio (w / w) of the polymer is 1 to 20.

7. A nucleic acid delivery vehicle according to claim 4, having a size of 20 to 150 nm.

8. A nucleic acid delivery vehicle that promotes proliferation specifically of immune cells according to claim 4.

9. A nucleic acid delivery vehicle according to claim 8, wherein the immune cell is a T cell, a B cell, an NK cell or a hematopoietic progenitor cell.

10. A vaccine composition comprising a nucleic acid vector according to any one of claims 4 to 9.

11. A vaccine composition according to claim 10, wherein the vaccine is for preventing or treating one or more infections selected from the group consisting of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and influenza virus.

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