Lipid nanoparticles for nucleic acid delivery

Abstract

The present invention pertains to lipid nanoparticles which contains a nucleic acid, a cationic lipid, and a phospholipid and in which: the cationic lipid is one or more selected from the group consisting of cKK-E15, Lipid A4, AA-T3A-C12, Dlin-KC2-DMA, OF-C4-Deg-Lin, and C14-4; the phospholipid is one or more selected from the group consisting of DOPC, DOPE, and DSPC; and the molar ratio of the cationic lipid to the phospholipid content is 0.2-15.0. The present invention also pertains to a method for producing the lipid nanoparticles, the method comprising: a step for preparing a lipid solution containing the cationic lipid, the phospholipid, sterol, and PEG lipid; a step for preparing a nucleic acid solution containing a nucleic acid; and a step for mixing the lipid solution and the nucleic acid solution.

Description

Lipid Nanoparticles for Nucleic Acid Delivery

[0001] The present invention relates to lipid nanoparticles, and more particularly to lipid nanoparticles for nucleic acid delivery, which contain a cationic lipid and a phospholipid in a specific ratio. This application claims priority based on Japanese Patent Application Nos. 2024-193102, 2024-193171, 2024-193204, 2024-193259, 2024-193224, and 2024-193083, filed in Japan on November 1, 2024, and incorporates the contents thereof herein.

[0002] In recent years, RNA has attracted attention as one of the modalities in gene therapy. RNA has a low risk of integration into the genome and is safer compared to DNA. In addition, once the sequence is determined, RNA is easy to design, can target any gene, or can express any protein in vivo. Therefore, RNA can lead to safe and versatile drug discovery.

[0003] RNAs such as mRNA and siRNA (small interfering RNA) have immunogenicity, are easily degraded by enzymes, and are not taken up by cells, so simply administering them in vivo cannot exert the desired therapeutic effect. Therefore, as a means for efficiently delivering RNA into target tissues or target cells, the means of using lipid nanoparticles (Lipid Nano Particle, LNP) as a carrier has been studied.

[0004] LNPs used in gene therapy and the like are a complex of a nucleic acid as an active ingredient and a lipid that protects the nucleic acid. Examples of the nucleic acid constituting the LNP include nucleic acids such as mRNA, siRNA, antisense oligonucleotide (Antisense Oligonucleotide, ASO), and DNA. As a result of the delivery of the nucleic acid into the target tissue or target cell, the desired biological activity is exerted. In addition, LNPs are basically composed of four lipid components: a cationic lipid, a phospholipid, cholesterol, and a PEG lipid.

[0005] Cationic lipids are one of the main components of LNPs (Low Nucleoplasmic Cells). They are a type of lipid molecule that exhibits charge neutrality at physiological pH and protonates in acidic regions. Cationic lipids are classified into unsaturated, multi-tailed, and biodegradable types based on their structural characteristics. These structural characteristics contribute to the efficiency of intracellular release of nucleic acids (the payload) and the reduction of LNP cytotoxicity. Phospholipids, also known as structural helper lipids in LNPs, contribute to the effectiveness of LNP formulations by promoting membrane fusion between LNPs and cells and facilitating endosomal escape. Cholesterol, like phospholipids, is also a structural helper lipid for LNPs and contributes to promoting the binding of LNPs to cells. PEG lipids play a role in extending the circulating half-life of LNPs in vivo and also affect the size (particle diameter) of LNPs. Numerous improved and derivative forms of these four types of lipids have been created to date.

[0006] Various LNPs have been developed by using diverse lipid molecules in various combinations and ratios. For example, Patent Documents 1 and 2 disclose LNPs characterized by containing a specific combination of lipids in a specific ratio. However, the LNPs developed so far are not sufficient in terms of safety and therapeutic efficacy, and there remains a strong need for the development of novel LNPs that are safer and have higher therapeutic efficacy.

[0007] Strength Patent No. 11191849, Strength Patent No. 11684577

[0008] Lee et al., International Journal of Molecular Sciences, 2011, vol.12(5), p.3263-3287.

[0009] The object of this invention is to provide a novel LNP that can effectively exert the function of the encapsulated nucleic acid within the cell.

[0010] As a result of diligent research into the above-mentioned problems, the inventors of the present invention have found that LNPs containing a cationic lipid having a specific structure and DOPC, DOPE, or DSPC, which are types of phospholipids, in a specific ratio exhibit high cellular activity, that is, that the encapsulated nucleic acids exert high activity within cells. Based on this finding, further research has led to the completion of the present invention. In other words, the present invention is as follows.

[0011] [1] Lipid nanoparticles comprising nucleic acid, cationic lipid, and phospholipid, wherein the cationic lipid is one or more selected from the group consisting of cKK-E15, Lipid A4, AA-T3A-C12, Dlin-KC2-DMA, OF-C4-Deg-Lin, and C14-4, the phospholipid is one or more selected from the group consisting of DOPC, DOPE, and DSPC, and the molar ratio of the cationic lipid to the phospholipid content is 0.2 to 15.0. [2] Lipid nanoparticles according to [1], wherein the molar ratio of the cationic lipid to the phospholipid content is 0.4 to 12.0. [3] Lipid nanoparticles according to [1] or [2], wherein the lipid nanoparticles further comprise PEG lipid. [4] The lipid nanoparticles of [3] wherein the PEG lipid is at least one selected from the group consisting of DMG-PEG5000, DMG-PEG2000, and ALC-0159. [5] The lipid nanoparticles of any of [1] to [4] wherein the lipid nanoparticles further contain a sterol. [6] The lipid nanoparticles of [5] wherein the sterol is cholesterol. [7] The lipid nanoparticles of any of [1] to [6] wherein the lipid nanoparticles further contain a sterol and a PEG lipid, and the content ratio of the cationic lipid to the total amount of constituent lipids in the lipid nanoparticles is 5 to 65 mol%, the phospholipid is 5 to 30 mol%, the sterol is 15 to 50 mol%, and the PEG lipid is 0.5 to 3 mol%. [8] The lipid nanoparticles of [7] wherein the content ratio of the cationic lipid to the total amount of constituent lipids in the lipid nanoparticles is 10 to 60 mol%. [9] The lipid nanoparticles of [7] or [8], wherein the content ratio of the phospholipid to the total amount of constituent lipids in the lipid nanoparticles is 5 to 25 mol%.

[10] Any of the lipid nanoparticles of [7] to [9], wherein the content ratio of the cationic lipid to the total amount of constituent lipids in the lipid nanoparticles is 10 to 60 mol%, and the content ratio of the phospholipid to the total amount of constituent lipids in the lipid nanoparticles is 5 to 25 mol%.

[11] Any of the lipid nanoparticles of [1] to

[10] , wherein the nucleic acid is mRNA or siRNA.

[12] Lipid nanoparticles according to any of [1] to

[11] , wherein the average particle size of the lipid nanoparticles is 30 to 250 nm.

[13] A method for producing lipid nanoparticles according to any of [7] to

[12] , comprising the steps of: preparing a lipid solution containing the cationic lipid, the phospholipid, sterol and PEG lipid; preparing a nucleic acid solution containing nucleic acid; and mixing the lipid solution and the nucleic acid solution.

[14] The method according to

[13] , wherein the solvent in the lipid solution is an alcoholic solvent.

[15] The method according to

[14] , wherein the alcoholic solvent is ethanol.

[16] The method according to any of

[13] to

[15] , wherein the solvent in the nucleic acid solution is an acetate buffer.

[17] The method according to any of

[13] to

[16] , wherein the pH of the nucleic acid solution is 3.0 to 7.0.

[18] A pharmaceutical composition comprising lipid nanoparticles according to any of [1] to

[12] .

[0012] According to the present invention, it is possible to provide a novel LNP that can more effectively exert the function of the encapsulated nucleic acid within the cell.

[0013] The present invention will be described in detail below.

[0014] <Lipid Nanoparticles> The lipid nanoparticles of this embodiment (hereinafter sometimes referred to as "the lipid nanoparticles of the present invention" or "the LNPs of the present invention") contain nucleic acids, cationic lipids, and phospholipids, wherein the cationic lipid is one or more selected from the group consisting of cKK-E15, Lipid A4, AA-T3A-C12, Dlin-KC2-DMA, OF-C4-Deg-Lin, and C14-4, the phospholipid is one or more selected from the group consisting of DOPC, DOPE, and DSPC, and the molar ratio of the cationic lipid to the phospholipid content is 0.2 to 15. By using a combination of a specific phospholipid and a specific cationic lipid as the lipids constituting the LNPs, LNPs with excellent cell activity can be obtained. The reason why the LNPs of the present invention have excellent cell activity is not clear, but it is presumed that the sharp particle size distribution is one of the contributing factors. LNPs with extremely large or extremely small particle sizes are difficult for target cells to take up. The LNPs of the present invention have a narrow particle size distribution, resulting in fewer particles that are too small for target cells to take up, and thus are presumed to have a higher uptake efficiency by target cells.

[0015] In this specification, LNP cellular activity refers to the strength of the physiological function exerted by the nucleic acid encapsulated in the LNP after it is introduced into the cell. For example, if the nucleic acid contained in the LNP is mRNA, the LNP cellular activity is the strength of the expression of the protein encoded by the mRNA within the cell, and the higher the expression level of the protein, the stronger the cellular activity is considered to be. If the nucleic acid contained in the LNP is a functional nucleic acid for RNA interference, such as siRNA, the LNP cellular activity is the strength of the suppression of the expression of the gene targeted by the siRNA, and the lower the expression level of the protein encoded by the gene, the stronger the cellular activity is considered to be. The cellular activity of an LNP is influenced by various factors, such as the stability of the LNP until it reaches the target cell, the efficiency of LNP uptake into the target cell, and the ease with which nucleic acid is released from the LNP within the target cell (endosomal escape efficiency).

[0016] The LNP of the present invention contains nucleic acid. The nucleic acid contained in the LNP of the present invention may be DNA, RNA, or a chimeric nucleic acid of DNA and RNA. Examples of DNA include genomic DNA, cDNA, plasmid DNA, and antisense oligonucleotides (ASOs). Examples of RNA include mRNA, siRNA, miRNA, and antisense RNA. The nucleic acid contained in the LNP of the present invention may be single-stranded or double-stranded. It may also be linear or circular.

[0017] The nucleic acid contained in the LNP of the present invention may be a nucleic acid composed of natural nucleic acid bases, an artificial nucleic acid, or a nucleic acid containing both natural and artificial nucleic acid bases. Examples of artificial nucleic acids include peptide nucleic acid (PNA), LNA (Locked Nucleic Acid), and alkynyl nucleic acid. For example, the nucleic acid contained in the LNP of the present invention may be unmodified, or it may be modified or altered in any part of the nucleic acid by a known method for purposes such as nucleic acid stabilization.

[0018] In one embodiment, the nucleic acid contained in the LNP of the present invention may be contained in a vector. The term "vector" as used in this specification encompasses all types of vectors. Examples include plasmid vectors, cosmid vectors, artificial chromosome vectors (such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs)), phage vectors, or viral vectors (such as adenovirus vectors or baculovirus vectors), and any other vector known to those skilled in the art.

[0019] The LNP of the present invention contains cationic lipids. The cationic lipids contained in the LNP of the present invention are cKK-E15(3,6-bis[4-[bis(2-hydroxypentadecyl)amino]butyl]-2,5-piperazinedione, Cas number: 1432494-71-7), Lipid A4(1,1'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxydodecyl)amino)ethoxy)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethoxy)ethyl)azanediyl)bis(dodecan-2-ol), Cas number: 2639634-71-0), AA-T3A-C12(N-(3-(bis(3-(bis(2-hydroxydodecyl)amino)propyl)amino)propyl)-4-methoxybenzamide, Cas number: 2938207-23-7), Dlin-KC2-DMA (1,3-Dioxolane-4-ethanamine, N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-N,N-Dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethanamine2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane XTC), Cas number: 1190197-97-7), OF-C4-Deg-Lin(9,12-octadecadienoic acid, It is one or more compounds selected from the group consisting of (9Z,12Z)-1,1′,1′′,1′′′-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1-butanediylnitrilodi-4,1-butanediyl)] ester (Cas number: 1853203-01-6) and C14-4 (Cas number: 2639634-80-1).

[0020] Hereafter, "cKK-E15, Lipid A4, AA-T3A-C12, Dlin-KC2-DMA, OF-C4-Deg-Lin, and C14-4" may be collectively referred to as "cationic lipid A".

[0021] The LNP of the present invention contains one or more phospholipids selected from the group consisting of DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, Cas number: 4235-95-4), DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, Cas number: 4004-05-1), and DSPC (1,2-distearoyl-sn-glycero-3-phosphatidylcholine, Cas number: 816-94-4). DOPC, DOPE, and DSPC are all phospholipids that are commonly used as constituent lipids of LNPs. Cationic lipid A can be combined with any of these to produce LNPs that can effectively exert the function of the encapsulated nucleic acid within the cell. In particular, preferred LNPs of the present invention include combinations of DOPC and cKK-E15, DOPC and Lipid A4, DOPE and Lipid A4, DOPC and AA-T3A-C12, DOPC and Dlin-KC2-DMA, DOPC and OF-C4-Deg-Lin, DOPE and OF-C4-Deg-Lin, DSPC and C14-4, and DOPE and C14-4.

[0022] Hereafter, DOPC, DOPE, and DSPC may be collectively referred to as "phospholipid A."

[0023] The molar ratio of cationic lipid A and phospholipid A constituting the LNP of the present invention is usually 0.2 to 15.0 ([Ratio of cationic lipid A content to total constituent lipids of the LNP (mol%)] / [Ratio of phospholipid A content to total constituent lipids of the LNP (mol%)]) (hereinafter sometimes referred to as the "cationic lipid / phospholipid ratio"). When the LNP of the present invention contains two or more types of cationic lipid A, the "Ratio of cationic lipid A content to total constituent lipids of the LNP (mol%)" means the total content of all cationic lipid A contained in the LNP.

[0024] The LNP of the present invention exhibits superior cell activity by containing cationic lipid A and phospholipid A in the ratios within the above range. The lower limit of the cationic lipid / phospholipid ratio of the LNP of the present invention may be 0.2 or higher, 0.3 or higher, 0.4 or higher, 0.5 or higher, 0.6 or higher, 0.7 or higher, 0.8 or higher, 0.9 or higher, 1.0 or higher, 1.5 or higher, 2.0 or higher, 2.5 or higher, 3.0 or higher, 3.5 or higher, 4.0 or higher, 4.5 or higher, 5.0 or higher, 7.0 or higher, 9.0 or higher, 11.0 or higher, or 13.0 or higher. Preferably, it is 0.3 or higher, more preferably 0.4 or higher, even more preferably 0.5 or higher, and even more preferably 0.6 or higher. Furthermore, the upper limit of the cationic lipid / phospholipid ratio of the LNP of the present invention may be 15.0 or less, 14.0 or less, 13.0 or less, 12.0 or less, 11.0 or less, 10.0 or less, 9.0 or less, 8.0 or less, 7.0 or less, 6.0 or less, 5.0 or less, 4.5 or less, 4.0 or less, 3.5 or less, 3.0 or less, 2.5 or less, 2.0 or less, 1.5 or less, 1.0 or less, 0.9 or less, 0.7 or less, or 0.5 or less. Preferably, it is 14.0 or less, more preferably 13.0 or less, and even more preferably 12.0 or less. The cationic lipid / phospholipid ratio of the LNP of the present invention is preferably 0.2 to 15.0, more preferably 1.0 to 10.0, even more preferably 0.2 to 8.0, 0.2 to 6.0, 0.8 to 6.0, and even more preferably 2.0 to 6.0.

[0025] The content ratio of cationic lipid A in the LNP of the present invention is not particularly limited, as long as the cationic lipid / phospholipid ratio is within the range of 0.2 to 15.0. From the viewpoint of obtaining higher cell activity, the content ratio of cationic lipid A to the total amount of constituent lipids in the LNP of the present invention is preferably 10 to 70 mol%, more preferably 10 to 60 mol%, even more preferably 10 to 50 mol%, and still more preferably 30 to 50 mol%.

[0026] The content ratio of phospholipid A in the LNP of the present invention is not particularly limited, as long as the cationic lipid / phospholipid ratio is within the range of 0.2 to 15.0. From the viewpoint of obtaining higher cell activity, the content ratio of phospholipid A to the total amount of constituent lipids in the LNP of the present invention is preferably 5 to 30 mol%, more preferably 5 to 25 mol%, even more preferably 5 to 20 mol%, and still more preferably 5 to 15 mol%.

[0027] In the LNP of the present invention, the content ratio of cationic lipid A to phospholipid A relative to the total amount of constituent lipids is preferably 10 to 70 mol% for cationic lipid A and 5 to 30 mol% for phospholipid A, more preferably 10 to 60 mol% for cationic lipid A and 5 to 25 mol%, and even more preferably 10 to 50 mol% for cationic lipid A and 5 to 15 mol% for phospholipid A. By having the content ratio of cationic lipid A to phospholipid A within the above range, the cellular activity exhibited by the LNP can be further enhanced. In particular, when the content ratio of cationic lipid A is 30 to 50 mol% and the content ratio of phospholipid A is 5 to 15 mol%, excellent cellular activity can be achieved regardless of the composition of constituent lipids other than cationic lipid A and phospholipid A.

[0028] Furthermore, in one embodiment, the LNP of the present invention may contain lipids other than cationic lipid A and phospholipid A as constituent lipids of the LNP. Examples of such other lipids include PEG lipids and sterols.

[0029] In one embodiment, the LNP of the present invention may further contain PEG lipids (also referred to as "PEG-modified lipids"). In this specification, "PEG lipid" means any lipid modified with a PEG (polyethylene glycol) group. Examples of PEG lipids that may be included in the LNP of the present invention include 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000 (DMG-PEG5000), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2000), and Cremophor EL (CREMOPHOR Examples include, but are not limited to, polyoxyethylene sorbitan monooleate (EL), etc. In one preferred embodiment, the PEG lipid may be DMG-PEG5000, DMG-PEG2000, and ALC-0159.

[0030] In one embodiment, the LNP of the present invention may contain sterols (also referred to as "steroid alcohols"). In this specification, "sterol" means a subgroup of steroids that can be produced by plants, animals, or fungi. Examples of sterols that may be included in the LNP of the present invention include, but are not limited to, cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol, sitosterol, and stigmasterol. In one preferred embodiment, the sterol may be cholesterol.

[0031] When the LNP of the present invention contains cationic lipid A, phospholipid A, sterol, and PEG lipid, the content ratio of each lipid component to the total amount of constituent lipids in the LNP may be as follows. The lower limit of the content ratio of cationic lipid A may be 5 mol% or more, 6 mol% or more, 7 mol% or more, 8 mol% or more, 9 mol% or more, 10 mol% or more, 15 mol% or more, 20 mol% or more, 25 mol% or more, 30 mol% or more, 35 mol% or more, 40 mol% or more, or 50 mol% or more. The upper limit of cationic lipid A may be 70 mol% or less, 65 mol% or less, 60 mol% or less, 55 mol% or less, 50 mol% or less, 45 mol% or less, 40 mol% or less, 35 mol% or less, 30 mol% or less, 25 mol% or less, 20 mol% or less, 15 mol% or less, or 10 mol% or less. The content ratio of cationic lipid A is preferably 5 to 70 mol%, more preferably 10 to 65 mol%, even more preferably 10 to 60 mol%, even more preferably 10 to 50 mol%, and particularly preferably 30 to 50 mol. The lower limit of the phospholipid A content ratio may be 5 mol% or more, 6 mol% or more, 7 mol% or more, 8 mol% or more, 9 mol% or more, 10 mol% or more, 15 mol% or more, 20 mol% or more, 25 mol% or more, 30 mol% or more, or 35 mol% or more. The upper limit of the phospholipid A content ratio may be 40 mol% or less, 35 mol% or less, 30 mol% or less, 25 mol% or less, 20 mol% or less, 15 mol% or less, or 10 mol% or less. The phospholipid A content ratio is preferably 5 to 30 mol%, more preferably 5 to 25 mol%, and even more preferably 5 to 20 mol%. The lower limit of the sterol content ratio may be 15 mol% or more, 20 mol% or more, 25 mol% or more, 30 mol% or more, 35 mol% or more, 40 mol% or more, 45 mol% or more, 50 mol% or more, 55 mol% or more, 60 mol% or more, 65 mol% or more, 70 mol% or more, or 75 mol% or more. The upper limit of the sterol content ratio may be 90 mol% or less, 85 mol% or less, 80 mol% or less, 75 mol% or less, 70 mol% or less, 65 mol% or less, 60 mol% or less, 55 mol% or less, 50 mol% or less, 45 mol% or less, 40 mol% or less, 35 mol% or less, 30 mol% or less, 25 mol% or less, or 20 mol% or less.The sterol content is preferably 15 to 90 mol%, more preferably 15 to 85%, and may also be 20 to 90 mol%. The lower limit of the PEG lipid content may be 0.5 mol% or more, 1.0 mol% or more, 1.5 mol% or more, 2.0 mol% or more, 2.5 mol% or more, 3.0 mol% or more, 3.5 mol% or more, or 4.0 mol% or more. The upper limit of the PEG lipid content may be 5.0 mol% or less, 4.5 mol% or less, 4.0 mol% or less, 3.5 mol% or less, 3.0 mol% or less, 2.5 mol% or less, 2.0 mol% or less, 1.5 mol% or less, or 1.0 mol% or less. The PEG lipid content is preferably 0.5 to 5.0 mol%, more preferably 0.5 to 4.0 mol%, even more preferably 0.5 to 3.0 mol%, and even more preferably 0.5 to 2.0 mol%.

[0032] When the LNP of the present invention contains cationic lipid A, phospholipid A, sterols, and PEG lipids, higher cell activity can be obtained. Therefore, the content ratio of cationic lipid A to the total amount of constituent lipids in the LNP is preferably 10 to 70 mol%, phospholipid A to 5 to 25 mol%, sterols to 15 to 85 mol%, and PEG lipids to 0.5 to 5 mol%, and more preferably 10 to 60 mol%, phospholipid A to 5 to 20 mol%, sterols to 15 to 85 mol%, and PEG lipids to 0.5 to 5 mol%, and cationic lipids It is more preferable that the content ratio of A is 10 to 50 mol%, the content ratio of phospholipid A is 5 to 20 mol%, the content ratio of sterols is 15 to 85 mol%, and the content ratio of PEG lipids is 0.5 to 3.0 mol%, and it is more preferable that the content ratio of cationic lipid A is 10 to 50 mol%, the content ratio of phospholipid A is 5 to 20 mol%, the content ratio of sterols is 15 to 85 mol%, and the content ratio of PEG lipids is 0.5 to 5 mol%, and it is more preferable that the content ratio of cationic lipid A is 10 to 50 mol%, the content ratio of phospholipid A is 5 to 20 mol%, the content ratio of sterols is 15 to 85 mol%, and the content ratio of PEG lipids is 0.5 to 3.0 mol%. By having the content ratio of cationic lipid A and phospholipid A within the above range, the cellular activity exhibited by the LNP can be further enhanced.

[0033] The ratio of lipids to nucleic acids in the LNP of the present invention is not particularly limited as long as the desired effects of the present invention are obtained. For example, the N / P ratio (total number of positively charged ionizable lipid amine groups (N) to total number of negatively charged nucleic acid phosphate groups (P)) is usually 2 to 48, preferably 4 to 32, more preferably 4 to 12, and also preferably 2 to 10, but is not limited to these values. The N / P ratio in the LNP of the present invention may preferably be 4 to 8, more preferably 5 to 7 (for example, 6).

[0034] Furthermore, in one embodiment, the LNP of the present invention may contain components other than nucleic acids and lipids. Examples of such components include, but are not limited to, surfactants, hyaluronic acid, or derivatives thereof.

[0035] In one embodiment, the LNP of the present invention may contain a surfactant. Examples of surfactants that may be included in the LNP of the present invention include, but are not limited to, polyoxyethylene sorbitan monooleate (e.g., polysorbate 80), polyoxyethylene polyoxypropylene glycol (e.g., Pluronic F68), sorbitan fatty acid esters (e.g., sorbitan monolaurate, sorbitan monooleate), polyoxyethylene derivatives (e.g., polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol), glycerin fatty acid esters, or polyethylene glycol alkyl ethers. In one preferred embodiment, the surfactant included in the LNP of the present invention is polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester, or polyethylene glycol alkyl ether.

[0036] In one embodiment, the LNP of the present invention may further contain hyaluronic acid or a derivative thereof in addition to nucleic acids and lipids. Examples of hyaluronic acid derivatives include compounds obtained by dehydrating and condensing hyaluronic acid on the hydroxyl group of fatty acid glyceryl.

[0037] In one embodiment, the average particle diameter of the LNP of the present invention may have average particle diameters of about 30 nm to about 250 nm, about 30 nm to about 200 nm, about 30 nm to about 170 nm, about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, and about 70 nm to about 80 nm. In a preferred embodiment, the average particle diameter of the LNP of the present invention may be about 30 nm to about 200 nm.

[0038] Where used herein, the term "approximately" refers to a value similar to the reference value when applied to the value of interest. The term "approximately" means a range of values ​​that fall within ±10% of the reference value.

[0039] In one embodiment, the lower limit of the polydispersity index (PDI) of the LNP of the present invention may be 0.001 or higher, but is not limited thereto. The upper limit of the PDI of the LNP of the present invention is preferably 0.5 or lower, more preferably 0.35 or lower, even more preferably 0.30 or lower, even more preferably 0.20 or lower, particularly preferably 0.15 or lower, and also preferably 0.12 or lower.

[0040] In the present invention and this specification, the average particle size of LNP is measured by dynamic light scattering and is a volume-based D ５０ This refers to the particle size (nm). The PDI of LNPs is calculated from the particle size distribution measured by dynamic light scattering.

[0041] <Method for Producing Lipid Nanoparticles> The method for producing lipid nanoparticles according to this embodiment (hereinafter sometimes referred to as "the method for producing the present invention") is a method for producing LNPs of the present invention that include at least cationic lipid A, phospholipid A, sterol, and PEG lipid as constituent lipids, and includes the following steps: a step of preparing a lipid solution containing cationic lipid A, phospholipid A, sterol, and PEG lipid; a step of preparing a nucleic acid solution containing nucleic acid; and a step of mixing the lipid solution and the nucleic acid solution.

[0042] Cationic lipid A, phospholipid A, sterol, and PEG lipid are used to be included in the LNP of the present invention described above.

[0043] In the production method of the present invention, first, a lipid solution containing cationic lipid A, phospholipid A, sterol, and PEG lipid is prepared. Specifically, cationic lipid A, phospholipid A, sterol, and PEG lipid are contained in an organic solvent at a desired molar ratio. The organic solvent is not particularly limited as long as it can dissolve all of cationic lipid A, phospholipid A, sterol, and PEG lipid. Examples of the organic solvent include alcoholic solvents such as methanol, ethanol, propanol, isopropanol, and butanol, ester solvents such as ethyl acetate and butyl acetate, ketone solvents such as acetone, methyl ethyl ketone, and methyl isobutyl ketone, and chlorine solvents such as chloroform. Further, a mixed solvent of two or more of these organic solvents, or a mixed solvent of one or two or more of these organic solvents and water may be used. In the production method of the present invention, an alcoholic solvent or a mixed solvent of water and an alcoholic solvent is preferable, an ethanol or a mixed solvent of ethanol and water is more preferable, and ethanol is particularly preferable, from the viewpoint of relatively high safety to a living body.

[0044] Further, the nucleic acid solution in the production method of the present invention can be prepared by mixing a nucleic acid such as DNA or RNA and an aqueous solvent capable of dissolving the nucleic acid. The aqueous solvent is not particularly limited as long as the nucleic acid can be dissolved, and any solvent may be used. Examples of the aqueous solvent include water and buffer solutions (for example, citrate buffer solution, acetate buffer solution, etc.), but are not limited thereto. In a preferred embodiment, the aqueous solvent is an acetate buffer solution.

[0045] In one embodiment, the pH of the aqueous solvent used in the production method of the present invention is usually 3.0 to 10.0, preferably 3.0 to 8.0, more preferably 3.0 to 7.0, and even more preferably 3.5 to 5.5.

[0046] In the manufacturing method of the present invention, the mixing of the lipid solution and the nucleic acid solution may be carried out by any means as long as the lipid solution and the nucleic acid solution are uniformly mixed. Examples of means for mixing the lipid solution and the nucleic acid solution include, but are not limited to, pipetting and microfluidic mixing devices. In a preferred embodiment, a microfluidic mixing device is used in the manufacturing method of the present invention.

[0047] In one embodiment, the lipid solution and the nucleic acid solution are mixed using a microfluidic mixing apparatus. By using a microfluidic mixing apparatus, the lipid solution and the nucleic acid solution can be mixed quickly and thoroughly. As the microfluidic mixing apparatus used in the manufacturing method of the present invention, for example, a microfluidic mixing apparatus outlined in Non-Patent Document 1 can be used. A suitable microfluidic mixing apparatus used in the manufacturing method of the present invention is any apparatus that can mix the lipid solution and the nucleic acid solution and thereby produce the desired LNPs. One example is, but is not limited to, NanoAssemblr (manufactured by Precision Nanosystems).

[0048] In microfluidic mixing, rapid and thorough mixing is typically achieved by bringing multiple sample solutions into contact at different flow rates, thereby enhancing the diffusion effect between the flows of the sample solutions. The flow rate ratio between the two solutions can affect the size of the LNPs prepared. In one embodiment, in the mixing of a lipid solution and a nucleic acid solution, the flow rate ratio of the lipid solution to the nucleic acid solution ([flow rate of lipid solution (mL / min)] : [flow rate of nucleic acid solution (mL / min)]) is typically 1:10 to 10:1, preferably 1:2 to 3:1, but is not limited to these values.

[0049] In one embodiment, the mixture of lipid solution and nucleic acid solution mixed by a microfluidic mixing device may be subjected to downsizing or purification as needed. Such downsizing or purification can be carried out using known methods such as ultrafiltration or filtration using filters.

[0050] <Pharmaceutical composition containing lipid nanoparticles> The present invention also provides a pharmaceutical composition containing the LNP of the present invention (hereinafter sometimes referred to as "the pharmaceutical composition of the present invention").

[0051] The amount of LNP of the present invention included in the pharmaceutical composition of the present invention is not particularly limited. The lower limit of the amount of LNP of the present invention included in the pharmaceutical composition of the present invention is usually 0.01% by mass or more, preferably 0.1% by mass or more, 0.5% by mass or more, 1.0% by mass or more, 1.5% by mass or more, 2.0% by mass or more, 2.5% by mass or more, 3.0% by mass or more, 3.5% by mass or more, 4.0% by mass or more, 4.5% by mass or more, 5.0% by mass or more, 5.5% by mass or more, 6.0% by mass or more, 6.5% by mass or more, 7.0% by mass or more, 7.5% by mass or more, 8.0% by mass or more, 8.5% by mass or more, 9.0% by mass or more, or 9.5% by mass or more, but is not limited to these. Furthermore, the upper limit of the blending amount is usually 100% by mass or less, and preferably may be 99.9% by mass or less, 99.0% by mass or less, 95.0% by mass or less, 90.0% by mass or less, 85.0% by mass or less, 80.0% by mass or less, 75.0% by mass or less, 70.0% by mass or less, 65.0% by mass or less, 60.0% by mass or less, 55.0% by mass or less, 50.0% by mass or less, 45.0% by mass or less, 40.0% by mass or less, 35.0% by mass or less, 30.0% by mass or less, 25.0% by mass or less, 20.0% by mass or less, 15.0% by mass or less, or 10.0% by mass or less, but is not limited to these.

[0052] The pharmaceutical composition of the present invention may contain components other than the LNP of the present invention. Examples of such components include pharmaceutically acceptable carriers. A pharmaceutically acceptable carrier generally means an inert and non-toxic solid or liquid filler, diluent, or encapsulating material that does not react with the active ingredient. Examples of pharmaceutically acceptable carriers used in the pharmaceutical composition of the present invention include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and mixtures thereof. Furthermore, in order to bring the physical properties of the pharmaceutical composition of the present invention closer to physiological conditions, the pharmaceutical composition of the present invention may contain pharmaceutically acceptable auxiliary substances. Examples of such auxiliary substances include, but are not limited to, pH adjusters, buffers, isotonic adjusters, wetting agents, etc.

[0053] The pharmaceutical composition of the present invention may be administered orally or parenterally. Parenteral administration may include, but is not limited to, transdermal, subcutaneous, intravenous, intra-arterial, intramuscular, intraperitoneal, vaginal, and intranasal administration. The number of administrations may be a single dose or multiple doses.

[0054] The target population to which the pharmaceutical composition of the present invention can be applied is not particularly limited. Examples of target populations to which the pharmaceutical composition of the present invention can be applied include, but are not limited to, mammals (humans, chimpanzees, dogs, cats, horses, cattle, sheep, goats, rats, mice, rabbits, pigs, etc.). In a preferred embodiment, the target population may be humans.

[0055] The present invention will be described in more detail in the following examples, but the present invention is not limited in any way by these examples.

[0056] [Example 1] LNPs encapsulating mRNA were prepared using cKK-E15, Lipid A4, AA-T3A-C12, Dlin-KC2-DMA, OF-C4-Deg-Lin, and C14-4 as cationic lipids, DOPC, DOPE, or DSPC as phospholipids, cholesterol as sterols, and DMG-PEG5000, DMG-PEG2000, or ALC-0159 as PEG lipids, and their physical properties and cellular activity were investigated. The mRNA used was the mRNA encoding green fluorescent protein GFP (SEQ ID NO: 1).

[0057] (Preparation of LNP) Cationic lipids, phospholipids, cholesterol, and PEG lipids were mixed in the molar ratios shown in Tables 1 to 3 to obtain a lipid mixture. This mixture was dissolved in 99.5% ethanol to obtain a 10 mmol / L lipid solution. mRNA encoding GFP (SEQ ID NO: 1) was dissolved in acetate buffer (25 mM sodium acetate, pH 3.5 to 5.5) to obtain a 0.167 mg / mL nucleic acid solution. The obtained lipid solution and nucleic acid solution were mixed at room temperature using a microfluidic mixer (Nanoassemblr, Precision Nanosystems) at a flow rate ratio of 1:2 ([flow rate of lipid solution (4 mL / min)]:[flow rate of nucleic acid solution (8 mL / min)]) to obtain a dispersion. The resulting dispersion was diluted 20-fold with D-PBS(-) buffer (Fujifilm Wako Pure Chemical Industries, Ltd.) and subjected to centrifugal ultrafiltration using a centrifugal filter unit (Amicon Ultra-4, Millipore). Subsequently, the solution after centrifugal ultrafiltration was further filtered using a 0.22 μm syringe filter (Millipore) to prepare LNPs. The obtained LNPs were stored at 4°C.

[0058] (Measurement of mRNA encapsulation rate) A portion of the prepared LNP solution was taken, and 0.5% Triton X-100 was added to dissolve the LNPs. The nucleic acid concentration was measured using the nucleic acid quantification kit "Quant-iT RiboGreen RNA Assay Kit" (Thermo Fisher Scientific). In addition, a portion of the prepared LNP solution was taken, and the nucleic acid concentration was measured in the same manner without adding Triton X-100 to determine the concentration of mRNA that was not encapsulated in the LNPs. From these measurement results, the mRNA encapsulation rate (%) in the LNPs was calculated.

[0059] Specifically, the reagents included in the nucleic acid quantification kit were added to the sample and reacted. Afterward, an excitation wavelength of 485 nm was applied, and the fluorescence intensity at 528 nm was measured. Based on two pre-prepared standard curves, depending on the presence or absence of a surfactant (Triton X-100), the total mRNA concentration in each sample (fluorescence intensity of the sample with surfactant added) and the concentration of mRNA not encapsulated in LNPs (fluorescence intensity of the sample without surfactant added) were calculated from the fluorescence intensity of each sample. Based on these, the mRNA inclusion rate (%) and mRNA yield (%) were calculated using the following formula. "Total mRNA concentration at the start of the reaction" refers to the total mRNA concentration used in the reaction after adding the reagents from the nucleic acid quantification kit.

[0060] [mRNA encapsulation rate (%)] = ([Total mRNA concentration in sample (%)] - [Concentration of mRNA not encapsulated in LNPs (%)]) / [Total mRNA concentration in sample (%)] × 100 (%) [mRNA yield (%)] = [Total mRNA concentration in sample (%)] / [Total mRNA concentration in preparation (%)] × 100 (%)

[0061] (Measurement of average particle size of LNPs) The average particle size of LNPs was measured by dynamic light scattering. Specifically, diluted samples were added to a measurement cuvette, and laser light (633 nm) was irradiated using a dynamic light scattering device (Zetasizer, Malvern Panalytical) to measure the particle size (nm) and PDI.

[0062] (Cell assay) AAVpro 293T cells, pre-cultured using FBS-DMEM (DMEM containing 10% FBS and 1 mM SP), were placed in a 24-well plate at a rate of 80,000 cells / cm². ２ Seeds were sown and cultured until 70-80% confluence (at 37°C, 5% CO2). ２ ). Twenty-four hours after seeding the cells, the culture medium was changed (FBS-DMEM). Next, AAVpro 293T cells were transfected by adding the prepared LNP to each well and then cultured further. Twenty-four hours after transfection, the cells were detached from the wells and collected by adding a trypsin-like enzyme (TrypLE select, Thermo Fisher Scientific).

[0063] (Measurement of Cell Activity (%)) The collected cells were passed through a strainer, and then the cell count and GFP emission intensity were measured using a FACS Verse flow cytometer (Becton Dickinson). The measurement results were analyzed using the analysis software "FlowJo" to determine the percentage of eGFP-positive cells ([number of GFP-expressing cells] / [total number of cells] × 100%) and MFI (Mean Fluorescence Intensity: average value of GFP emission intensity). The percentage of eGFP-positive cells (%) was defined as cell activity (%). LNPs with cell activity of 1% or more were evaluated as useful carriers, i.e., carriers that can effectively exert the function of the encapsulated nucleic acid within the cell.

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[0067] Tables 1-3 show the results of measuring the average particle size (nm), mRNA inclusion rate (%), and cell activity (%) of each prepared LNP. All of the test groups 1-18 showed a cell activity of 1% or more. These results indicate that when delivering nucleic acids for protein expression, such as mRNA, to cells using LNPs as carriers, the efficiency of nucleic acid delivery to cells can be increased by combining one or more lipids selected from the group consisting of DOPC, DOPE, and DSPC as constituent lipids of the LNPs, with one or more lipids selected from the group consisting of cKK-E15, Lipid A4, AA-T3A-C12, Dlin-KC2-DMA, OF-C4-Deg-Lin, and C14-4.

[0068] Furthermore, among test groups 1 to 3, test group 3 showed particularly high cell activity of over 40%. Among test groups 4 to 6, test groups 5 and 6 showed particularly high cell activity of over 50%. Among test groups 7 to 9, test group 9 showed particularly high cell activity of over 20%. Among test groups 10 to 12, test group 12 showed particularly high cell activity of over 70%. Among test groups 13 to 15, test groups 14 and 15 showed particularly high cell activity of over 68%. Among test groups 16 to 18, test groups 16 and 17 showed particularly high cell activity of over 40%. These results demonstrate that LNPs containing DOPC and cKK-E15, LNPs containing DOPC and Lipid A4, LNPs containing DOPE and Lipid A4, LNPs containing DOPC and AA-T3A-C12, LNPs containing DOPC and Dlin-KC2-DMA, LNPs containing DOPC and OF-C4-Deg-Lin, LNPs containing DOPE and OF-C4-Deg-Lin, LNPs containing DSPC and C14-4, and LNPs containing DOPE and C14-4 are particularly useful as carriers for introducing nucleic acids for protein expression, such as mRNA, into cells.

[0069] [Example 2] LNPs containing mRNA were prepared using one of the following as cationic lipids: cKK-E15, Lipid A4, AA-T3A-C12, Dlin-KC2-DMA, OF-C4-Deg-Lin, and C14-4; DOPC, DOPE, or DSPC as phospholipids; cholesterol (Chol) as sterols; and DMG-PEG2000 as PEG lipids. Their physical properties and cellular activity were then investigated. The mRNA used was the mRNA encoding the luciferase protein FLuc (SEQ ID NO: 2).

[0070] (Preparation of LNPs) Cationic lipids, phospholipids, cholesterol, and PEG lipids were mixed in the molar ratios shown in Tables 4 to 71 to obtain a lipid mixture. This mixture was dissolved in 99.5% ethanol to obtain a 10 mmol / L lipid solution. In addition, mRNA encoding FLuc was dissolved in acetate buffer (25 mM sodium acetate, pH 3.5 to 5.0) to obtain a 0.167 mg / mL nucleic acid solution. The obtained lipid solution and nucleic acid solution were mixed at room temperature in a volume ratio of 1:2 by pipetting to obtain a dispersion. The obtained dispersion was diluted 20-fold with D-PBS(-) buffer (Fujifilm Wako Pure Chemical Industries, Ltd.) and subjected to centrifugal ultrafiltration using a centrifugal filter unit (Amicon Ultra-4, Millipore). Next, the solution obtained after centrifugal ultrafiltration was further filtered using a 0.22 μm syringe filter (Millipore) to prepare LNPs. The obtained LNPs were stored at 4°C.

[0071] (Measurement of mRNA inclusion rate) A portion of the prepared LNP solution was taken, and the LNPs were dissolved by adding 0.5% Triton X-100. The nucleic acid concentration was measured using the nucleic acid quantification kit "Quant-iT RiboGreen RNA Assay Kit" (Thermo Fisher Scientific). In addition, a portion of the prepared LNP solution was taken, and the nucleic acid concentration was measured in the same manner without adding Triton X-100 to determine the concentration of mRNA that was not encapsulated in the LNPs. From these measurement results, the mRNA inclusion rate (%) in the LNPs was calculated.

[0072] (Measurement of average particle size of LNPs) The average particle size of LNPs was measured by dynamic light scattering. Specifically, diluted samples were added to a measurement plate, and laser light (633 nm) was irradiated using a dynamic light scattering device (DynaPro PlateReader III, Wyatt Technology) to measure the particle size (nm) and PDI.

[0073] (Cell assay) AAVpro 293T cells, pre-cultured using FBS-DMEM (DMEM containing 10% FBS and 1 mM SP), were seeded in 96-well plates at a rate of 80,000 cells / cm2 and cultured until 70-80% confluence (37°C, 5% CO2). 24 hours after seeding, the AAVpro 293T cells were transfected by adding the prepared LNP to each well and cultured further. 24 hours after transfection, Steady-Glo® Luciferase Assay System (Promega) was added, and the luminescence intensity was analyzed using BioTek Synergy Neo2 (Agilent Technologies).

[0074] (Cytotoxicity) AAVpro 293T cells, pre-cultured using FBS-DMEM (DMEM containing 10% FBS and 1 mM SP), were placed in a 96-well plate at a rate of 80,000 cells / cm². ２ The cells were seeded at the following ratio and cultured until 70-80% confluence (37°C, 5% CO2). 24 hours after seeding, the prepared LNPs were added to each well to transfect AAVpro 293T cells, which were then cultured further. 24 hours after transfection, Cell Counting Kit-8 (Dojin Chemical Co., Ltd.) was added, and the cells were cultured for approximately 60 minutes before absorption was analyzed using BioTek Synergy Neo2 (Agilent Technologies). As a control, LNPs prepared in the same manner except without nucleic acid encapsulation were transfected and absorbance was analyzed in the same way. The relative absorbance value (%), with the absorbance value of the control cells set to 100%, was defined as the cell viability (%) for each cell type.

[0075] The average particle size (D) of each prepared LNP ５０ Tables 4 to 71 show the measurement results for (nm), PDI, mRNA inclusion rate (%), luminescence intensity, and cell viability (%). Under the experimental conditions of this experiment, a luminescence intensity of 20,000 or higher is preferable, 500,000 or higher is more preferable, and 950,000 or higher is even preferable. In the PDI measurement results, "Multimodal" means that multiple peaks due to aggregates, etc., were observed.

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[0144] While preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments. Additions, omissions, substitutions, and other modifications are possible without departing from the spirit of the invention. The present invention is not limited by the foregoing description, but only by the scope of the appended claims.

[0145] The LNPs of the present invention can more effectively exert the function of the nucleic acids they contain within cells. For this reason, the LNPs of the present invention are particularly useful as carriers for transporting functional nucleic acids such as mRNA and siRNA to target cells, and are especially useful in fields such as nucleic acid drugs.

Claims

1. Lipid nanoparticles comprising nucleic acid, cationic lipid, and phospholipid, wherein the cationic lipid is one or more selected from the group consisting of cKK-E15, Lipid A4, AA-T3A-C12, Dlin-KC2-DMA, OF-C4-Deg-Lin, and C14-4, the phospholipid is one or more selected from the group consisting of DOPC, DOPE, and DSPC, and the molar ratio of the cationic lipid to the phospholipid content is 0.2 to 15.

0.

2. The lipid nanoparticle according to claim 1, wherein the molar ratio of the cationic lipid to the phospholipid content is 0.4 to 12.

0.

3. The lipid nanoparticles according to claim 1, wherein the lipid nanoparticles further comprise PEG lipids.

4. The lipid nanoparticle according to claim 3, wherein the PEG lipid is at least one selected from the group consisting of DMG-PEG5000, DMG-PEG2000, and ALC-0159.

5. The lipid nanoparticles according to claim 1, wherein the lipid nanoparticles further comprise sterols.

6. The lipid nanoparticle according to claim 5, wherein the sterol is cholesterol.

7. The lipid nanoparticles according to claim 1, wherein the lipid nanoparticles further comprise sterols and PEG lipids, and the content ratio of the cationic lipids to the total amount of constituent lipids in the lipid nanoparticles is 5 to 65 mol%, the phospholipids to 5 to 30 mol%, the sterols to 15 to 50 mol%, and the PEG lipids to 0.5 to 3 mol%.

8. The lipid nanoparticle according to claim 7, wherein the content ratio of the cationic lipid to the total amount of constituent lipids in the lipid nanoparticle is 10 to 60 mol%.

9. The lipid nanoparticle according to claim 7, wherein the content ratio of the phospholipid to the total amount of constituent lipids in the lipid nanoparticle is 5 to 25 mol%.

10. The lipid nanoparticle according to claim 7, wherein the content ratio of the cationic lipid to the total amount of constituent lipids in the lipid nanoparticle is 10 to 60 mol%, and the content ratio of the phospholipid to the total amount of constituent lipids in the lipid nanoparticle is 5 to 25 mol%.

11. The lipid nanoparticle according to any one of claims 1 to 10, wherein the nucleic acid is mRNA or siRNA.

12. The lipid nanoparticles according to any one of claims 1 to 10, wherein the average particle size of the lipid nanoparticles is 30 to 250 nm.

13. A method for producing lipid nanoparticles according to any one of claims 7 to 10, comprising the following steps: preparing a lipid solution containing the cationic lipid, the phospholipid, sterol and PEG lipid; preparing a nucleic acid solution containing nucleic acid; and mixing the lipid solution and the nucleic acid solution.

14. The method according to claim 13, wherein the solvent in the lipid solution is an alcoholic solvent.

15. The method according to claim 14, wherein the alcoholic solvent is ethanol.

16. The method according to claim 13, wherein the solvent in the nucleic acid solution is an acetate buffer.

17. The method according to claim 13, wherein the pH of the nucleic acid solution is 3.0 to 7.

0.

18. A pharmaceutical composition comprising lipid nanoparticles according to any one of claims 1 to 10.

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