Nucleic acid-encapsulating lipid nanoparticle production method

Abstract

The present invention provides a production method that makes it possible to obtain nucleic acid-encapsulating lipid nanoparticles having improved particle size distribution uniformity and improved gene expression efficiency.　A nucleic acid-encapsulating lipid nanoparticle production method according to the present invention comprises mixing a lipid solution containing a PEG-lipid, an ionizable lipid, a phospholipid, a structural lipid, and a solvent with an aqueous nucleic acid solution, wherein at least part of the solvent in the lipid solution is pyridine.

Description

Method for producing nucleic acid-encapsulated lipid nanoparticles

[0001] The present invention relates to a method for producing nucleic acid-encapsulated lipid nanoparticles. This application claims priority to Japanese Patent Application No. 2024-12999, filed with the Japan Patent Office on January 31, 2024, the entire contents of which are incorporated by reference.

[0002] Nucleic acid-encapsulated nanoparticles can be formed by encapsulating nucleic acids inside lipid nanoparticles (LNPs). For example, mRNA-encapsulated lipid nanoparticles, in which mRNA is encapsulated in lipid nanoparticles, have attracted attention in the fields of medicine and drug discovery as a technology for delivering mRNA to target cells (e.g., Patent Document 1, Non-Patent Document 1).

[0003] US Patent Application Publication No. 2018 / 0153822

[0004] PNAS 2023 Vol. 120 No. 33 e2303567120

[0005] Although the techniques for producing nucleic acid-encapsulated lipid nanoparticles are widely known, little research has been done on the solvents used to dissolve lipids. According to the inventor's research, there is room for improvement in the uniformity of particle size distribution and the gene expression efficiency of the encapsulated nucleic acid in conventional methods for producing nucleic acid-encapsulated lipid nanoparticles.

[0006] The present invention provides a production method that can yield nucleic acid-encapsulating lipid nanoparticles with improved uniformity in particle size distribution and gene expression efficiency.

[0007] The present invention has the following aspects. [1] A method for producing nucleic acid-encapsulating lipid nanoparticles, comprising mixing a lipid solution containing PEG lipids, ionized lipids, phospholipids, structured lipids, and a solvent with an aqueous nucleic acid solution, wherein at least a portion of the solvent in the lipid solution is pyridine. [2] The method according to [1], wherein a vortex or microfluidic device is used when mixing the lipid solution with the aqueous nucleic acid solution. [3] The method according to [1] or [2], further comprising removing the pyridine from the nucleic acid-encapsulating lipid nanoparticles after mixing the lipid solution and the aqueous nucleic acid solution. [4] The method according to [3], wherein the residual pyridine concentration of the nucleic acid-encapsulating lipid nanoparticles is less than 200 ppm. [5] The method according to any one of [1] to [4], wherein the aqueous nucleic acid solution contains at least one selected from the group consisting of mRNA and plasmid DNA. [6] The method according to any one of [1] to [5], wherein the structured lipid is cholesterol. [7] The method of any one of [1] to [6], wherein the number average particle diameter of the nucleic acid-encapsulating lipid nanoparticles is less than 150 nm. [8] The method of any one of [1] to [7], wherein the content of pyridine in the solvent of the lipid solution is 75% by volume or more.

[0008] According to the present invention, there is provided a production method that can obtain nucleic acid-encapsulating lipid nanoparticles with improved uniformity in particle size distribution and gene expression efficiency.

[0009] FIG. 1 shows the results of protein expression levels measured in Experiment 1. FIG. 2 shows the results of protein expression levels measured in Experiment 1. FIG. 3 shows the results of protein expression levels measured in Experiment 1. FIG. 4 shows the results of protein expression levels measured in Experiment 2. FIG. 5 shows the results of protein expression levels measured in Experiment 3. FIG. 6 shows the results of protein expression levels measured in Experiment 3. FIG. 7 shows the results of protein expression levels measured in Experiment 3. FIG. 8 shows the results of protein expression levels measured in Experiment 4. FIG. 9 shows the results of protein expression levels measured in Experiment 5. FIG. 10 shows the results of confocal laser scanning microscopy obtained in Experiment 6. FIG. 11 shows the results of GC-MS measured in Experiment 7. FIG. 12 shows the results of GC-MS measured in Experiment 7. FIG. 13 shows the results of GC-MS measured in Experiment 7. FIG. 14 shows the results of protein expression levels in mice measured in Experiment 8. FIG. 15 shows the results of protein expression levels in mice measured in Experiment 8. FIG. 16 shows the results of protein expression levels in mice measured in Experiment 8.

[0010] Ethanol has traditionally been widely used as a solvent for lipid solutions. The present inventors have investigated various solvents suitable for lipid solutions in order to improve the uniformity of the particle size distribution and gene expression efficiency of nucleic acid-encapsulated lipid nanoparticles. As a result, the present inventors have found that the use of pyridine as a lipid solution solvent improves the uniformity of the particle size distribution and gene expression efficiency of nucleic acid-encapsulated lipid nanoparticles compared to conventional techniques.

[0011] Representative embodiments of the method for producing nucleic acid-encapsulated lipid nanoparticles of the present invention will be described below. The following description is an example (representative example) of an embodiment, and the present invention is not limited to these, and can be implemented with any modifications within the scope of the gist of the present invention.

[0012] [Method for producing nucleic acid-encapsulating lipid nanoparticles] The method for producing nucleic acid-encapsulating lipid nanoparticles of the present invention comprises mixing an aqueous solution of nucleic acid with a lipid solution containing PEG lipids, ionizable lipids, phospholipids, structural lipids, and a solvent, at least a portion of which is pyridine.

[0013] (Lipid solution) The lipid solution contains PEG lipids, ionized lipids, phospholipids, structured lipids, and a solvent. However, the lipid solution may contain other components other than PEG lipids, ionized lipids, phospholipids, structured lipids, and solvents, and is not particularly limited. The other components referred to here may be various additives and are not particularly limited. For example, vitamins, peptides, aptamers, and antibodies for intracellular localization and targeting are included.

[0014] PEG lipids are lipids having a structure derived from polyethylene glycol. PEG lipids may be PEG-modified lipids. In one example, PEG lipids are also called PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol.

[0015] Examples of PEG lipids include PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, folic acid-modified PEG-DSPE, and peptide-modified PEG-DSPE. However, the PEG lipid is not limited to these examples. One type of PEG lipid may be used alone, or two or more types may be used in combination.

[0016] Commercially available PEG lipids may be used. For example, NOF Corporation's SUNBRIGHT GM-020, NOF Corporation's SUNBRIGHT DSPE-020CN, Avanti's DSPE-PEG (2000) amine, and Avanti's DSPE-PEG (2000) maleimide are examples. However, PEG lipids are not limited to these examples.

[0017] An ionizable lipid is a lipid or lipid analogue that is ionized by being protonated under acidic conditions. The ionizable lipid may be a cationic lipid. Examples of the ionizable lipid include DLin-KC2-DMA, DLin-MC3-DMA, SM-102, ALC-0315, and cKK-E12. However, the ionizable lipid is not limited to these examples. One type of ionizable lipid may be used alone, or two or more types may be used in combination.

[0018] Commercially available ionizable lipids may be used. Examples include SM-102 manufactured by BroadPharm, ALC-0315 manufactured by MedChemExpress, cKK-E12 manufactured by Echelon Biosciences, and SS-OP manufactured by NOF Corporation. However, the ionizable lipids are not limited to these examples.

[0019] Phospholipids are lipids whose structure is derived from phosphate esters. Phospholipids can assemble into one or more lipid bilayers. Generally, phospholipids can have a phospholipid moiety and one or more fatty acid moieties.

[0020] Examples of phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diunde octadecyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine Examples of phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. However, the phospholipids are not limited to these examples. One type of phospholipid may be used alone, or two or more types may be used in combination.

[0021] The structured lipid is used to stabilize the nanoparticle structure. Examples of the structured lipid include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, and α-tocopherol. However, the structured lipid is not limited to these examples. One type of structured lipid may be used alone, or two or more types may be used in combination.

[0022] The solvent is for dissolving the PEG lipid, ionized lipid, phospholipid, and structured lipid. At least a part of the solvent may be pyridine, or a solvent other than pyridine may be used in combination, or the entire solvent may be pyridine.

[0023] Examples of the solvent other than pyridine include ethanol, methanol, 2-propanol, dimethyl sulfoxide, acetone, acetonitrile, dioxane, N,N-dimethylformamide, tetrahydrofuran, and 3-methylpyridine. However, the other solvent is not limited to these examples. One type of the other solvent may be used alone, or two or more types may be used in combination.

[0024] (Aqueous solution of nucleic acid) An aqueous solution of nucleic acid is a solution in which nucleic acid such as mRNA or a plasmid is dissolved in water. The aqueous solution of nucleic acid may further contain other components such as a buffer. Details of the buffer will be described later.

[0025] Nucleic acids may be naturally occurring, synthetic, or chemically modified. Nucleic acids may be purified or unpurified. Nucleic acids may be polynucleotides.

[0026] For example, genetic information in an mRNA molecule is encoded by the sequence of nucleotide bases of the mRNA molecule, which are arranged into codons, each consisting of three nucleotide bases. Each codon encodes a specific amino acid of a polynucleotide, except for stop codons, which terminate translation (protein synthesis). In living cells, mRNA is transported to the ribosome, the site of protein synthesis, where it provides the genetic information for protein synthesis (translation). For a more detailed description, see Alberts B et al. (2007) Molecular Biology of the Cell, Fifth Edition, Garland Science.

[0027] In eukaryotes, mRNA is translated in vivo in chromosomes by the cellular enzyme RNA polymerase. During or after in vivo translation, a 5' cap is added to the 5' end of the mRNA in vivo. The 5' cap is a terminal 7-methylguanosine residue joined to the transcribed nucleotide by a 5'-5'-triphosphate linkage. In addition, most eukaryotic mRNA molecules have a polyadenylyl moiety ("poly(A) tail") at the 3' end of the mRNA molecule. In vivo, the poly(A) tail in eukaryotic cells is approximately 250 adenosine residues long, and the poly(A) tail is added post-transcriptionally.

[0028] Thus, a typical mature eukaryotic mRNA has a structure beginning at the 5' end with an mRNA cap, followed by a 5' untranslated region (5'UTR) of nucleotides, then an open reading frame beginning with a start codon which is an AUG triplet of nucleotide bases, and ending with a stop codon which may be a UAA, UAG, or UGA triplet of nucleotide bases, which is the coding sequence for the protein, followed by a 3' untranslated region (3'UTR) of nucleotides, and ending with a polyadenosine tail.

[0029] While typical mature eukaryotic mRNA is made naturally in eukaryotic cells in vivo, identical or structurally and functionally equivalent mRNAs can be made in vitro using molecular biology methods. Thus, any RNA with a structure similar to typical mature eukaryotic mRNA can function as an mRNA.

[0030] mRNA molecules generally have a size that allows them to be encapsulated in the nucleic acid-encapsulating lipid nanoparticles of the present invention. While the size of mRNA molecules can vary substantially depending on the identity of the mRNA species encoding a particular protein, the average size for an mRNA molecule can be between 500 and 10,000 bases.

[0031] The nucleic acid in the nucleic acid aqueous solution is not limited to mRNA. Other examples include siRNA, shRNA, aiRNA, miRNA, dsRNA, antisense RNA, ribozyme, catalytic DNA, RNA that induces triple helix formation, aptamer, vector, and plasmid DNA encoding these. However, the nucleic acid is not limited to these examples. One type of nucleic acid may be used alone, or two or more types may be used in combination.

[0032] (Mixing of lipid solution and nucleic acid aqueous solution) By mixing the lipid solution and the nucleic acid aqueous solution, a dispersion of nucleic acid-encapsulated lipid nanoparticles is obtained, in which nucleic acids derived from the nucleic acid aqueous solution are encapsulated. For example, by mixing using a vortex, the lipids self-organize, resulting in the production of nucleic acid-encapsulated lipid nanoparticles. For more detailed explanations, please refer to Patent Document 1 and Non-Patent Document 1.

[0033] A microfluidic device may be used when mixing a lipid solution and an aqueous nucleic acid solution. A microfluidic device has multiple flow channels on the order of micrometers, and mixes the liquids flowing through each channel at the confluence or connection point of these multiple flow channels. Commercially available microfluidic devices may be used. Examples of commercially available products include NanoAssembler from Precision NanoSystems, Reagent Droplet Chip Kit from Dolomite, KeyChem series from YMC, and iLiNP from Lilac Pharma. However, microfluidic devices are not limited to these examples.

[0034] (Removal of pyridine) After mixing the lipid solution and the nucleic acid aqueous solution, pyridine can be removed from the nucleic acid-encapsulated lipid nanoparticles.The method of removing pyridine is not particularly limited.For example, various methods well known to those skilled in the art, such as drying removal, chromatographic separation, dialysis, ultrafiltration, gel filtration, etc., can be mentioned, but are not limited thereto.

[0035] (Properties of nucleic acid-encapsulating lipid nanoparticles) From the viewpoint of uniformity, the polydispersity index (PDI) of the nucleic acid-encapsulating lipid nanoparticles is preferably 0.3 or less, more preferably 0.2 or less, and even more preferably 0.1 or less. The PDI of the nucleic acid-encapsulating lipid nanoparticles is a value determined by dynamic light scattering.

[0036] The number average particle diameter of the nucleic acid-encapsulating lipid nanoparticles is not particularly limited, and may be, for example, 1 to 1000 nm, 50 to 200 nm, 70 to 150 nm, etc. The number average particle diameter of the nucleic acid-encapsulating lipid nanoparticles is a value determined by dynamic light scattering.

[0037] The residual pyridine concentration of the nucleic acid-encapsulated lipid nanoparticles is preferably less than 200 ppm, more preferably less than 100 ppm, even more preferably less than 50 ppm, particularly preferably less than 10 ppm, and most preferably below the detection limit. If the residual pyridine concentration is less than 200 ppm, it is believed that the standard value of the guidelines in various countries and regions can be met.

[0038] The nucleic acid-encapsulating lipid nanoparticles obtained by the production method of the present invention are obtained in the form of a dispersion liquid, which may further contain water and other additives in addition to the nucleic acid-encapsulating lipid nanoparticles.

[0039] For example, the dispersion of nucleic acid-encapsulated lipid nanoparticles may further contain pharmaceutically acceptable additives. Pharmaceutically acceptable additives include, but are not limited to, diluents, dispersants, granulating agents, disintegrating agents, fillers, lubricants, liquid vehicles, binders, surfactants, isotonic agents, thickeners, emulsifiers, buffers, lubricants, and preservatives. One type of additive may be used alone, or two or more types may be used in combination.

[0040] Examples of diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, and powdered sugar. However, the diluents are not limited to these examples. One type of diluent may be used alone, or two or more types may be used in combination.

[0041] Examples of granulating agents and dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, wood products, natural sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM (registered trademark)), sodium lauryl sulfate, and quaternary ammonium compounds. However, the granulating agents and dispersing agents are not limited to these examples. One type of granulating agent or dispersing agent may be used alone, or two or more types may be used in combination.

[0042] Examples of emulsifiers include acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, carboxypolymethylene, polyacrylic acid, acrylic acid polymer, carrageenan, sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, polyoxyethylene sorbitan monolaurate [TWEEN (registered trademark) 20], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, and docusate sodium, although the emulsifier is not limited to these examples. The emulsifiers may be used alone or in combination of two or more.

[0043] Examples of buffers include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, amino-sulfonic acid buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free distilled water, isotonic saline, Ringer's solution, and ethyl alcohol. However, the buffer is not limited to these examples. A single buffer may be used, or two or more may be used in combination.

[0044] (Mechanism of action) In one embodiment described above using one example, at least a part of the solvent of the lipid solution is pyridine. Therefore, the uniformity of the particle size distribution of the nucleic acid-encapsulated lipid nanoparticles and the gene expression efficiency encoded by the nucleic acid are improved. Here, gene expression typically refers to protein expression, but is not limited to this. For example, when the nucleic acid is a plasmid DNA, it may be the expression of various RNAs such as miRNA, shRNA, siRNA, tRNA, etc.

[0045] (Advantages and Applications) The advantages offered by the method for producing nucleic acid-encapsulated lipid nanoparticles of the present invention are as follows: - A dispersion of nucleic acid-encapsulated lipid nanoparticles with a narrow particle size distribution can be obtained. - The efficiency of gene expression encoded by the plasmid or mRNA encapsulated in the lipid nanoparticles is improved. - Because pyridine and ethanol can be used in combination as solvents for the lipid solution, the barrier to application to already established methods that use ethanol is low.

[0046] Examples of applications of nucleic acid-encapsulated lipid nanoparticles are shown below. However, the applications of nucleic acid-encapsulated lipid nanoparticles are not particularly limited to the following descriptions. - The method for producing nucleic acid-encapsulated lipid nanoparticles of the present invention can be applied to the production of mRNA vaccines and mRNA pharmaceuticals. - The method for producing nucleic acid-encapsulated lipid nanoparticles of the present invention can also be suitably applied to the field of protein function analysis.

[0047] [Experimental Results] Experiments conducted by the inventors and their results will be described in detail below, but the present invention is not limited to the following description.

[0048] (Explanation of Terms) The terms used in the following description of the experimental results have the following meanings. PDI: Polydispersity index SM-102: Ionized lipid (BroadPharm product, model number: BP-25499) ALC-0315: Ionized lipid (Echelon Biosciences product, model number: N-1020) DLin-MC3-DMA: Ionized lipid (BroadPharm product, model number: BP-25497) DOPC: Phospholipid (NOF Corporation, model number: MC-8181) DSPC: Phospholipid (NOF Corporation, model number: MC-8080) DOPE: Phospholipid (NOF Corporation, model number: ME-8181) DMG-PEG2000: PEG lipid (NOF Corporation, model number: GM-020) Fluorescent dye DiD: Carbocyanine dye (Setareh Biotech product, model number 6330) PBS: Phosphate buffered saline A549 cells: Riken BRC product, model number RCB3677 purchased. HepG2 cells: Riken BRC product, model number RCB164 purchased.

[0049] (Experiment 1) In Experiment 1, we screened for solvents superior to ethanol in the preparation of mRNA-encapsulated lipid nanoparticles. We measured the properties of the mRNA-encapsulated lipid nanoparticles obtained using various solvents and the amount of protein expressed in vitro. The detailed experimental method is described below.

[0050] A lipid mixture (SM-102 / DOPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5 (molar ratio)) was dissolved in various organic solvents at a concentration of 1.45 mM to prepare a lipid solution. Furthermore, firefly luciferase mRNA (SEQ ID NO: 1) was dissolved in 50 mM citrate buffer (pH 3) at a concentration of 12 μg / mL to prepare an mRNA aqueous solution. The lipid solution and mRNA aqueous solution were mixed at a volume ratio of 1:2.5 using a vortex mixer. Subsequently, mRNA-encapsulated lipid nanoparticles were purified by dialysis against phosphate-buffered saline (pH 7.5). The particles in the mixture were analyzed using dynamic light scattering. Furthermore, the mRNA encapsulation rate was measured by a fluorimetric method (QuantiFluor RNA System).

[0051] After diluting with medium to a RNA concentration of 200 ng / mL, mRNA-encapsulated lipid nanoparticles were added to A549 cells and HepG2 cells. After 24 hours of culture, protein extraction was performed to measure the amount of firefly luciferase expression. The results are shown in Table 1. A549 cells were cultured in D-MEM medium containing 10% fetal bovine serum at 37°C and 5% CO. ２ HepG2 cells were cultured for 24 hours under the conditions below. HepG2 cells were cultured in D-MEM medium containing 10% fetal bovine serum at 37°C and 5% CO. ２ The cells were cultured under the conditions for 24 hours and then used.

[0052]

[0053] The number average particle size (Size), PDI, and mRNA encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles are shown in Table 1. PDI is smaller with pyridine than with ethanol. This confirms that the use of pyridine resulted in mRNA-encapsulated lipid nanoparticles with improved uniformity of particle size distribution.

[0054] The amounts of luciferase expressed in A549 cells and HepG2 cells are shown in Figures 1, 2, and 3. The highest amount of expression was achieved when pyridine was used as the solvent.

[0055] (Experiment 2) In Experiment 2, mRNA-encapsulated lipid nanoparticles were prepared using various ionized lipids currently in clinical use, and the usefulness of pyridine as a solvent was confirmed. The detailed experimental method is described below.

[0056] A lipid mixture (ionized lipid / DOPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5 (molar ratio)) was dissolved in ethanol and pyridine at a concentration of 1.45 mM to prepare a lipid solution. SM-102, ALC-0315, and DLin-MC3-DMA were used as the ionized lipids. Except for these, mRNA-encapsulated lipid nanoparticles were prepared under the same conditions as in Experiment 1, and then the properties and protein expression levels were evaluated. The results are shown in Table 2 and Figure 4.

[0057]

[0058] The mRNA-encapsulated lipid nanoparticles prepared using pyridine have a smaller PDI. This confirmed that the use of pyridine improves the uniformity of the particle size distribution of mRNA-encapsulated lipid nanoparticles. Figure 4 shows the luciferase expression levels in A549 cells and HepG2 cells. In both ionized lipids, the mRNA-encapsulated lipid nanoparticles prepared using pyridine showed higher luciferase expression levels.

[0059] (Experiment 3) In Experiment 3, the relationship between the pyridine concentration of the lipid solution and the function of mRNA-encapsulating lipid nanoparticles was mainly evaluated. The detailed experimental method is described below.

[0060] A lipid mixture (SM-102 / phospholipid / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5 (molar ratio)) was dissolved in a mixed solvent at a concentration of 1.45 mM. The solvent was evaluated for five pyridine ratios: 0 vol% (ethanol:pyridine = 100:0), 25 vol% (ethanol:pyridine = 75:25), 50 vol% (ethanol:pyridine = 50:50), and 100 vol% (ethanol:pyridine = 0:100). DOPC, DSPC, and DOPE were used as phospholipids. After preparing mRNA-encapsulated lipid nanoparticles under the same conditions as in Experiment 1, their properties and protein expression levels were evaluated. The results are shown in Table 3, Figures 5, 6, and 7, respectively. Figure 5 shows the measurement results when DOPC was used as the phospholipid. Figure 6 shows the measurement results when DSPC was used as the phospholipid. FIG. 7 shows the measurement results when DOPE was used as the phospholipid.

[0061]

[0062] In any lipid, the higher the pyridine concentration, the lower the PDI. From this, it was confirmed that the higher the pyridine concentration of the lipid solution, the more uniform the particle size distribution of mRNA-encapsulated lipid nanoparticles. In the case of DSPC, when the pyridine concentration exceeded 75% by volume, the encapsulation rate decreased. As shown in Figures 5, 6 and 7, in any lipid, DOPC, DSPC, DOPE, the higher the pyridine concentration of the lipid solution, the higher the luciferase expression level.

[0063] (Experiment 4) In Experiment 4, the usefulness of pyridine in the solvent dilution method using a microfluidic device was evaluated. The detailed experimental method is described below.

[0064] A lipid mixture (ionized lipid / DOPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5 (molar ratio)) was dissolved in ethanol and pyridine at a concentration of 1.45 mM to prepare a lipid solution. SM-102, ALC-0315, and DLin-MC3-DMA were used as ionized lipids. A firefly luciferase mRNA aqueous solution, prepared under the same conditions as in Experiment 1, was used as the nucleic acid aqueous solution. The lipid solution and mRNA aqueous solution were filled into a syringe and delivered to a glass microchannel using a syringe pump. The delivery conditions were as follows: total flow rate: 1 mL / min, flow rate ratio: 1:2.5 (lipid solution:mRNA aqueous solution). The delivered solution was stirred with a stirrer and then dialyzed against phosphate-buffered saline (pH 7.5) to produce mRNA-encapsulated lipid nanoparticles. The properties and protein expression levels were evaluated under the same conditions as in Experiment 1. The results are shown in Table 4 and FIG.

[0065]

[0066] The mRNA-encapsulated lipid nanoparticles prepared using pyridine have a smaller PDI. This confirmed that the use of pyridine improves the uniformity of the particle size distribution of mRNA-encapsulated lipid nanoparticles. Figure 8 shows the luciferase expression levels in A549 cells and HepG2 cells. In both ionized lipids, the mRNA-encapsulated lipid nanoparticles prepared using pyridine showed higher luciferase expression levels. These results confirmed the usefulness of pyridine in the solvent dilution method using a microfluidic device.

[0067] (Experiment 5) In Experiment 5, the usefulness of pyridine in preparing lipid nanoparticles encapsulating plasmid DNA was confirmed. The detailed experimental method is described below.

[0068] A lipid mixture (SM-102 / DOPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5 (molar ratio)) was dissolved in ethanol or pyridine at a concentration of 1.45 mM to prepare a lipid solution. Furthermore, firefly luciferase expression plasmid DNA (SEQ ID NO: 2) was dissolved in 50 mM citrate buffer (pH 3) at a concentration of 12 μg / mL to prepare an aqueous plasmid DNA solution. The lipid solution and the mRNA aqueous solution were mixed by vortexing at a volume ratio of 1:2.5, and then dialyzed against phosphate-buffered saline (pH 7.5) to produce lipid nanoparticles encapsulating plasmid DNA. The properties and protein expression levels were evaluated under the same conditions as in Experiment 1. The results are shown in Table 5 and Figure 9.

[0069]

[0070] The PDI of the plasmid DNA-encapsulated lipid nanoparticles prepared using pyridine was smaller. This confirmed that the use of pyridine improved the uniformity of the particle size distribution of the plasmid DNA-encapsulated lipid nanoparticles. Figure 9 shows the luciferase expression levels in A549 cells and HepG2 cells. In both ionized lipids, the mRNA-encapsulated lipid nanoparticles prepared using pyridine showed higher luciferase expression levels. These results confirmed the usefulness of pyridine as a solvent in the preparation of plasmid DNA-encapsulated lipid nanoparticles.

[0071] (Experiment 6) In Experiment 6, the efficiency of protein expression by mRNA-encapsulated lipid nanoparticles was qualitatively evaluated using a confocal laser microscope. The detailed experimental method is described below.

[0072] A lipid mixture (SM-102 / DOPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5 (molar ratio)) and the fluorescent dye DiD were dissolved in ethanol or pyridine at a concentration of 1.45 mM to prepare a lipid solution. Furthermore, tdTomato mRNA (SEQ ID NO: 3) was dissolved in 50 mM citrate buffer (pH 3) at a concentration of 12 μg / mL to prepare an mRNA aqueous solution. Subsequently, mRNA-encapsulated lipid nanoparticles encapsulating tdTomato mRNA were produced under the same conditions as in Experiment 1. The mRNA-encapsulated lipid nanoparticles were added to A549 and HepG2 cells, respectively. The localization of tdTomato and DiD fluorescence was then observed using a confocal laser microscope. The results are shown in Figure 10.

[0073] As shown in FIG. 10, the mRNA-encapsulated lipid nanoparticles prepared using pyridine showed a higher expression level of tdTomato mRNA.

[0074] (Experiment 7) In Experiment 7, the amount of residual pyridine was measured by GC-MS. The detailed experimental method is described below.

[0075] A lipid mixture (SM-102 / DOPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5 (molar ratio)) was dissolved in pyridine to prepare a lipid solution. Except for this, mRNA-encapsulated lipid nanoparticles were prepared under the same conditions as in Experiment 1. After dialysis (1 / 100,000,000) against PBS, the RNA concentration was concentrated to 100 μg / mL by ultrafiltration. After further dilution 10-fold with PBS, the pyridine concentration was measured by GC-MS. The results are shown in Figures 11, 12, and 13.

[0076] As shown in Figures 11, 12, and 13, the pyridine concentration was lower than the limit of quantitation (5 ppm). This result confirmed that pyridine can be easily removed from mRNA-encapsulated lipid nanoparticles. It was confirmed that by properly removing pyridine, the residual pyridine concentration can be reduced to below the guideline standard value. This suggests the possibility of increasing the concentration of mRNA-encapsulated lipid nanoparticles in the final pharmaceutical formulation.

[0077] (Experiment 8) In Experiment 8, the amount of protein expressed in mice using mRNA-encapsulated lipid nanoparticles prepared using pyridine was evaluated. The detailed experimental method is described below.

[0078] mRNA-encapsulated lipid nanoparticles encapsulating firefly luciferase mRNA (SEQ ID NO: 1) were prepared under the same conditions as in Experiment 4. The mRNA-encapsulated lipid nanoparticles were administered intravenously to mice (RNA dose: 60 μg / kg). Separately, the mRNA-encapsulated lipid nanoparticles were administered intramuscularly to mice (RNA dose: 625 ng). Six hours later, organs were removed and the expression level of firefly luciferase in each organ was measured. The results of intravenous administration are shown in Figure 14. The results of intramuscular administration are shown in Figure 15. Furthermore, mRNA-encapsulated lipid nanoparticles encapsulating secretory Gaussia luciferase mRNA (SEQ ID NO: 4) were prepared under the same conditions. These mRNA-encapsulated lipid nanoparticles were administered intravenously to mice under the same conditions. The expression level of luciferase in plasma at each time point was then measured. The results are shown in Figure 16.

[0079] As shown in Figures 14 and 15, mRNA-encapsulated lipid nanoparticles prepared using pyridine also showed high luciferase expression levels in mouse organs. As shown in Figure 16, mRNA-encapsulated lipid nanoparticles prepared using pyridine also showed high luciferase expression levels when secreted into the blood. These results confirmed that mRNA-encapsulated lipid nanoparticles prepared using pyridine can also improve protein expression levels in vivo.

[0080] (Sequence Designation) The sequences used in the above experiments are shown below, where Ψ is N1-Methyl-Pseudouridine.

[0081]

[0082]

[0083]

[0084] Sequence number 4: GAGAAAAAACAA CAAAAAAAAAHHAAAAHCHAAAAHAAAAAAAHHAAAAAAAHHAHCAHHHGACCH - GGG - ΨCAAA@HHHHCAAHHHCAAHHH"HBAAAHAHCAAAAHHGG"HHCHBAAAHHHAAAHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH AAHCCCHCGAGCHGGΨACΨGAAHGAGAAAHGGGGHGAGAHHHAACHA"HGGGΨACAAAHHGHAAAHAHHAHAHAAAA CΨCΨGCHAHHCCAGGHGGGAHHGGGGGLGHGGGGHGGGGHGGGAGHHHGGGHGGGAA AΨAC

[0085] According to the present invention, there is provided a production method that can obtain nucleic acid-encapsulating lipid nanoparticles with improved uniformity in particle size distribution and gene expression efficiency.

Claims

1. A method for producing nucleic acid-encapsulated lipid nanoparticles, comprising mixing an aqueous solution of nucleic acid with a lipid solution containing PEG lipids, ionizable lipids, phospholipids, structural lipids, and a solvent, wherein at least a portion of the solvent in the lipid solution is pyridine.

2. The method of claim 1, wherein a vortex or a microfluidic device is used to mix the lipid solution and the aqueous nucleic acid solution.

3. The method of claim 1, further comprising removing the pyridine from the nucleic acid-encapsulated lipid nanoparticles after mixing the lipid solution and the aqueous nucleic acid solution.

4. The method of claim 3, wherein the residual pyridine concentration of the nucleic acid-encapsulated lipid nanoparticles is less than 200 ppm.

5. The method of any one of claims 1 to 4, wherein the aqueous nucleic acid solution contains at least one selected from the group consisting of mRNA and plasmid DNA.

6. The method according to any one of claims 1 to 4, wherein the structured lipid is cholesterol.

7. The method of any one of claims 1 to 4, wherein the number average particle diameter of the nucleic acid-encapsulating lipid nanoparticles is less than 150 nm.

8. A manufacturing method according to any one of claims 1 to 4, wherein the content of pyridine in the solvent of the lipid solution is 75% by volume or more.

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