Composition for transferring nucleic acids comprising cationic liposomes and uses thereof

Abstract

The present invention relates to a composition for transferring nucleic acids comprising cationic liposomes, wherein a complex (lipoplex) is formed by mixing liposomes prepared by mixing cationic lipids and neutral lipids with nucleic acids. The lipoplex thus prepared constitutes highly stable particles, effectively introduces a target nucleic acid into cells, and has a high expression rate of the target nucleic acid. In addition, it was confirmed that, when liposomes are prepared, an immune enhancer such as a lipopeptide is included, thereby enhancing an immune activity response through the lipoplex of the present invention. When a nucleic acid for a target antigen expressed in tumor cells is included in the lipoplex, it was observed that tumor formation occurs late, and thus, it was confirmed that the lipoplex can have efficacy as a vaccine for anticancer as well as an infectious disease.

Description

Composition for nucleic acid delivery comprising cationic liposomes and use thereof

[0001] Cross-reference to related applications

[0002] This disclosure claims priority to Patent Application No. 10-2023-0144497, filed October 26, 2023, and Patent Application No. 10-2024-0107641, filed August 12, 2024, the entire contents of which are incorporated herein by reference.

[0003] Technology field

[0004] The present invention relates to a lipoplex for delivery of nucleic acids such as DNA and / or RNA, and more particularly, to a composition for delivery of nucleic acids comprising a cationic lipid-based liposome and a method for preparing the same.

[0005] Gene therapy and genetic vaccines are proven and widely applied technologies in the medical field. They can treat not only genetic diseases but also autoimmune diseases, infectious diseases, cancer or tumor-related diseases, and inflammatory diseases. The development of genetic vaccines began with the report that direct injection of DNA and RNA encoding target genes into living animals can result in the expression of the target genes in living animals, thereby inducing immunity (Wolff JA et al. Science, 247:1465-8, 1990).

[0006] In gene therapy or genetic vaccination, both DNA and RNA can be used as nucleic acid molecules for gene delivery, and DNA is known to be relatively stable and easier to handle than RNA. However, DNA poses a potential risk if the administered DNA fragment inserts into an unintended location within the patient's genome, potentially damaging the gene. Additionally, unwanted anti-DNA antibodies may develop. Another potential problem is the limited expression levels of peptides or proteins produced by DNA administration and subsequent transcription / translation. The presence or absence of specific transcription factors significantly influences the expression levels of administered DNA. In the absence of specific transcription factors, sufficient RNA is not produced by DNA transcription, resulting in limited levels of peptides or proteins produced through translation. In contrast, RNA, when used as a gene delivery tool, does not require transcription and can synthesize proteins directly within the cytoplasm without the need for DNA entry into the nucleus. Therefore, there is no risk of RNA incorporation into cellular chromosomes, potentially causing unwanted genetic damage. Additionally, compared to DNA, it has a shorter half-life and does not induce long-term genetic modification (Sayour EJ et al., J Immunother Cancer 2015;3:13, 2015). When delivered into cells, typical RNA vaccines are activated for only a short period of time, causing target protein expression, and are destroyed within a few days by enzymatic reactions, while a specific immune response to the expressed target antigen (protein) remains.

[0007] However, the use of nucleic acids for therapeutic and prophylactic purposes currently faces two challenges. First, free RNA is vulnerable to nuclease digestion in plasma. Second, free RNA has limited access to intracellular compartments where relevant translational machinery resides. Lipid nanoparticles formed from cationic lipids and other lipid components, such as neutral lipids, cholesterol, PEGylated lipids, and nucleic acids, are being explored to block RNA degradation in plasma and promote cellular uptake of oligonucleotides. When using lipid-based carriers such as liposomes or lipid nanoparticles, mRNA is typically either adsorbed externally or encapsulated internally. When mRNA is adsorbed externally, it typically exists as an aggregate of liposome and nucleic acid, not as a single liposome. Adsorption and stability vary depending on the lipid composition, nucleic acid state, and nucleic acid-liposome ratio, necessitating optimization.

[0008] Furthermore, even if the expression of a carrier has been verified in vitro, there is a problem that the expression results in vivo may differ in terms of mRNA expression ability. In vivo, there are many factors that reduce the efficiency of delivery into cells, such as reduced half-life and biodistribution due to plasma protein aggregation depending on the lipid composition and physicochemical properties of the carrier, differences in cell uptake methods depending on lipid composition, and barriers by the extracellular matrix (ECM). Therefore, unlike in vitro delivery, carrier optimization is essential for in vivo delivery.

[0009] As a prior art, Korean Patent Publication No. 10-2022-0126235 discloses the preparation of an mRNA delivery composition using liposomes containing the cationic lipid DOTAP, the neutral lipid DOPE, and cholesterol. However, this disclosure fails to disclose how to replace DOTAP and cholesterol by changing the type of cationic lipid and maintain stability. Furthermore, since the purpose is to induce an immune response by delivering nucleic acid material, methods to enhance such immunogenicity are needed.

[0010] Accordingly, the inventors of the present invention conducted repeated experiments using various cationic lipids including DOTAP, and confirmed that stability and efficacy as a vaccine can be improved by including an immune enhancer other than cholesterol, thereby completing the present invention.

[0011] The purpose of the present invention is to provide a nucleic acid delivery platform technology using cationic liposomes with excellent safety and stability.

[0012] An object of the present invention is to provide a composition for nucleic acid delivery comprising liposomes formed from a mixture of cationic lipids and neutral lipids.

[0013] Another object of the present invention is to provide a vaccine comprising the nucleic acid delivery composition as an active ingredient.

[0014] Another object of the present invention is to provide an anticancer vaccine comprising the nucleic acid delivery composition as an active ingredient.

[0015] Another object of the present invention is to provide a method for preparing a composition for nucleic acid delivery, comprising the step of mixing a liposome formed from a mixture of a cationic lipid and a neutral lipid and a nucleic acid.

[0016] The present invention relates to a composition for nucleic acid delivery comprising cationic liposomes. A complex (lipoplex) is formed by mixing liposomes prepared by mixing cationic lipids and neutral lipids with nucleic acids. The lipoplexes prepared in this manner form highly stable particles, effectively introduce target nucleic acids into cells, and exhibit high expression rates of the target nucleic acids. Furthermore, it has been confirmed that the lipoplexes of the present invention can enhance immune response by including an immunostimulant such as a lipopeptide during liposome preparation. In addition, it has been observed that when a nucleic acid for a target antigen expressed on tumor cells is included in the lipoplex, tumor formation occurs late, confirming that the lipoplexes can be effective not only for infectious diseases but also as anticancer vaccines.

[0017] Figure 1 is a schematic diagram showing the process of manufacturing a lipoplex.

[0018] Figure 2 shows the results of evaluating the stability of lipoplexes manufactured using DOTAP and DOPE. Figure 2a shows the results of particle size and polydispersity index (PDI) according to the ratio of DOTAP, presence or absence of cholesterol, or N / P ratio, Figure 2b shows the results of zeta-potential values, and Figure 2c shows the results of visual observation.

[0019] Figure 3 shows the results of confirming the in vitro expression efficiency using lipoplexes manufactured using DOTAP and DOPE. Figure 3a shows the results using the HeLa cell line, and Figure 3b shows the results using the RAW264.7 cell line.

[0020] Figure 4 shows the results of evaluating the stability of lipoplexes manufactured using DOTAP, DOPE, or DSPC. Figure 4a shows the results showing the particle size and polydispersity index (PDI) according to the type of neutral lipid, the presence or absence of cholesterol, or the N / P ratio, Figure 4b shows the results showing the zeta-potential value, and Figure 4c shows the results observed with the naked eye.

[0021] Figure 5 shows the results of confirming the in vitro expression efficiency using lipoplexes manufactured using DOTAP, DOPE, or DSPC. Figure 5a shows the results using the HeLa cell line, and Figure 5b shows the results using the RAW264.7 cell line.

[0022] Figure 6 shows the results of evaluating the stability of lipoplexes manufactured using DOTAP, DOPE, or DSPC, and confirming the stability of lipoplexes in the presence of serum.

[0023] Figure 7 shows the results of evaluating the stability of lipoplexes manufactured using different types of cationic lipids. Figure 7a shows the results showing the particle size and polydispersity index (PDI) according to the type of cationic lipid, presence or absence of cholesterol, or N / P ratio, and Figure 7b shows the results showing the zeta-potential value.

[0024] Figure 8 shows the results of confirming in vitro expression efficiency using lipoplexes manufactured using different types of cationic lipids.

[0025] Figure 9 shows the results of evaluating the stability of a lipoplex prepared by mixing Pam3CSK4 with DDAB and DOPE. Figure 9a shows the results showing the particle size and polydispersity index (PDI) according to the amount of Pam3CSK4 or the N / P ratio, and Figure 9b shows the results showing the zeta-potential value.

[0026] Figure 10 shows the results comparing the mRNA adsorption levels for liposomes prepared by mixing Pam3CSK4 with DDAB and DOPE.

[0027] Figure 11 shows the results of confirming in vitro expression efficiency using a lipoplex prepared by mixing Pam3CSK4 with DDAB and DOPE.

[0028] Figure 12 shows the results of confirming the stability of liposomes or lipoplexes (CVI-LPX-1 and CVI-LPX-2) manufactured with DDAB, DOPE, and Pam3CSK4. Figure 12a is a schematic diagram showing the shape of the lipoplex, Figure 12b shows the size and dispersion of each particle, and Figure 12c shows the zeta fraction value.

[0029] Figure 13 shows the results comparing the mRNA adsorption levels for liposomes prepared by mixing Pam3CSK4 with DDAB and DOPE. Figure 13a shows the results under conditions not including triton X-100, and Figure 13b shows the results under conditions including triton X-100.

[0030] Figure 14 shows the results of confirming the in vitro expression efficiency of CVI-LPX-1 and CVI-LPX-2.

[0031] Figure 15 shows the results of measuring the immune response induction effect of CVI-LPX-1 and CVI-LPX-2. Figure 15a shows the results of confirming the level of antigen-specific antibody production, Figure 15b shows the results of performing ELIspot to measure the number of activated cells, and Figure 15c is a graph analyzing the results of Figure 15b.

[0032] Figure 16 shows the results of measuring the immune response induction effect of CVI-LPX-1 and CVI-LPX-2. Figures 16a and 16b are graphs showing the results of measuring the proportion of CD8+ T cells and analyzing the results, Figures 16c and 16d are graphs showing the results of measuring the proportion of MHCⅡ macrophages and analyzing the results, and Figures 16e and 16f are graphs showing the results of measuring the proportion of M1 macrophages and analyzing the results.

[0033] Figure 17 shows the results of measuring the level of cytokine secretion by CVI-LPX-1 and CVI-LPX-2. Figure 17a is a graph measuring the level of cytokine secretion involved in the immune response, and Figure 17b is a graph measuring the level of cytokine secretion involved in the inflammatory response.

[0034] Figure 18 is a graph summarizing the time required for tumor formation in mice injected with B16F10 mouse melanoma expressing OVA.

[0035] Figure 19 shows the results of evaluating the safety of lipoplexes CVI-LPX-1 and CVI-LPX-2. Figure 19a shows the results of measuring the body weight of mice after lipoplex administration, Figure 19b shows the results of measuring the concentration of ALT (alanine aminotransferase) in mice after lipoplex administration, Figure 19c shows the results of measuring the concentration of AST (aspartate aminotransferase) in mice after lipoplex administration, and Figure 19d shows the results of measuring the concentration of bilirubin in mice after lipoplex administration.

[0036] Figure 20 shows the results of characterization after lyophilization of lipoplex CVI-LPX-1. Figure 20a shows the results of visual observation after lyophilization, Figure 20b shows the results of confirming the changes in particle size and dispersion after re-dissolving in solution after lyophilization, and Figure 20c shows the results of measuring the zeta localization value after re-dissolving in solution after lyophilization. Figure 20d shows the results showing the mRNA adsorption level in the lyophilized formulation, and Figure 20e shows the results showing the in vitro expression level in Hela cells.

[0037] Hereinafter, the present invention will be described in detail.

[0038]

[0039] According to one aspect of the present invention, the present invention provides a composition for nucleic acid delivery comprising a liposome based on a mixture of cationic lipids and neutral lipids.

[0040] The above cationic lipid includes a lipid that continuously exhibits cationicity without being affected by a pH change or an ionic lipid that is converted to cationicity by a pH change.

[0041] The cationic lipids include 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), dimethyldioctadecylammonium bromide salt (DDAB), 3β-[N-(N',N'-dimethylaminoethane) carbamoyl cholesterol (DC-Chol), 1,2-dioleoyloxy-3-dimethylammonium propane (DODAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 Etyl PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 Ethyl PC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 Ethyl PC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholin (18:0 Ethyl PC), 1,2-Dipalmitoyl-sn-glycero-3-ethylphosphocholine (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-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dimyristoyl-3-dimethylammonium propane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP), 1,2-distearoyl-3-dimethylammonium-propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 1,2-stearoyl-3-trimethylammoniumpropane (18:0 TAP), It may be at least one selected from the group consisting of 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP) and N4-cholesteryl-spermine (GL67), preferably DOTAP, 18:0 TAP, 14:0 TAP,and may be at least one selected from the group consisting of DDAB, but is not limited thereto.

[0042] The above DOTAP is a cationic lipid having a structure represented by the following chemical formula 1.

[0043]

[0044] The above DDAB is a cationic lipid having a structure represented by the following chemical formula 2.

[0045]

[0046] The neutral lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), It may be at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), phosphatidylserine (PS), phosphoethanolamine (PE), phosphatidylglycerol (PG), phosphoric acid (PA), and phosphatidylcholine (PC), and preferably DOPE, DPPC, or DSPC, but is not limited thereto.

[0047] The above DOPE is a neutral lipid having a structure represented by the following chemical formula 3.

[0048]

[0049] The above DSPC is a neutral lipid having a structure represented by the following chemical formula 4.

[0050]

[0051] The above liposome may be formed by including cationic lipids and neutral lipids in a ratio of 0.5 to 2:1, 0.6 to 1.5:1, 0.7 to 1.3:1, 0.8 to 1.3:1, 0.8 to 1.2:1, or 0.9 to 1.1:1.

[0052] The liposome may further comprise a lipopeptide. The lipopeptide may be composed of a fatty acid and multiple amino acids bound to a glycerol molecule. The number of fatty acids within the glycerol molecule or the amino acids constituting the lipopeptide may be one or more. The fatty acids and amino acids may be chemically modified. The lipopeptide may be a lipoprotein, either a portion of a molecule derived from a gram-positive or gram-negative bacterium or mycoplasma, or in the form of an entire molecule.

[0053] For example, the lipopeptide may be at least one selected from the group consisting of Pam3-CSKKKK, PHC-SKKKK, Ole2PamCys-SKKKK, Pam2Cys-SKKKK, PamCys(Pam)-SKKKK, Ole2Cys-SKKKK, Myr2Cys-SKKKK, PamDhc-SKKKK, Pam-CSKKKK, Dhc-SKKKK and FSL-1. The above lipopeptide can be inserted into the liposome at a concentration of 20 to 250, 20 to 50, 50 to 250, 150 to 250, 50 to 150, 20 to 2500, 20 to 500, 50 to 2500, 150 to 2500 or 50 to 1500 μg / dose.

[0054] The lipopeptide may be included in an amount of 1 to 10% (w / w), 2 to 8% (w / w), 2 to 6% (w / w), or 2 to 5% (w / w) by weight of the cationic lipid and neutral lipid mixture.

[0055] The nucleic acid included in the above composition encodes a peptide or protein that can act as an immunogen, and the nucleic acid may be DNA, mRNA (messenger RNA), circRNA (circular RNA), or saRNA (self-amplifying RNA).

[0056] The mRNA may typically be an mRNA having at least one open reading frame (ORF) that can be translated by a cell or organism provided with the mRNA. The product of this translation is an antigen, preferably a peptide or protein that can act as an immunogen. The product may also be a fusion protein comprising two or more immunogens, for example, a fusion protein comprising two or more epitopes, peptides, or proteins derived from the same or different viral proteins, wherein the epitopes, peptides, or proteins may be linked by a linker sequence.

[0057] Furthermore, the mRNA may be understood as an artificial mRNA, i.e., an mRNA molecule that does not occur naturally. An artificial mRNA molecule may be understood as a non-natural mRNA molecule. Such mRNA molecules may be non-natural due to individual sequences (that do not occur naturally) and / or other non-natural modifications, such as structural modifications of nucleotides. An artificial mRNA molecule may be designed and / or produced by genetic engineering methods corresponding to a desired artificial nucleotide sequence (a heterologous sequence).

[0058] Furthermore, the mRNA may exhibit modifications that increase its resistance to in vivo degradation (e.g., degradation by exo- or endo-nucleases) and / or in vitro degradation (e.g., during the manufacturing process prior to vaccine administration, e.g., during the preparation of the vaccine solution to be administered). Stabilization of the RNA may be achieved, for example, by providing a 5'-CAP structure, a poly-A tail, or any other UTR modification. Stabilization of the RNA may also be achieved by chemical modification or by altering the G / C content of the nucleic acid. Various other methods are known in the art and may be applied to the present invention.

[0059] The above circRNA is a type of single-stranded RNA. Unlike the widely known linear RNA, it is a continuous closed loop structure formed by reverse splicing during the splicing process. This structure makes it resistant to nucleic acid degradation by terminal nucleases and highly stable, as it lacks a 5' or 3' terminus.

[0060] The above saRNA is a type of RNA that self-amplifies upon entering a host cell. This amplification occurs multiple times within the cell, increasing RNA production and producing target peptides and / or proteins within the cell. This property increases the likelihood of higher production and prolonged immune responses, making it a promising candidate for vaccine development.

[0061] The mixing ratio of liposomes and nucleic acids in the above composition can be expressed as the N / P ratio, and the expression and stability of the complex are affected depending on the N / P ratio.

[0062] The N / P ratio of the liposome and nucleic acid may be, but is not limited to, 0.6 to 1.4, 0.6 to 1.2, 0.7 to 1.3, 0.8 to 1.3, 0.8 to 1.2, 0.5 to 1.0, or 1.0 to 1.2.

[0063]

[0064] According to another aspect of the present invention, the present invention provides a vaccine comprising the nucleic acid delivery composition as an active ingredient.

[0065] According to another aspect of the present invention, the present invention provides an anticancer vaccine comprising the nucleic acid delivery composition as an active ingredient.

[0066] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a disease selected from the group consisting of cancer, tumor, autoimmune disease, genetic disease, inflammatory disease, viral infection, and bacterial infection, comprising the above-described nucleic acid delivery composition as an active ingredient.

[0067] The above prevention means any action to suppress the occurrence of the above-described disease or delay its progression by administering the pharmaceutical composition according to the present invention.

[0068] The above treatment means any action that improves or beneficially changes the symptoms of the above-described disease by administering the pharmaceutical composition according to the present invention.

[0069] The pharmaceutical composition may be combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. The pharmaceutical composition may include buffers, such as neutral buffered saline, phosphate buffered saline, citric acid buffered solution, and the like; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0070] The pharmaceutical composition may be administered orally or parenterally, and may be administered by, but is not limited to, intravenous administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intratumoral administration, intracerebral administration, intracranial administration, intrapulmonary administration, and rectal administration.

[0071] The above pharmaceutical composition is administered in a pharmaceutically effective amount. The pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective amount may be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route, and excretion rate, treatment duration, concurrent medications, and other factors well known in the medical field.

[0072] The above pharmaceutical composition can be administered as an individual treatment or in combination with other treatments. It can be administered sequentially or simultaneously with conventional treatments, or as a single or multiple dose. It is important to consider all of the above factors and administer the amount that achieves maximum effect with the minimum amount possible without causing side effects. This can be readily determined by those skilled in the art.

[0073] According to another aspect of the present invention, the present invention provides a method for treating a disease, comprising administering a pharmaceutically effective amount of the nucleic acid delivery composition described above.

[0074] The above disease may be selected from the group consisting of cancer, tumor, autoimmune disease, genetic disease, inflammatory disease, viral infection and bacterial infection.

[0075] The cancer may be selected from breast cancer, kidney cancer, testicular cancer, prostate cancer, ovarian cancer, uterine cancer, cervical cancer, vaginal cancer, fallopian tube cancer, rectal cancer, lung cancer, stomach cancer, liver cancer, esophageal cancer, small intestine cancer, pancreatic cancer, oral cancer, melanoma, or sarcoma.

[0076] According to another aspect of the present invention, a method for inducing immunity comprises administering the nucleic acid delivery composition described above to a subject. The subject includes humans, cows, dogs, guinea pigs, rabbits, chickens, insects, and the like, and refers to any individual, patient, or group thereof requiring treatment. Furthermore, any subject participating in a clinical research trial without any clinical findings of a disease, a subject participating in an epidemiological study, or a subject used as a control may also be included.

[0077] Another object of the present invention is to provide a method for preparing a composition for nucleic acid delivery, comprising the step of mixing a liposome formed from a mixture of a cationic lipid and a neutral lipid and a nucleic acid.

[0078] The above method may be performed in the order of mixing cationic lipids and neutral lipids to prepare liposomes and mixing the liposomes with nucleic acids.

[0079] Additionally, the process of preparing liposomes by mixing cationic lipids and neutral lipids may additionally include a step of mixing an immunostimulant.

[0080] The above-mentioned immunostimulant may be a lipopeptide-based immunostimulant, and the lipopeptide may be at least one selected from the group consisting of Pam3CSK4, Pam3-CSKKKK, PHC-SKKKK, Ole2PamCys-SKKKK, Pam2Cys-SKKKK, PamCys(Pam)-SKKKK, Ole2Cys-SKKKK, Myr2Cys-SKKKK, PamDhc-SKKKK, Pam-CSKKKK, Dhc-SKKKK and FSL-1, but is not limited thereto.

[0081] The nucleic acid and / or liposome, or lipoplex, may be provided in a lyophilized powder form, or may be provided dissolved in an appropriate solution or buffer. If the mRNA and / or liposome is provided in a lyophilized form, it may be used by dissolving it in an appropriate solution or buffer.

[0082]

[0083] In specific embodiments and experimental examples of the present invention, liposomes were formed using a mixture of cationic lipids and neutral lipids, and liposomes were added to an mRNA solution to prepare a lipoplex, which is an mRNA-liposome complex (Fig. 1). As a result of evaluating the stability of the particles under different conditions, it was observed that the N / P ratio, rather than the ratio of cationic lipids to neutral lipids, the addition of cholesterol, or the type of neutral lipid, affected the stability such as particle size, polydispersity index (PDI), and zeta position of the particles (Figs. 2 to 5). However, it was confirmed that there may be differences in stability to serum depending on the type of neutral lipid (Fig. 6), and it was confirmed that differences in the stability and effectiveness of the particles occurred depending on the type of cationic lipid (Figs. 7 and 8).

[0084] To enhance the immune response, lipoplexes were manufactured by mixing lipopeptides, which are immune-enhancing agents, during the liposome manufacturing process, and as the ratio (amount) of the mixed lipopeptides increased, the stability and effectiveness of the particles were impaired (Figs. 9 and 11).

[0085] Based on this, the inventors of the present invention manufactured two types of lipoplexes containing DDAB, DOPE, Pam3CSK4, and mRNA, and confirmed that the lipoplexes manufactured under the conditions of the present invention maintained an appropriate size, had high stability, and stably observed mRNA binding affinity (Figs. 12 and 13). It was also observed that antigen expression and antigen-specific immune responses were excellent (Figs. 14 to 17). In addition, it was observed that tumor formation was inhibited due to the enhanced immune response to the antigen expressed on tumor cells (Fig. 18). Despite these excellent effects, the lipoplexes of the present invention are safe without toxicity (Fig. 19), and their stability is not impaired even when manufactured in a lyophilized formulation, so they can be used in various formulations (Fig. 20).

[0086] Through this, it was confirmed that the lipoplex constituting the nucleic acid delivery composition of the present invention can be stably manufactured, has high antigen expression efficiency, and induces humoral and cellular immune responses, so that it can be used as a vaccine for infectious diseases as well as a preventive or therapeutic vaccine for cancer and tumors depending on the type of antigen.

[0087] Hereinafter, the present invention will be described in detail through examples and experimental examples.

[0088] However, the following examples and experimental examples are illustrative of the present invention, and the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the claims also fall within the scope of the present invention.

[0089]

[0090] <Example 1> Preparation of lipoplex containing liposomes and RNA

[0091] <1-1> Manufacture of liposomes using the thin film method

[0092] A mixture of cationic lipids and neutral lipids dissolved in chloroform (CHCl3) was dried with nitrogen gas at 50°C for 30 minutes to form a lipid film. The lipid mixture was mixed with cholesterol or Pam3CSK4 at a certain ratio according to the examples performed below, and then dried. After that, a hydration buffer (5% sucrose, 20 mM HEPES) was added to the lipid film, vortexed for 10 seconds, and left at 50°C for 10 minutes. This process was repeated three times to hydrate the lipid film. The hydrated solution was prepared and extruded 21 times in one direction in an extruder placed in a 70°C heat block to homogenize the particles. After that, the homogenized suspension was transferred to a 1 ml syringe and sterilized by using a 0.2 um PVDF filter. The filtered solution was sequentially cooled from room temperature to 4°C and stored in a refrigerator.

[0093] <1-2> Preparation of RNA-liposome complex (lipoplex)

[0094] The liposome suspension prepared in <1-1> was added to the mRNA solution at a 1:1 ratio and left on ice for 30 minutes to form a complex of mRNA and liposomes. The liposome formation and complex preparation method is roughly illustrated in Figure 1.

[0095]

[0096] <Example 2> Lipoplex using DOTAP and DOPE and / or cholesterol

[0097] Liposomes were formed using DOTAP as a cationic lipid and DOPE as a neutral lipid, and cholesterol was added to this, and the stability and effectiveness of the lipoplexes were confirmed according to the ratio of DOTAP and the ratio between mRNA and liposomes.

[0098] <2-1> Lipoplex production according to DOTAP ratio, cholesterol presence, and N / P ratio

[0099] Liposomes were prepared using the method described in Example 1, but with different ratios of DOTAP, and were named LP-1 to LP-6 depending on the ratio of DOTAP and the presence or absence of cholesterol addition (Table 1).

[0100] Name compositionLP-1DOTAP:DOPE (40:60)LP-2DOTAP:DOPE (40:60):CholLP-3DOTAP:DOPE (50:50)LP-4DOTAP:DOPE (50:50):CholLP-5DOTAP:DOPE (60:40)LP-6DOTAP:DOPE (60:40):Chol

[0101] mRNA (CleanCap) in these six types of liposomes ® Lipoplexes were prepared by adding RLucmRNA, TriLink#L7204) and varying the ratio of liposomes and mRNA. The N / P ratio, which is the mixing ratio of liposomes and mRNA, was calculated using the following formula.

[0102]

[0103] The composition ratio and manufacturing capacity are as shown in Table 2.

[0104] 시료정보Sample 제조Label조성N / P ratiomRNA(1 mg / ml)LP(2 mg / ml)BufferTotal volumeRNALPR1-1RLuc mRNA + LP-1 (N / P=1.0)1056.61.01028.361.7100R1-2RLuc mRNA + LP-1 (N / P=1.2)1067.91.21034.056.1100R1-3RLuc mRNA + LP-1 (N / P=1.4)1079.21.41039.650.4100R1-4RLuc mRNA + LP-1 (N / P=1.6)1030.61.61045.344.7100R1-5RLuc mRNA + LP-1 (N / P=1.8)10101.91.81051.039.1100R1-6RLuc mRNA + LP-1 (N / P=2.0)10113.22.01056.633.4100R2-1RLuc mRNA + LP-2 (N / P=1.0)1056.61.01028.361.7100R2-2RLuc mRNA + LP-2 (N / P=1.2)1067.91.21034.056.1100R2-3RLuc mRNA + LP-2 (N / P=1.4)1079.21.41039.650.4100R2-4RLuc mRNA + LP-2 (N / P=1.6)1030.61.61045.344.7100R2-5RLuc mRNA + LP-2 (N / P=1.8)10101.91.81051.039.1100R2-6RLuc mRNA + LP-2 (N / P=2.0)10113.22.01056.633.4100R3-1RLuc mRNA + LP-3 (N / P=1.0)10451.01022.567.5100R3-2RLuc mRNA + LP-3 (N / P=1.2)10541.21027.063.0100R3-3RLuc mRNA + LP-3 (N / P=1.4)10631.41031.558.5100R3-4RLuc mRNA + LP-3 (N / P=1.6)10721.61036.054.0100R3-5RLuc mRNA + LP-3 (N / P=1.8)10811.81040.549.5100R3-6RLuc mRNA + LP-3 (N / P=2.0)10902.01045.045.0100R4-1RLuc mRNA + LP-4 (N / P=1.0)10451.01022.567.5100R4-2RLuc mRNA + LP-4 (N / P=1.2)10541.21027.063.0100R4-3RLuc mRNA + LP-4 (N / P=1.4)10631.41031.558.5100R4-4RLuc mRNA + LP-4 (N / P=1.6)10721.61036.054.0100R4-5RLuc mRNA + LP-4 (N / P=1.8)10811.81040.549.5100R4-6RLuc mRNA + LP-4 (N / P=2.0)10902.01045.045.0100R5-1RLuc mRNA + LP-5 (N / P=1.0)1037.31.01018.771.4100R5-2RLuc mRNA + LP-5 (N / P=1.2)1044.71.21022.467.7100R5-3RLuc mRNA + LP-5 (N / P=1.4)1052.21.41026.163.9100R5-4RLuc mRNA + LP-5 (N / P=1.6)1059.61.61029.860.2100R5-5RLuc mRNA + LP-5 (N / P=1.8)1067.11.81033.656.5100R5-6RLuc mRNA + LP-5 (N / P=2.0)1074.52.01037.352.8100R6-1RLuc mRNA + LP-6 (N / P=1.0)1037.31.01018.771.4100R6-2RLuc mRNA + LP-6 (N / P=1.2)1044.71.21022.467.7100R6-3RLuc mRNA + LP-6 (N / P=1.4)1052.21.41026.163.9100R6-4RLuc mRNA + LP-6 (N / P=1.6)1059.61.61029.860.2100R6-5RLuc mRNA + LP-6 (N / P=1.8)1067.11.81033.656.5100R6-6RLuc mRNA + LP-6 (N / P=2.0)1074.52.01037.352.8100.

[0105]

[0106] <2-2> Analysis of physicochemical properties of lipoplexes containing DOTAP

[0107] To determine the stability of various lipoplexes prepared with varying DOTAP ratios, cholesterol content, and N / P ratios, physicochemical properties such as size, polydispersity index (PDI), and zeta potential were analyzed. Physicochemical properties were measured and analyzed using dynamic light scattering equipment.

[0108] The size of the mRNA-liposome complex was confirmed to increase with the addition of mRNA, regardless of the presence or absence of cholesterol and the ratio of DOTAP, and to increase rapidly when the N / P ratio was 1.6 or higher (Fig. 2a).

[0109] The dispersion index (PDI) of the complex was not affected by the presence or absence of cholesterol or the ratio of DOTAP, but it was confirmed that the PDI value increased rapidly when the N / P ratio was 1.6 or higher (Fig. 2a).

[0110] The zeta potential of the complex was also not affected by the presence or absence of cholesterol or the ratio of DOTAP, but it was confirmed that liposomes without added mRNA had a value of +40 mV or higher, and a negative value when mRNA was added (Fig. 2b). In addition, when the N / P ratio was 1.8 or higher, the value was -30 mV or higher, indicating that the particles were unstable when the N / P ratio was 1.8 or higher (Fig. 2b).

[0111] Additionally, the manufactured lipoplexes were visually observed to determine transparency and the degree of aggregation formation. Similar to the previous results, it was observed that aggregation was formed when the N / P ratio was 1.6 or higher (Fig. 2c). This indicates that the stability of the lipoplexes is low when the N / P ratio is 1.6 or higher.

[0112] Visual analysis, size, dispersion, and zeta potential analysis results showed that the stability of lipoplexes containing DOTAP and DOPE was not affected by the ratio of DOTAP or the presence or absence of cholesterol, but that the N / P ratio was important, and that the particle stability was relatively high when the N / P ratio was 1.4 or less.

[0113]

[0114] <2-3> Confirmation of RNA expression in lipoplexes

[0115] To compare the function as an mRNA delivery vehicle, the level of mRNA expression was confirmed using the lipoplex manufactured in <2-1>.

[0116] Specifically, 20,000 HeLa cells or 50,000 RAW264.7 cells were seeded per well in a 96-well plate, and then transfected with RLuc. mRNA (positive control), liposomes, or lipoplexes so that each well contained 0.5 μg of mRNA. As a control, transfection was performed using lipofectamine (Lipofectamine 2000 or Lipofectamine Max). The transfected cells were harvested one day later, and the Renila luciferase assay system (Promega #E2820) was processed according to the manufacturer's protocol, and the luminescence level was measured using SpectraMax. The total protein amount of the cell lysate was measured using the BCA protein assay kit (Pierce #23225), and the expression level was compared by calculating the luminescence value per protein amount.

[0117] As a result, when comparing the N / P ratios of 1.0 to 1.4, excluding formulations in which aggregates were observed at an N / P ratio of 1.6 or higher, it was confirmed that the highest expression efficiency was achieved when the N / P ratio was 1.4 (Figs. 3a and 3b). In addition, expression efficiency showed a similar trend regardless of cell type.

[0118]

[0119] <Example 3> Analysis of lipoplex characteristics according to the type of neutral lipid

[0120] In order to compare whether there is a difference in the stability and effectiveness of lipoplexes depending on the type of neutral lipid forming the liposome, lipoplexes were manufactured using the method of Example 1 using DOTAP as the cationic lipid and DOPE or DSPC as the neutral lipid, and the physicochemical characteristics and effectiveness were evaluated.

[0121] <3-1> Manufacturing of lipoplex containing DSPC

[0122] DSPC is a helper lipid for Moderna's mRNA-1273 and BioNTech / Pfizer's BNT126b2, and is widely used in the composition of lipid nano-particles (LNPs) for mRNA delivery. DSPC is a saturated lipid, and when forming liposomes with DOTAP, an unsaturated lipid, it has the effect of lowering membrane fluidity compared to DOPE, an unsaturated lipid. Therefore, cholesterol, which plays a role in lowering membrane fluidity and increasing the Tm value, was not added to DOTAP:DSPC liposomes.

[0123] Three liposomes were formed by varying the types of lipids forming the liposomes, and CleanCap® FLuc mRNA (TriLink#L7204) was added as mRNA to prepare lipoplexes. The compositions of the three liposomes are shown in Table 3, and information on lipoplexes with different liposome types and N / P ratios is presented in Table 4.

[0124] Name compositionLP-1DOTAP:DOPE (50:50)LP-2DOTAP:DOPE (50:50):CholLP-3DOTAP:DSPC (50:50)

[0125] 시료정보Sample 제조Label조성N / P ratiomRNA(1 mg / ml)LP(2 mg / ml)BufferTotal volumeRNALPF1-1FLuc mRNA + LP-1 (N / P=1.0)10451.01022.567.5100F1-2FLuc mRNA + LP-1 (N / P=1.2)10541.21027.063.0100F1-3FLuc mRNA + LP-1 (N / P=1.4)10641.41031.558.5100F1-4FLuc mRNA + LP-1 (N / P=1.6)10721.61036.054.0100F1-5FLuc mRNA + LP-1 (N / P=1.8)10811.81040.549.5100F1-6FLuc mRNA + LP-1 (N / P=2.0)10902.01045.045.0100F2-1FLuc mRNA + LP-2 (N / P=1.0)10451.01022.567.5100F2-2FLuc mRNA + LP-2 (N / P=1.2)10541.21027.063.0100F2-3FLuc mRNA + LP-2 (N / P=1.4)10641.41031.558.5100F2-4FLuc mRNA + LP-2 (N / P=1.6)10721.61036.054.0100F2-5FLuc mRNA + LP-2 (N / P=1.8)10811.81040.549.5100F2-6FLuc mRNA + LP-2 (N / P=2.0)10902.01045.045.0100F3-1FLuc mRNA + LP-3 (N / P=1.0)1046.41.01023.266.8100F3-2FLuc mRNA + LP-3 (N / P=1.2)1055.71.21027.962.2100F3-3FLuc mRNA + LP-3 (N / P=1.4)10651.41032.557.5100F3-4FLuc mRNA + LP-3 (N / P=1.6)1074.31.61037.252.9100F3-5FLuc mRNA + LP-3 (N / P=1.8)1083.61.81041.848.2100F3-6FLuc mRNA + LP-3 (N / P=2.0)1092.92.01046.543.6100

[0126]

[0127] <3-2> Analysis of physicochemical properties of lipoplexes containing DSPC

[0128] The size, dispersion, and zeta position of the lipoplex prepared as in Example 2 were measured, and the stability was evaluated by visually observing the suspension containing the lipoplex.

[0129] As a result, similar to Example 2, the size and dispersion of the complex increased depending on the N / P ratio regardless of the type of neutral lipid, and a stable size was maintained when the N / P ratio was 1.4 or lower (Fig. 4a), and the zeta localization value increased as the N / P ratio increased (Fig. 4b). In addition, the visual analysis results also showed that aggregates tended to be observed when the N / P ratio was 1.6 or higher (Fig. 4c).

[0130] This shows that the type of neutral lipid does not have a significant correlation with particle stability, and that particle stability varies depending on the N / P ratio. In particular, instability was confirmed when the N / P ratio was 1.6 or higher.

[0131] <3-3> Confirmation of RNA expression efficiency according to differences in neutral lipids

[0132] As performed in Example 2, the mRNA expression efficiency of lipoplexes containing DOPE and lipoplexes containing DSPC was compared using HeLa cells or RAW264.7 cells.

[0133] As a result, it was confirmed that the mRNA expression efficiency was higher when the lipoplex manufactured using DOPE was used than when the lipoplex manufactured using DSPC was used, and this result did not differ depending on the cell type, and it was confirmed that the addition of cholesterol to the lipoplex manufactured using DOPE did not affect the mRNA expression efficiency (Fig. 5a and Fig. 5b).

[0134]

[0135] <3-4> Confirmation of stability from serum according to differences in neutral lipids

[0136] Lipoplexes containing DOPE or DSPC were transfected into HeLa cells and cultured in serum-supplemented culture medium for 24 hours. The stability of the lipoplexes in the presence of serum was then determined by measuring luminescence levels. Serum stability was calculated using the following formula.

[0137]

[0138] As a result, it was confirmed that the lipoplex containing DSPC had a significantly reduced protective effect against serum, and the lipoplex containing DOPE also had a stability of less than 80% in the presence of serum. It was confirmed that the stability of the lipoplex containing DOPE could be relatively increased by adding cholesterol (Fig. 6).

[0139] Therefore, regardless of the type of neutral lipid, a stable size and dispersion were maintained when the N / P ratio was 1.0 to 1.4, and it was found that the type of neutral lipid did not affect particle formation and mRNA expression efficiency, but there may be a significant difference in the protective effect against serum.

[0140]

[0141] <Example 4> Analysis of lipoplex characteristics according to the type of cationic lipid

[0142] We confirmed that the type of neutral lipid does not affect particle formation and effectiveness, but may affect serum protective effect. Therefore, we used DOPE as a neutral lipid and wanted to determine whether the type of cationic lipid affects the stability and effectiveness of the complex.

[0143] <4-1> Lipoplex production according to cationic lipid type

[0144] The type of neutral lipid was fixed to DOPE, and the type of cationic lipid and the type of cholesterol were varied, and the ratio of cationic lipid and neutral lipid was mixed at 1:1, and 20% cholesterol was added, and liposomes were prepared by the method described in Example 1. Then, mRNA was added at an N / P ratio of 1.0, 1.2, or 1.4 to prepare mRNA-liposome complexes (lipoplexes). The cationic lipids used here were DDAB (18:0 DDAB), 14:0 TAP, 18:0 TAP, or DOTAP (18:1 TAP), and the characteristics of each lipid are as described in Table 5.

[0145] ChargeSaturationLipidsTm (℃)MW (g / mol)+SaturatedDDAB (18:0 DDAB)-630.952+Saturated14:0 TAP-590.361+Saturated18:0 TAP-702.574+UnsaturatedDOTAP (18:1 TAP)-11.9698.542

[0146]

[0147] Since saturated lipids have high Tm values, bath-type sonication was performed at 50°C for 20 minutes after hydration during the liposome manufacturing process, and a mini-extruder and a 200 nm PC membrane were used to homogenize the liposomes.

[0148]

[0149] <4-2> Confirmation of physicochemical properties of lipoplexes according to cationic lipid type

[0150] The size, dispersion, and zeta position of the lipoplex were measured through light scattering analysis as described in Example 2.

[0151] As a result, similar to Example 2, when mRNA was added, the size and dispersion of the complex increased, and when the N / P ratio was 1.4, the lipoplexes under 14:0 TAP, DDAB, and DDAB+cholesterol conditions were found to aggregate (Fig. 7a). When mRNA was added, the zeta localization value changed to a negative value (Fig. 7b).

[0152] Liposomes manufactured using cationic lipids were observed as cationic nanoparticles with a size of 90 to 150 nm and a dispersity of less than 0.3, and the complex with mRNA was observed to be stable with a size of less than 200 nm (dispersity of less than 0.3) and a zeta potential of less than -30 mV when the N / P ratio was 1.0 to 1.4.

[0153] Through this, it can be seen that, unlike neutral lipids, there is a difference in stability depending on the type of cationic lipid, and, as in the results of Example 2, stability is impaired when the N / P ratio increases above a certain level.

[0154] Additionally, when DDAB was used, it was found that liposomes with a positive charge of 130 nm were formed even without cholesterol, indicating that they were highly stable.

[0155]

[0156] <4-3> Confirmation of RNA expression efficiency according to the type of cationic lipid

[0157] As performed in Example 2, the mRNA expression efficiency of lipoplexes with different types of cationic lipids, presence or absence of cholesterol, and N / P ratios was compared using HeLa cells or RAW264.7 cells.

[0158] As a result, it was confirmed that the expression efficiency of lipoplexes containing cationic lipids was high, equivalent to or higher than that of lipofectamine (Figs. 8a and 8b). In particular, high expression efficiency was observed when lipoplexes prepared using DOTAP or DDAB were used.

[0159]

[0160] <Example 5> Analysis of lipoplex characteristics containing Pam3CSK4

[0161] In the case of DOTAP, cholesterol needs to be included as an unsaturated lipid for stability, so DDAB, not DOTAP, was used as a cationic lipid, and DOPE was used as a neutral lipid. However, to increase the efficacy as a vaccine, lipoplexes were manufactured by adding lipopeptide Pam3CSK4, an adjuvant.

[0162] <5-1> Manufacturing of lipoplex containing Pam3CSK4

[0163] A cationic lipid (18:0 DDAB) and a neutral lipid (DOPE) were mixed in a 1:1 ratio, and Pam3CSK4 was added to prepare liposomes according to the method described in Example 1. mRNA was added to the prepared liposomes at different N / P ratios to prepare mRNA-liposome complexes (lipoplexes).

[0164]

[0165] <5-2> Confirmation of physicochemical properties of lipoplex containing Pam3CSK4

[0166] The size, dispersion, and zeta position of the lipoplex were measured through light scattering analysis as described in Example 2.

[0167] As a result, similar to Example 2, when mRNA was added, the size and dispersion of the complex increased, and it was confirmed that aggregation occurred when the N / P ratio was 1.6 or higher (Fig. 9a). When mRNA was added, the zeta percentile value changed to a negative value, and it was confirmed that as the N / P ratio increased, the zeta percentile value approached 0 (Fig. 9b).

[0168] In addition, as the ratio of Pam3CSK4 increases, the size and dispersion of the complex rapidly increase even at low N / P ratios, and it can be confirmed that the zeta localization value approaches 0 (Fig. 9a and Fig. 9b).

[0169] Through this, we can see that an increase in the ratio of Pam3CSK4 affects stability.

[0170]

[0171] <5-3> Confirmation of RNA binding level in lipoplex containing Pam3CSK4

[0172] Since the lipoplex was manufactured in a form that adsorbs mRNA to the outside of the liposome, the degree of mRNA binding may vary depending on the type of liposome-forming material. Therefore, to compare the degree of mRNA adsorption according to the presence or absence or amount of Pam3CSK4 added, the manufactured lipoplex was subjected to electrophoresis on a 1.25% agarose gel and the nucleic acid band was observed to confirm the amount of mRNA.

[0173] As a result, it was confirmed that free mRNA was observed when Pam3CSK4 was included, and that the adsorption characteristics increased as the N / P ratio increased, and that the adsorption characteristics became stronger as the amount of Pam3CSK4 increased (Fig. 10).

[0174]

[0175] <5-4> Confirmation of RNA expression efficiency of lipoplex containing Pam3CSK4

[0176] As performed in Example 2, the effectiveness of lipoplexes according to the Pam3CSK4 ratio was evaluated by comparing the mRNA expression efficiency of lipoplexes containing different amounts of Pam3CSK4 using HeLa cells.

[0177] As a result, it was confirmed that the expression efficiency of the lipoplex containing Pam3CSK4 increased as the N / P ratio increased, but the expression efficiency decreased as the amount of Pam3CSK4 increased (Fig. 11).

[0178] However, it was found that lipoplexes containing 20 µg to 100 µg of Pam3CSK4 per 2 mg of lipid and containing mRNA at an N / P ratio of 1.0 to 1.4 showed an effect similar to that of lipofectamine (Fig. 11).

[0179]

[0180] <Example 6> In vitro efficacy confirmation of lipoplex containing Pam3CSK4

[0181] Based on the results of Example 5, a formulation of a lipoplex containing Pam3CSK4 was selected, and a lipoplex under the selected conditions was manufactured to evaluate its physicochemical properties and efficacy.

[0182] <6-1> Manufacturing of lipoplex containing Pam3CSK4

[0183] The ratio of cationic lipid (18:0 DDAB) and neutral lipid (DOPE) was mixed at 1:1, Pam3CSK4 was added at 50 µg / ml or 100 µg / ml, and the N / P ratio was fixed at 1.2, and liposomes were prepared by the method described in Example 1. Depending on the amount of Pam3CSK4, they were named CVI-LPX-1 or CVI-LPX-2, and the information for each is as shown in Table 6, and the structure of the formed lipoplex is as shown in Fig. 12a.

[0184] NameCation lipidNeutral lipidMixed ratioAdjuvantN / P ratioCVI-LPX-1DDAB(18:0 Dimethyldioctadecylammonium bromide)DOPE[18:1 (49-Cis) PE]1:1Pam3CSK450 μg / ml1.2CVI-LPX-2DDAB(18:0 Dimethyldioctadecylammonium bromide)DOPE[18:1 (49-Cis)PE]1:1Pam3CSK4100 μg / ml1.2

[0185] <6-2> Characterization of lipoplexes containing Pam3CSK4

[0186] The lipoplex manufactured in <6-1> was analyzed by light scattering analysis to measure the size, dispersion, and zeta position of the lipoplex, thereby confirming its physicochemical properties.

[0187] As a result, both formulations had a size of 120 to 160 nm, a dispersity of less than 0.3, and a zeta potential of 30 mV or more, confirming that the particles were stable (Figs. 12b and 12c).

[0188]

[0189] To evaluate the mRNA adsorption level of the lipoplex, electrophoresis was performed as described in Example 6. In addition, the stability of the adsorbed mRNA was evaluated by treating it with Triton X-100.

[0190] As a result, CVI-LPX-1 was measured to have an mRNA binding level of 56.1±2.9%, and CVI-LPX-2 was measured to have an mRNA binding affinity of 57.4±1.3%, confirming that both formulations had an mRNA binding affinity of 40 to 70% and that mRNA degradation did not occur (Fig. 13a and Fig. 13b).

[0191]

[0192] After transfecting HEK293 cells with CVI-LPX-1 and CVI-LPX-2 conjugated with OVA mRNA, cells were harvested one day later, and Western blotting for OVA was performed with the resulting cell lysates. As a result, it was found that both CVI-LPX-1 and CVI-LPX-2 exhibited expression efficiencies similar to those of lipofectamine (Fig. 14).

[0193]

[0194] <Example 7> In vivo efficacy of lipoplex containing Pam3CSK4

[0195] <7-1> Confirmation of immunogenicity of lipoplex containing Pam3CSK4

[0196] To evaluate the in vivo efficacy of the lipoplexes (CVI-LPX-1 and CVI-LPX-2) manufactured in Example 6, C57BL / 6 (male, 5 weeks old) mice were prepared and administered intramuscular injection (IM) once a week with the negative control (vehicle), OVA-mRNA, and lipoplex groups adjusted in dosage to contain 5 μg of mRNA. A total of three immunizations were performed, and one week later, the formation level of OVA-specific antibodies and the number of IFNγ-secreting spleen cells were measured.

[0197] Blood was transferred to a microtube, centrifuged at 5,500 rpm at 4°C for 5 minutes, and the supernatant was transferred to a new microtube to obtain plasma. Then, OVA antigen was diluted to 1 μg / mL using PBS (phosphate buffered saline), dispensed into a plate at 100 μl / well, covered with a sealing film, and left overnight at 4°C to coat the OVA antigen. Then, the plate was washed and blocked according to the ELISA kit manufacturer's protocol, and the obtained plasma was diluted and ELISA (Enzyme-linked immune) was performed to measure the absorbance.

[0198] As a result, it was confirmed that when OVA-mRNA was administered alone, no OVA-specific antibodies were formed, but when it was administered in the form of a lipoplex, a large number of OVA-specific antibodies were formed (Figure 15a).

[0199]

[0200] Additionally, enzyme-linked immune absorbent spot (ELISpot) assay was performed to determine the number of IFNγ-secreting cells. Anti-IFNγ was used as the capture antibody, and spleens were isolated from mice to obtain spleen cells, which were then used for analysis.

[0201] As a result, in the group administered mRNA alone, no spleen cells secreting IFNγ were observed, but in the group administered in the form of lipoplex, the number of spleen cells secreting IFNγ significantly increased (Figures 15b and 15c).

[0202]

[0203] In addition, FACS was performed to analyze quantitative changes in specific cell populations using whole blood from mice that had completed three rounds of immunization.

[0204] As a result, the number of total T cells and CD8+ T cells significantly increased in the group administered lipoplexes (2 types of CVI-LPX) compared to the negative control group and the mRNA-only administration group (Fig. 16a and Fig. 16b).

[0205] The results of analyzing the number of MHC class II macrophages also confirmed that the number of MHC class II macrophages significantly increased (more than 2.5 times) in the lipoplex administration group (Figures 16c and 16d).

[0206] The analysis results for CD86, a marker of M1 type macrophages, also showed that the number of M1 macrophages significantly increased in the lipoplex-administered group (Fig. 16e and Fig. 16f).

[0207]

[0208] To determine the level of T cell immune response activity within an individual following immunization, plasma cytokine concentrations were measured using ELISA. As in the previous experiment, plasma was isolated from mice after three immunizations, and the experiment was performed according to the ELISA kit protocol.

[0209] As a result, the mRNA-only treatment group showed no significant difference from the negative control group, but in the lipoplex-treated group, the secretion of IL-12 and INFγ significantly increased, and in the CVI-LPX-2 administration group, the concentration of IL-2 was also observed to be high (Fig. 17a).

[0210] The results of measuring the concentration of inflammatory cytokines using the plasma of the same mice showed that, as in the previous results, there was no difference between the mRNA-only treatment group and the negative control group, but in the group administered lipoplex, the secretion of IL-1β was confirmed to increase to a significant level (Fig. 17b).

[0211] This shows that lipoplexes can enhance not only humoral immunity but also cellular immunity.

[0212]

[0213] <7-2> Confirmation of anticancer efficacy of lipoplex containing Pam3CSK4

[0214] C57BL / 6 (male, 5 weeks old) mice were prepared and administered intramuscular injection (IM) with the negative control group (vehicle), OVA-mRNA, and lipoplex groups once a week. A total of three immunizations were performed, and 1 week later, B16F10 mouse melanoma cells expressing OVA (1 × 10 per mouse) were injected. 5 Tumor cells) were mixed with Matrigel in a 1:1 ratio to make a total volume of 100 µl and injected subcutaneously into the right thigh of mice. The degree of tumor formation was observed one day after the injection of tumor cells, and the time point at which tumor tissue was formed was recorded.

[0215] As a result, tumors were formed on average on the 3rd day in the negative control group and the mRNA-only administration group, but tumors were formed on average on the 27th day in the lipoplex administration group (Fig. 18 and Table 7).

[0216] Group average tumor formation time (days) Non-administered group 3Vehicle 3mRNA only 3CVI-LPX-127CVI-LPX-227

[0217] This suggests that immunization using lipoplexes can prevent tumor formation and exhibit anticancer efficacy.

[0218]

[0219] <Example 8> Safety verification of lipoplex containing Pam3CSK4

[0220] To determine whether there was a toxic reaction due to lipid-based delivery vehicles, we administered the vehicle to animals and observed the toxic reaction.

[0221] Specifically, immunization was performed three times as in the previous example, but the body weights of the mice were measured one week before the start of immunization, and the weight measurements were repeated every week together with the immunization. One week after the three immunizations were performed, whole blood was collected through heart puncture, and the plasma was separated by centrifugation for 5 minutes at 5,500 rpm and 4℃. The separated plasma was used to measure the levels of ALT, AST, and bilirubin, which are markers related to liver damage.

[0222] As a result, it can be confirmed that the body weight change of the lipoplex administration group was no different from that of the negative control group (vehicle) or mRNA only treatment group (Fig. 19a), and that there was no difference in ALT, AST, and bilirubin levels (Figs. 19b, 19c, and 19d).

[0223] Through this, it can be seen that no toxic reaction occurs due to the lipid-based carrier manufactured in the present invention, and it can be inferred that CVI-LPX-1 and CVI-LPX-2 are safe.

[0224]

[0225] <Example 9> Preparation of a freeze-dried formulation of a lipoplex containing Pam3CSK4

[0226] CVI-LPX-1, a lipoplex manufactured with the composition described in Table 6, was lyophilized to prepare a lyophilized formulation. A lyophilized vial containing 250 μl of CVI-LPX-1 was placed on a lyophilizer shelf, and a lyophilized formulation was prepared through the process described in Table 8.

[0227] Protocol Pre-freezing SV01 SV02 SV03 SV04 SV05 SV06 SV07 SV08 Temp. (℃) -40 - 40 - 30 - 20 - 100 - 102 - 20 mTorr Atm 00000000 Time (min) 360 - 120 - 120 - 360 - 360 - 360 - 120 - 120 - 9999

[0228] As a result of observing the shape with the naked eye, it was confirmed that the cake was well formed without collapse, whether it was a liposome alone or a lipoplex, which is an mRNA-liposome complex (Fig. 20a).

[0229] Lyophilized liposomes or lipoplexes were rehydrated and compared with non-lyophilized liquid formulations of liposomes or lipoplexes in terms of size, particle dispersity index (PDI), and zeta position.

[0230] As a result, in the case of liposomes, it was confirmed that they were unstable, with the size increasing to more than 100 nm and the dispersity increasing to more than 0.3 after lyophilization, but in the case of lipoplexes (CVI-LPX-1), the size increased to about 20 nm and the dispersity was maintained at 0.2 or less, so it was found that they still maintained stability (Fig. 20b). The zeta localization value was 30 mV or more for liposomes regardless of whether they were lyophilized, and -30 mV or less for lipoplexes regardless of whether they were lyophilized, indicating that stable particles were formed (Fig. 20c).

[0231]

[0232] Electrophoresis was performed to measure the degree of mRNA binding using lyophilized lipoplexes, and the results also confirmed that even after lyophilization, the level of mRNA binding was similar and stable without damage to the mRNA (Fig. 20d).

[0233]

[0234] Additionally, expression levels were measured by transfecting HeLa cells with lipoplexes containing CleanCap® FLuc mRNA (TriLink#L7204) and observing the level of luminescence.

[0235] As a result, the lyophilized lipoplex showed an expression efficiency equal to or greater than that of the liquid formulation lipoplex (Fig. 20e).

[0236] Through this, it was confirmed that the lipoplex manufactured from the present invention can be used not only in a liquid formulation but also in a lyophilized formulation.

[0237]

[0238] The present invention synthesizes a complex (lipoplex) by mixing cationic and neutral lipids to form liposomes and binding nucleic acids to them, demonstrating that this complex can efficiently induce intracellular or in vivo nucleic acid delivery. Furthermore, it was confirmed that lipoplexes can efficiently deliver and immunize nucleic acids expressing target antigens. Therefore, it can be seen that lipoplexes can be effectively utilized in various nucleic acid-based vaccines by varying the target antigen.

[0239]

[0240]

Claims

1. As a composition for nucleic acid delivery, Nucleic acid to be delivered to the subject; and A composition comprising a liposome formed from a mixture of a cationic lipid and a neutral lipid.

2. In paragraph 1, A composition for nucleic acid delivery, wherein the cationic lipid is at least one selected from the group consisting of DOTAP (1,2-Dioleoyl-3-Trimethylammonium-Propane), 18:0 TAP (1,2-stearoyl-3-Trimethylammonium-Propane), 14:0 TAP (1,2-dimyristoyl)-3-Trimethylammonium-Propane), and 18:0 DDAB (Dimethyldioctadecylammonium Bromide Salt).

3. In paragraph 1, A composition for nucleic acid delivery, wherein the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

4. In paragraph 1, A composition for nucleic acid delivery, wherein the liposome further comprises a lipopeptide-based immunostimulant.

5. In paragraph 4, A composition for nucleic acid delivery, wherein the lipopeptide-based immune enhancer is at least one selected from the group consisting of Pam3-CSKKKK, PHC-SKKKK, Ole2PamCys-SKKKK, Pam2Cys-SKKKK, PamCys(Pam)-SKKKK, Ole2Cys-SKKKK, Myr2Cys-SKKKK, PamDhc-SKKKK, Pam-CSKKKK, Dhc-SKKKK and FSL-1.

6. In paragraph 1, A composition for delivering a nucleic acid, wherein the nucleic acid encodes a peptide or protein that can act as an immunogen in a subject.

7. In paragraph 1, A composition for delivering a nucleic acid, wherein the nucleic acid is DNA, mRNA (messenger RNA), circRNA (circular RNA), or saRNA (self-amplifying RNA).

8. In paragraph 1, A composition for nucleic acid delivery, wherein the N / P ratio of the liposome and nucleic acid is 0.6 to 1.

4.

9. In paragraph 1, A nucleic acid delivery composition, wherein the nucleic acid delivery composition forms a complex in the form of particles.

10. A vaccine comprising a nucleic acid delivery composition according to any one of claims 1 to 9 as an active ingredient.

11. An anticancer vaccine comprising a nucleic acid delivery composition according to any one of claims 1 to 9 as an active ingredient.

12. A method for preventing or treating cancer, comprising administering a pharmaceutically effective amount of a nucleic acid delivery composition according to any one of claims 1 to 9.

13. Use of a nucleic acid delivery composition according to any one of claims 1 to 9 in the manufacture of an anticancer vaccine.

14. A method for inducing an immune response, comprising administering to a subject a nucleic acid delivery composition according to any one of claims 1 to 9.

15. A method for preparing a nucleic acid delivery composition according to any one of claims 1 to 9, comprising the step of mixing a liposome formed from a mixture of a cationic lipid and a neutral lipid and a nucleic acid.

16. In paragraph 15, A method for producing a composition for delivering nucleic acids, wherein the method comprises mixing cationic lipids and neutral lipids to produce liposomes and mixing nucleic acids therein.

17. In paragraph 15, A method for producing a composition for delivering nucleic acid, wherein the method further comprises a step of mixing a lipopeptide-based immunostimulant.

18. In paragraph 17, A method for producing a composition for nucleic acid delivery, wherein the lipopeptide-based immune enhancer is at least one selected from the group consisting of Pam3-CSKKKK, PHC-SKKKK, Ole2PamCys-SKKKK, Pam2Cys-SKKKK, PamCys(Pam)-SKKKK, Ole2Cys-SKKKK, Myr2Cys-SKKKK, PamDhc-SKKKK, Pam-CSKKKK, Dhc-SKKKK and FSL-1.

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