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WO2025089649A1 - Composition de transfert d'acides nucléiques comprenant des liposomes cationiques et son utilisation - Google Patents

Composition de transfert d'acides nucléiques comprenant des liposomes cationiques et son utilisation Download PDF

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WO2025089649A1
WO2025089649A1 PCT/KR2024/014773 KR2024014773W WO2025089649A1 WO 2025089649 A1 WO2025089649 A1 WO 2025089649A1 KR 2024014773 W KR2024014773 W KR 2024014773W WO 2025089649 A1 WO2025089649 A1 WO 2025089649A1
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nucleic acid
composition
skkkk
mrna
lipoplex
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Korean (ko)
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염정선
안병철
전은영
오유정
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Cha Vaccine Research Institute Co Ltd
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Cha Vaccine Research Institute Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to lipoplexes for delivery of nucleic acids such as DNA and/or RNA, and more particularly, to a composition for delivery of nucleic acids comprising cationic lipid-based liposomes and a method for preparing the same.
  • Gene therapy and genetic vaccines are technologies that have already been proven and are generally applied in the medical field, and can be used to treat not only genetic diseases but also autoimmune diseases, infectious diseases, cancer or tumor-related diseases, and inflammatory diseases.
  • Genetic vaccines began to be developed when it was reported that when DNA and RNA encoding target genes are directly injected into animals, the target genes are expressed in living animals, and immunity is possible through this expression (Wolff JA et al. Science, 247:1465-8, 1990).
  • RNA when used as a tool for gene administration, RNA does not require transcription, so it can synthesize proteins directly in the cytoplasm without having to enter the nucleus like DNA, and there is no concern that it will be inserted into the cell chromosome and cause unintended gene damage.
  • RNA vaccine When a typical RNA vaccine is delivered into a cell, it is activated for a short period of time to express the target protein, and is destroyed by an enzymatic reaction within a few days, and the specific immune response to the expressed target antigen (protein) remains.
  • RNA is vulnerable to nuclease digestion in plasma.
  • 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 attempted to block RNA degradation in plasma and promote cellular uptake of oligonucleotides.
  • lipid-based carriers such as liposomes or lipid nanoparticles
  • mRNA is usually adsorbed to the outside or encapsulated inside.
  • mRNA when mRNA is adsorbed to the outside, it is known that it generally exists as an aggregate of liposome and nucleic acid rather than a single liposome, and since the adsorption capacity differs depending on the combination of lipids, the state of nucleic acid, and the ratio of nucleic acid to liposome, and stability is also affected, optimization of this is necessary.
  • Korean Patent Publication No. 10-2022-0126235 discloses that an mRNA delivery composition can be prepared using liposomes containing DOTAP, a cationic lipid, DOPE, a neutral lipid, and cholesterol.
  • DOTAP and cholesterol can be replaced and stability can be maintained by changing the type of cationic lipid.
  • the purpose is to induce an immune response by delivering a nucleic acid material, a method for improving such immunogenicity is necessary.
  • the inventors of the present invention performed 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.
  • the purpose of the present invention is to provide a nucleic acid delivery platform technology using cationic liposomes with excellent safety and stability.
  • 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.
  • Another object of the present invention is to provide a vaccine comprising the nucleic acid delivery composition as an active ingredient.
  • Another object of the present invention is to provide an anticancer vaccine comprising the nucleic acid delivery composition as an active ingredient.
  • 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.
  • the present invention relates to a composition for nucleic acid delivery comprising a cationic liposome, and a complex (lipoplex) is formed by mixing a liposome prepared by mixing a cationic lipid and a neutral lipid with a nucleic acid.
  • the lipoplex prepared in this way constitutes a particle with high stability, effectively introduces a target nucleic acid into cells, and a high expression rate of the target nucleic acid is observed.
  • an immune activation response through the lipoplex of the present invention can be enhanced by preparing the liposome by including an immunostimulant such as a lipopeptide, and when a nucleic acid for a target antigen expressed on a tumor cell is included in the lipoplex, tumor formation is observed to be delayed, confirming that it can have efficacy not only as an infectious disease vaccine but also as an anticancer vaccine.
  • an immunostimulant such as a lipopeptide
  • Figure 1 is a schematic diagram showing the process of manufacturing a lipoplex.
  • 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, the presence or absence of cholesterol, or the N/P ratio
  • Figure 2b shows the results of zeta-potential values
  • Figure 2c shows the results of visual observation.
  • PDI polydispersity index
  • 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
  • Figure 3b shows the results using the RAW264.7 cell line.
  • Figure 4 shows the results of evaluating the stability of lipoplexes manufactured using DOTAP and DOPE or DSPC.
  • Figure 4a shows the results of particle size and polydispersity index (PDI) according to the type of neutral lipid, presence or absence of cholesterol, or N/P ratio
  • Figure 4b shows the results of zeta-potential values
  • Figure 4c shows the results of visual observation.
  • PDI polydispersity index
  • Figure 5 shows the results of confirming the in vitro expression efficiency using lipoplexes manufactured using DOTAP and DOPE or DSPC.
  • Figure 5a shows the results using the HeLa cell line
  • Figure 5b shows the results using the RAW264.7 cell line.
  • Figure 6 shows the results of evaluating the stability of lipoplexes manufactured using DOTAP and DOPE or DSPC, confirming the stability of lipoplexes in the presence of serum.
  • Figure 7 shows the results of evaluating the stability of lipoplexes manufactured using different types of cationic lipids.
  • Figure 7a shows the results of particle size and polydispersity index (PDI) according to the type of cationic lipid, presence or absence of cholesterol, or N/P ratio
  • Figure 7b shows the results of zeta-potential values.
  • PDI polydispersity index
  • Figure 8 shows the results of confirming the in vitro expression efficiency using lipoplexes manufactured with different types of cationic lipids.
  • Figure 9 shows the results of evaluating the stability of lipoplexes manufactured by mixing Pam3CSK4 with DDAB and DOPE.
  • Figure 9a shows the results of particle size and polydispersity index (PDI) according to the amount of Pam3CSK4 or N/P ratio, and
  • Figure 9b shows the results of zeta-potential values.
  • PDI polydispersity index
  • Figure 10 shows the results comparing the mRNA adsorption levels for liposomes prepared by mixing Pam3CSK4 with DDAB and DOPE.
  • Figure 11 shows the results of confirming the in vitro expression efficiency using a lipoplex prepared by mixing Pam3CSK4 with DDAB and DOPE.
  • Figure 12 shows the results of confirming the stability of liposomes or lipoplexes (CVI-LPX-1 and CVI-LPX-2) manufactured with DDAB and DOPE, Pam3CSK4.
  • Figure 12a is a schematic diagram showing the morphology of the lipoplex
  • Figure 12b shows the size and dispersion of each particle
  • Figure 12c shows the zeta fraction value.
  • 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
  • Figure 13b shows the results under conditions including Triton X-100.
  • Figure 14 shows the results of confirming the in vitro expression efficiency of CVI-LPX-1 and CVI-LPX-2.
  • 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
  • Figure 15c is a graph analyzing the results of Figure 15b.
  • 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 their analysis
  • Figures 16c and 16d are graphs showing the results of measuring the proportion of MHCII macrophages and their analysis
  • Figures 16e and 16f are graphs showing the results of measuring the proportion of M1 macrophages and their analysis.
  • 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
  • Figure 17b is a graph measuring the level of cytokine secretion involved in the inflammatory response.
  • Figure 18 is a graph summarizing the time required for tumor formation in mice injected with B16F10 mouse melanoma expressing OVA.
  • 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
  • Figure 19d shows the results of measuring the concentration of bilirubin in mice after lipoplex administration.
  • 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
  • Figure 20c shows the results of measuring the zeta position value after re-dissolving in solution after lyophilization.
  • Figure 20d shows the results of mRNA adsorption level in lyophilized formulation
  • Figure 20e shows the results of in vitro expression level in Hela cells.
  • the present invention provides a composition for nucleic acid delivery comprising liposomes based on a mixture of cationic lipids and neutral lipids.
  • the above cationic lipids include lipids that continuously exhibit cationic properties without being affected by pH changes or ionic lipids that are converted to cationic properties by pH changes.
  • the cationic lipids are 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), dimethyldioctadecylammonium bromide salt (DDAB), 3 ⁇ -[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 Ethyl 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,
  • DOTAP is a cationic lipid having a structure represented by the following chemical formula 1.
  • the neutral lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3 phosphorylcholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), It may be 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.
  • DOPE 1,2-
  • the above DOPE is a neutral lipid having a structure represented by the following chemical formula 3.
  • the above DSPC is a neutral lipid having a structure represented by the following chemical formula 4.
  • the above liposome may be formed by containing 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.
  • the liposome may further comprise a lipopeptide.
  • the lipopeptide may be composed of a fatty acid and several amino acids bound to a glycerol molecule.
  • the number of fatty acids in the glycerol molecule or 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 in the form of a part of a molecule derived from a gram-positive or gram-negative bacterium or mycoplasma or in the form of a whole molecule.
  • 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 ⁇ g/dose.
  • the lipopeptide may be present 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 mixture of cationic lipids and neutral lipids.
  • the nucleic acid included in the above composition encodes a peptide or protein capable of acting as an immunogen, and the nucleic acid may be DNA, mRNA (messenger RNA), circRNA (circular RNA), or saRNA (self-amplifying RNA).
  • mRNA messenger RNA
  • circRNA circular RNA
  • saRNA self-amplifying RNA
  • the mRNA may 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 consisting of two or more immunogens, for example a fusion protein consisting of two or more epitopes, peptides or proteins derived from the same or different viral proteins, wherein the epitopes, peptides or proteins may be connected by a linker sequence.
  • 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 alterations, such as structural alterations of nucleotides.
  • An artificial mRNA molecule may be designed and/or produced by genetic engineering methods corresponding to a desired artificial sequence of nucleotides (heterologous sequence).
  • the mRNA may exhibit modifications that increase its resistance to in vivo degradation (e.g., degradation by exo- or endo-nucleases) and/or to in vitro degradation (e.g., during the manufacturing process prior to vaccine administration, e.g., during the manufacturing 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 modifications.
  • Stabilization of the RNA may also be achieved by chemical modifications or by modifying the G/C content of the nucleic acid.
  • Various other methods are known in the art and may be applied to the present invention.
  • the above circRNA is a type of single-stranded RNA, and unlike the widely known linear RNA, it is a continuous closed loop structure formed by reverse splicing during the splicing process. Due to this structure, it has no 5' or 3' end, so it is resistant to nucleic acid degradation by exonuclease and is highly stable.
  • the above saRNA is an RNA that has the property of amplifying itself when introduced into a host cell, and increases RNA production by amplifying multiple times within the cell and produces target peptides and/or proteins within the cell. Due to this property, it is likely to exhibit higher production and can maintain an immune response for a long time, and is thus attracting attention in the vaccine field.
  • 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.
  • 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.
  • the present invention provides a vaccine comprising the nucleic acid delivery composition as an active ingredient.
  • the present invention provides an anticancer vaccine comprising the nucleic acid delivery composition as an active ingredient.
  • 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.
  • the above prevention means any act of inhibiting the occurrence of the above-described disease or delaying its progression by administering the pharmaceutical composition according to the present invention.
  • the above treatment means any action in which the symptoms of the above-described disease are improved or beneficially changed by administering the pharmaceutical composition according to the present invention.
  • the pharmaceutical composition may comprise in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • the pharmaceutical composition may comprise a buffer, such as neutral buffered saline, phosphate buffered saline, citric acid buffered solution, and the like; a carbohydrate, such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid, such as glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative.
  • a buffer such as neutral buffered saline, phosphate buffered saline, citric acid buffered solution, and the like
  • a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol
  • a protein such as glycine
  • a chelating agent such as EDTA
  • 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.
  • the above pharmaceutical composition is administered in a pharmaceutically effective amount.
  • the pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective amount can be determined based on the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field.
  • the above pharmaceutical composition can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.
  • the present invention provides a method for treating a disease comprising a step of administering a pharmaceutically effective amount of the nucleic acid delivery composition described above.
  • the above disease may be selected from the group consisting of cancer, tumor, autoimmune disease, genetic disease, inflammatory disease, viral infection and bacterial infection.
  • the above 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.
  • liposomes were formed using a mixture of cationic lipids and neutral lipids, and liposomes were added to an mRNA solution to prepare lipoplexes, which are mRNA-liposome complexes (Fig. 1).
  • the N/P ratio rather than the ratio between cationic lipids and neutral lipids, whether cholesterol was added, 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).
  • PDI polydispersity index
  • Figs. 7 and 8 it was confirmed that there may be differences in stability with respect to serum depending on the type of neutral lipid
  • the liposome suspension prepared in ⁇ 1-1> was added to the mRNA solution at a ratio of 1:1, and left on ice for 30 minutes to form a complex of mRNA and liposomes.
  • the liposome formation and complex preparation method is roughly shown in Fig. 1.
  • Liposomes were formed using DOTAP as a cationic lipid and DOPE as a neutral lipid, to which cholesterol was added, and the stability and effectiveness of the lipoplexes according to the ratio of DOTAP and the ratio between mRNA and liposomes were confirmed.
  • 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 added (Table 1).
  • Lipoplexes were prepared by adding mRNA (CleanCap ® RLucmRNA, TriLink#L7204) to these six types of liposomes at different ratios of liposomes and mRNA.
  • the N/P ratio which is the mixing ratio of liposomes and mRNA, was calculated using the following formula.
  • composition ratio and manufacturing capacity are as shown in Table 2.
  • physicochemical properties such as size, polydispersity (PDI), and zeta potential of lipoplexes were analyzed. Physicochemical properties were measured and analyzed using dynamic light scattering equipment.
  • the size of the mRNA-liposome complexes 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).
  • the polydispersity 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).
  • 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).
  • the manufactured lipoplexes were visually observed to determine transparency and the degree of aggregation formation, and 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 shows that the stability of the lipoplexes is low when the N/P ratio is 1.6 or higher.
  • the level of mRNA expression was confirmed using the lipoplex manufactured in ⁇ 2-1>.
  • 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 ug of mRNA.
  • transfection was performed using lipofectamine (Lipofectamine 2000 or Lipofectamine Max) for comparison.
  • 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. Then, the total protein amount of the cell lysate was measured using a BCA protein assay kit (Pierce #23225), and the expression level was compared by calculating the luminescence value versus the protein amount.
  • lipoplexes were prepared 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.
  • DSPC is a helper lipid for Moderna's mRNA-1273 and BioNTech/Pfizer's BNT126b2, and is widely used when forming lipid nano-particles (LNPs) for mRNA delivery. Since DSPC is a saturated lipid, it has the effect of lowering membrane fluidity compared to DOPE, an unsaturated lipid, when forming liposomes with DOTAP, an unsaturated lipid. Therefore, cholesterol, which lowers membrane fluidity and increases the Tm value, was not added to DOTAP:DSPC liposomes.
  • 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 shown in Table 4.
  • composition LP-1 DOTAP:DOPE (50:50)
  • LP-2 DOTAP:DOPE 50:50
  • Chol LP-3 DOTAP:DSPC 50:50
  • the size, dispersion, and zeta position of the lipoplexes manufactured as in Example 2 were measured, and the stability was evaluated by visually observing the suspension containing the lipoplexes.
  • the type of neutral lipid does not have a significant correlation with the stability of the particle, and that the stability of the particle varies depending on the N/P ratio. In particular, it was confirmed that it was unstable when the N/P ratio was 1.6 or higher.
  • Example 2 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.
  • Lipoplexes containing DOPE or DSPC were transfected into HeLa cells and cultured for 24 hours using culture medium containing serum. The stability of the lipoplexes in the presence of serum was then confirmed by checking the luminescence level. Serum stability was calculated using the following formula.
  • the type of neutral lipid was fixed to DOPE, and the types of cationic lipid and cholesterol were varied, 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 are 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.
  • the size, dispersion, and zeta position of lipoplexes were measured by light scattering analysis as described in Example 2.
  • Liposomes manufactured using cationic lipids were observed as cationic nanoparticles with a size of 90 to 150 nm and a polydispersity of less than 0.3, and the complex with mRNA was observed to be stable with a size of less than 200 nm (polydispersity 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.
  • Example 2 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.
  • the expression efficiency of the lipoplex containing cationic lipids was high, equivalent to or higher than that of lipofectamine (Figs. 8a and 8b).
  • the expression efficiency was observed to be high when the lipoplex prepared using DOTAP or DDAB was used.
  • DOTAP since it was necessary to include cholesterol as an unsaturated lipid for stability, DDAB, not DOTAP, was used as a cationic lipid, and DOPE was used as a neutral lipid.
  • the lipoplex was manufactured by adding the lipopeptide Pam3CSK4, which is an adjuvant.
  • the cationic lipid (18:0 DDAB) and neutral lipid (DOPE) were mixed in a 1:1 ratio and Pam3CSK4 was added to prepare liposomes using the method described in Example 1.
  • mRNA was added to the prepared liposomes at different N/P ratios to prepare mRNA-liposome complexes (lipoplexes).
  • the size, dispersion, and zeta position of lipoplexes were measured by light scattering analysis as described in Example 2.
  • 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 material forming the liposome. Therefore, in order 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.
  • Example 2 As performed in Example 2, the mRNA expression efficiency of lipoplexes containing different amounts of Pam3CSK4 was compared using HeLa cells to evaluate the effectiveness of lipoplexes according to the Pam3CSK4 ratio.
  • lipoplexes containing 20 ⁇ g to 100 ⁇ g of Pam3CSK4 per 2 mg of lipid and mRNA at an N/P ratio of 1.0 to 1.4 showed a similar level of effectiveness as lipofectamine (Fig. 11).
  • Example 5 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.
  • the ratio of cationic lipid (18:0 DDAB) and neutral lipid (DOPE) was mixed at a 1:1 ratio, Pam3CSK4 was added at 50 ⁇ g/ml or 100 ⁇ g/ml, and the N/P ratio was fixed at 1.2, and liposomes were prepared by the method described in Example 1.
  • 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.
  • the lipoplex manufactured in ⁇ 6-1> was analyzed using light scattering analysis to measure the size, dispersion, and zeta position of the lipoplex, thereby confirming its physicochemical properties.
  • both formulations had a size of 120 to 160 nm, a dispersion of less than 0.3, and a zeta potential of 30 mV or more, confirming that the particles were stable (Figs. 12b and 12c).
  • CVI-LPX-1 and CVI-LPX-2 were prepared and the negative control group (vehicle), OVA-mRNA, and lipoplex groups were administered via intramuscular injection (IM) once a week at doses adjusted to contain 5 ⁇ g of mRNA.
  • IM intramuscular injection
  • Plasma Blood was transferred to a microtube, centrifuged for 5 minutes at 5,500 rpm, 4°C, and the supernatant was transferred to a new microtube to obtain plasma. Then, OVA antigen was diluted to 1 ⁇ g/mL using PBS (phosphate buffered saline), 100 ⁇ l/well was dispensed onto the plate, covered with a sealing film, and incubated at 4°C overnight to coat the OVA antigen. Then, plate washing and blocking were performed according to the protocol of the ELISA kit manufacturer, and the obtained plasma was diluted to perform ELISA (Enzyme-linked immune) and measure the absorbance.
  • PBS phosphate buffered saline
  • ELISPOT enzyme-linked immune absorbent spot
  • FACS was performed to analyze quantitative changes in specific cell populations using whole blood from mice that had been immunized three times.
  • 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).
  • the concentration of cytokines in plasma was measured using ELISA.
  • plasma of mice was separated after three immunizations and the experiment was performed according to the protocol of the ELISA kit.
  • 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 INF ⁇ significantly increased, and in the CVI-LPX-2 administration group, the concentration of IL-2 was also observed to be high (Fig. 17a).
  • lipoplexes can enhance not only humoral immunity but also cellular immunity.
  • mice C57BL/6 (male, 5 weeks old) mice were prepared and administered the negative control group (vehicle), OVA-mRNA, and lipoplex groups via intramuscular injection (IM) once a week.
  • IM intramuscular injection
  • a total of 3 immunizations were performed, and 1 week later, B16F10 mouse melanoma cells expressing OVA (1 ⁇ 10 5 cells per individual) were mixed 1:1 with Matrigel to prepare a total volume of 100 ⁇ l and injected subcutaneously into the right thigh of the mice. The degree of tumor formation was observed one day after the injection of tumor cells, and the time point of tumor tissue formation was recorded.
  • tumors were formed on average in the negative control group and mRNA-only administration group on day 3, whereas tumors were formed on average in the lipoplex administration group on day 27 (Fig. 18 and Table 7).
  • Non-administered group 3 Vehicle 3 mRNA only 3 CVI-LPX-1 27 CVI-LPX-2 27
  • 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.
  • whole blood was collected through heart puncture, and the plasma was separated by centrifugation for 5 minutes at 5,500 rpm and 4°C. The separated plasma was used to measure the levels of ALT, AST, and bilirubin, which are markers related to liver damage.
  • 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 it could be confirmed that there was no difference in the ALT, AST, and bilirubin levels (Figs. 19b, 19c, and 19d).
  • 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 ul of CVI-LPX-1 was placed on a lyophilizer shelf, and a lyophilized formulation was prepared through the process described in Table 8.
  • 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.
  • PDI particle dispersity index
  • Electrophoresis was performed to measure the degree of mRNA binding using lyophilized lipoplexes, and the results also confirmed that the mRNA binding level was similar and stable without damage to the mRNA even after lyophilization (Fig. 20d).
  • the lyophilized lipoplex showed an expression efficiency equal to or greater than that of the liquid formulation lipoplex (Fig. 20e).
  • the lipoplex manufactured from the present invention can be used not only in a liquid formulation but also in a lyophilized formulation.
  • the present invention formed a liposome by mixing cationic lipids and neutral lipids, and manufactured a complex (lipoplex) by binding nucleic acid thereto, and confirmed that this can efficiently induce nucleic acid delivery within a cell or in a living body.
  • nucleic acid for expressing a target antigen can be efficiently delivered and immunized by using lipoplexes, and therefore it can be seen that it can be effectively used in various vaccines using nucleic acids by varying the target antigen.

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Abstract

La présente invention concerne une composition de transfert d'acides nucléiques comprenant des liposomes cationiques, un complexe (lipoplexe) étant formé par un mélange de liposomes préparés au moyen d'un mélange de lipides cationiques et de lipides neutres avec des acides nucléiques. Le lipoplexe ainsi préparé constitue des particules hautement stables, introduit efficacement un acide nucléique cible dans des cellules et présente un taux d'expression élevé de l'acide nucléique cible. De plus, il a été confirmé que, lors de la préparation des liposomes, un activateur immunitaire tel qu'un lipopeptide est inclus, ce qui permet d'améliorer une réponse d'activité immunitaire à travers le lipoplexe de la présente invention. Lorsqu'un acide nucléique destiné à un antigène cible exprimé dans des cellules tumorales est inclus dans le lipoplexe, il a été observé que la formation de tumeur se produit tardivement et, ainsi, il a été confirmé que le lipoplexe peut présenter une efficacité en tant que vaccin contre le cancer ainsi que contre une maladie infectieuse.
PCT/KR2024/014773 2023-10-26 2024-09-27 Composition de transfert d'acides nucléiques comprenant des liposomes cationiques et son utilisation Pending WO2025089649A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007505954A (ja) * 2003-09-15 2007-03-15 プロチバ バイオセラピューティクス インコーポレイティッド ポリエチレングリコール修飾脂質化合物およびその使用
KR20170005499A (ko) * 2014-06-04 2017-01-13 엑시큐어, 인크. 예방 또는 치료 용도를 위한 리포솜성 구형 핵산에 의한 면역 조절인자의 다가 전달
KR20220126235A (ko) * 2021-03-08 2022-09-15 아이진 주식회사 Rna의 체내 전달용 조성물 및 이의 제조방법
KR20230008739A (ko) * 2020-04-10 2023-01-16 바이엘 애니멀 헬스 게엠베하 면역자극 조성물
JP2023520506A (ja) * 2020-04-01 2023-05-17 ユニバーシティ オブ フロリダ リサーチ ファンデーション インコーポレーティッド Ｓａｒｓ－ｃｏｖ－２に対する多層ｒｎａナノ粒子ワクチン

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007505954A (ja) * 2003-09-15 2007-03-15 プロチバ バイオセラピューティクス インコーポレイティッド ポリエチレングリコール修飾脂質化合物およびその使用
KR20170005499A (ko) * 2014-06-04 2017-01-13 엑시큐어, 인크. 예방 또는 치료 용도를 위한 리포솜성 구형 핵산에 의한 면역 조절인자의 다가 전달
JP2023520506A (ja) * 2020-04-01 2023-05-17 ユニバーシティ オブ フロリダ リサーチ ファンデーション インコーポレーティッド Ｓａｒｓ－ｃｏｖ－２に対する多層ｒｎａナノ粒子ワクチン
KR20230008739A (ko) * 2020-04-10 2023-01-16 바이엘 애니멀 헬스 게엠베하 면역자극 조성물
KR20220126235A (ko) * 2021-03-08 2022-09-15 아이진 주식회사 Rna의 체내 전달용 조성물 및 이의 제조방법

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