Lipid nanoparticles for nucleic acid delivery

Abstract

Lipid nanoparticles containing a nucleic acid, a cationic lipid, and DOPC, wherein the cationic lipid is at least one selected from the group consisting of CL4H6, Lipid_23, CL4F8-6, YK-009, Lipid_III-45, cKK-E12, C12-200, OF-02, OC2-K3-E10, 306-O12B, 113-O12B, 113-O16B, 306Oi10, CIN-16645, and Lipid_2,2(8,8)_4C_CH3, and the molar ratio of the cationic lipid to the content of the DOPC is 0.2-15.0; and a method for producing the lipid nanoparticles, the method comprising: a step for preparing a lipid solution containing the cationic lipid, DOPC, a sterol, and a 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 comprising cationic lipids and DOPC in specific proportions. This application claims priority based on Japanese Patent Applications No. 2024-192994, 2024-193062, 2024-193132, 2024-193156 and 2024-193210, filed in Japan on November 1, 2024, the contents of which are incorporated herein by reference.

[0002] In recent years, RNA has attracted attention as a modality in gene therapy. RNA has a low risk of integration into the genome and is safer than DNA. Furthermore, once the RNA sequence is determined, it is easy to design, and it can target any gene or express any protein in the body. For these reasons, RNA has the potential to lead to safe and versatile drug discovery.

[0003] RNAs such as mRNA and siRNA (small interfering RNA) are immunogenic, easily degraded by enzymes, and not taken up by cells; therefore, simply administering them into the body does not produce the desired therapeutic effect. As a means of efficiently delivering RNA into target tissues or target cells, methods using lipid nanoparticles (LNPs) as carriers have been investigated.

[0004] LNPs used in gene therapy and other applications are complexes of nucleic acids, which are the active ingredient, and lipids that protect those nucleic acids. Examples of nucleic acids that make up LNPs include mRNA, siRNA, antisense oligonucleotides (ASOs), and DNA. When these nucleic acids are delivered into target tissues or target cells, the desired biological activity is exerted. LNPs are basically composed of four types of lipid components: cationic lipids, phospholipids, cholesterol, and PEG lipids.

[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 this invention discovered that LNPs containing a cationic lipid having a specific structure and DOPC, a type of phospholipid, in a specific ratio exhibit high cellular activity, that is, that the encapsulated nucleic acid exerts high activity within the cell. Based on this finding, further research 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 DOPC, wherein the cationic lipid is CL4H6, Lipid_23, CL4F8-6, YK-009, Lipid_III-45, cKK-E12, C12-200, OF-02, OC2-K3-E10, 306-O12B, 113-O12B, 113-O16B, 306Oi10, CIN-16645, and Lipid_2,2(8,8)_4C_CH ３[1] Lipid nanoparticles selected from the group consisting of the following, wherein the molar ratio of the cationic lipid to the DOPC content is 0.2 to 15.0. [2] Lipid nanoparticles of [1], wherein the molar ratio of the cationic lipid to the DOPC content is 0.4 to 12.0. [3] Lipid nanoparticles of [1] or [2], wherein the lipid nanoparticles further comprise PEG lipids. [4] Lipid nanoparticles of [3], wherein the PEG lipid is selected from the group consisting of DMG-PEG5000, DMG-PEG2000 and ALC-0159. [5] Lipid nanoparticles of any of [1] to [4], wherein the lipid nanoparticles further comprise sterols. [6] Lipid nanoparticles of [5], wherein the sterol is cholesterol. [7] The lipid nanoparticles of any of [1] to [6], 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%, DOPC 5 to 30 mol%, sterols 15 to 50 mol%, and PEG lipids 0.5 to 3 mol%. [8] The lipid nanoparticles of [7], wherein the content ratio of the cationic lipids 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 DOPC to the total amount of constituent lipids in the lipid nanoparticles is 5 to 25 mol%.

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

[11] Lipid nanoparticles of any of [1] to

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

[12] Lipid nanoparticles of 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 of any of [7] to

[12] , comprising the following steps: preparing a lipid solution containing the cationic lipid, DOPC, 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 of

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

[15] The method of

[14] , wherein the alcoholic solvent is ethanol.

[16] Any of the methods of

[13] to

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

[17] Any of the methods of

[13] to

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

[18] A pharmaceutical composition comprising any of the lipid nanoparticles 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") include nucleic acids, cationic lipids, and DOPC, wherein the cationic lipids are CL4H6, Lipid_23, CL4F8-6, YK-009 and Lipid_III-45, cKK-E12, C12-200, OF-02, OC2-K3-E10, 306-O12B, 113-O12B, 113-O16B, 306Oi10, CIN-16645, and Lipid_2,2(8,8)_4C_CH ３ The present invention provides LNPs that are selected from the group consisting of the following, and the molar ratio of the cationic lipid to the DOPC content is 0.2 to 15. By using a combination of DOPC 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 one reason is the sharp particle size distribution. LNPs with extremely large particle sizes or, conversely, extremely small particle sizes are difficult for target cells to take up. It is presumed that the LNPs of the present invention have a narrow particle size distribution, with fewer particles of a size that is difficult for target cells to take up, and thus have a high uptake efficiency into 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 CL4H6 (9-octadecenoic acid, 1,1′-[7-[4-(dipropylamino)butyl]-7-hydroxy-1,13-tridecanediyl] ester, Cas number: 2256087-35-9), Lipid_23 (8-((2-((4-(dimethylamino)butanoyl)oxy)ethyl)(10-oxo-10-(tridecan-7-yloxy)decyl)amino)octyl 2-hexyldecanoate, Cas number: none), CL4F8-6 (Cas number: 2766493-12-1), YK-009 (6-[[4-(decyloxy)-4-oxobutyl](2-hydroxyethyl)amino]-hexanoic acid, 2-octyldecyl ester, Cas number: 2761458-86-8), Lipid_III-45 (2-butyl-octanoic acid, 1,1′-[[(3-hydroxypropyl)imino]di-9,1-nonanediyl] ester, Cas number: 2096984-25-5), cKK-E12 (3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, Cas number: 1432494-65-9), C12-200 (1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, Cas number: 1220890-25-4), OF-02 (3,6-bis[4-[bis[(9Z,12Z)-2-hydroxy-9,12-octadecadien-1-yl]amino]butyl]-2,5-piperazinedione, Cas number: 1883431-67-1), OC2-K3-E10 (I-28) (3,3'-((2-hydroxyethyl)azanediyl)bis(N-(3-(bis(2-hydroxydecyl)amino)propyl)propanamide), Cas number: 2933216-12-5), 306-O12B (Tetrakis(2-(octyldisulfaneyl)ethyl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate, Cas number: 2566523-06-4), 113-O12B (Tetrakis(2-(octyldisulfaneyl)ethyl) 3,3',3'',3'''-(((methylazanediyl)bis(ethane-2,1-diyl))bis(azanetriyl))tetrapropionate, Cas number: 2803699-72-9), 113-O16B (Tetrakis(2-(dodecyldisulfaneyl)ethyl) 3,3',3'',3'''-(((methylazanediyl)bis(ethane-2,1-diyl))bis(azanetriyl))tetrapropionate, Cas number: 2566523-07-5), 306Oi10 (tetrakis(8-methylnonyl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate, Cas number: 2322290-93-5), CIN-16645 (9,12-Octadecadienoic acid (9Z,12Z)-, 3-[4,4-bis(octyloxy)-1-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester, Cas number: 1799316-64-5), and Lipid_2,2(8,8)_4C_CH, ３The cationic lipid is at least one selected from the group consisting of (heptadecan-9-yl (Z)-N-(((4-(dimethylamino)butyl)thio)carbonyl)-N-(2-(non-2-en-1-yloxy)-2-oxoethyl)glycinate, CAS number: 2230647-30-8). In a preferred embodiment, the cationic lipid is Lipid_23, CL4F8-6, YK-009, cKK-E12, C12-200, OF-02, 113-O12B, 306Oi10, CIN-16645 and Lipid_2,2(8,8)_4C_CH ３ It may be at least one selected from the group consisting of Lipid_23, CL4F8-6, YK-009, cKK-E12, C12-200, OF-02, 113-O12B, and CIN-16645, and more preferably at least one selected from the group consisting of Lipid_23, CL4F8-6, YK-009, cKK-E12, C12-200, and CIN-16645.

[0020] Hereafter, "CL4H6, Lipid_23, CL4F8-6, YK-009, Lipid_III-45, cKK-E12, C12-200, OF-02, OC2-K3-E10, 306-O12B, 113-O12B, 113-O16B, 306Oi10, CIN-16645, and Lipid_2,2(8,8)_4C_CH" will be used. ３ This is sometimes referred to as "cationic lipid A".

[0021] The LNP of the present invention comprises DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, CAS number: 4235-95-4).

[0022] The molar ratio of cationic lipid A and DOPC constituting the LNP of the present invention is usually 0.2 to 15.0 (the molar ratio of cationic lipid A to DOPC content [percentage of cationic lipid A content relative to the total amount of constituent lipids in the LNP (mol%)] / [percentage of DOPC content relative to the total amount of constituent lipids in the LNP (mol%)]) (hereinafter sometimes referred to as the "cationic lipid / DOPC ratio"). When the LNP of the present invention contains two or more types of cationic lipid A, the "percentage of cationic lipid A content relative to the total amount of constituent lipids in the LNP (mol%)" means the total content of all cationic lipid A contained in the LNP.

[0023] The LNP of the present invention exhibits superior cell activity by containing cationic lipid A and DOPC in the ratios within the above range. The lower limit of the cationic lipid / DOPC 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 / DOPC 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 / DOPC ratio of the LNP in the present invention is preferably 0.2 to 15.0, more preferably 0.4 to 12.0, even more preferably 0.6 to 10.0, and even more preferably 0.8 to 10.0, with 1.0 to 10.0 also being preferred, 1.5 to 8.0 being more preferred, and 1.5 to 6.0 being even more preferred.

[0024] The content ratio of cationic lipid A in the LNP of the present invention is not particularly limited, as long as the cationic lipid / DOPC 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 5 to 70 mol%, more preferably 5 to 65 mol%, even more preferably 10 to 60 mol%, even more preferably 20 to 60 mol%, and particularly preferably 30 to 60 mol%.

[0025] The DOPC content ratio in the LNP of the present invention is not particularly limited, as long as the cationic lipid / DOPC ratio is within the range of 0.2 to 15.0. From the viewpoint of obtaining higher cell activity, the DOPC content ratio 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%, and even more preferably 10 to 20 mol%.

[0026] In the LNP of the present invention, the content ratio of cationic lipid A to DOPC relative to the total amount of constituent lipids is preferably 5 to 65 mol% for cationic lipid A and 5 to 30 mol% for DOPC, more preferably 10 to 60 mol% for cationic lipid A and 5 to 25 mol% for DOPC, even more preferably 20 to 60 mol% for cationic lipid A and 10 to 20 mol% for DOPC, and still more preferably 30 to 60 mol% for cationic lipid A and 10 to 20 mol% for DOPC. By having the content ratio of cationic lipid A to DOPC within the above range, the cellular activity exhibited by the LNP can be further enhanced.

[0027] Furthermore, in one embodiment, the LNP of the present invention may contain lipids other than cationic lipids and DOPC as constituent lipids of the LNP. Examples of such other lipids include PEG lipids and sterols.

[0028] 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.

[0029] 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.

[0030] When the LNP of the present invention contains cationic lipid A, DOPC, sterols, and PEG lipids, 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 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 may be 5 to 70 mol%, preferably 5 to 65 mol%, more preferably 10 to 65 mol%, even more preferably 20 to 65 mol%, and even more preferably 35 to 65 mol%. The content ratio of cationic lipid A may also be 10 to 60 mol%, more preferably 20 to 60 mol%, and even more preferably 30 to 60 mol%. The lower limit of the DOPC 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 DOPC 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 DOPC content is preferably 5 to 30 mol%, more preferably 5 to 25 mol%, and even more preferably 10 to 20 mol%. The lower limit of the sterol content can 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.Furthermore, 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 ratio is preferably 15 to 90 mol%, more preferably 20 to 90 mol%. The lower limit of the PEG lipid content ratio 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. Furthermore, 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. Preferably, the PEG lipid content is 0.5 to 5 mol%, more preferably 0.5 to 4 mol%.

[0031] When the LNP of the present invention contains cationic lipid A, DOPC, sterols, and PEG lipids, higher cell activity can be obtained. Therefore, the content ratio of the constituent lipids of the LNP to the total amount of constituent lipids is preferably 5 to 65 mol% for cationic lipid A, 5 to 30 mol% for DOPC, 15 to 90 mol% for sterols, and 0.5 to 5.0 mol% for PEG lipids. It is more preferably 10 to 60 mol% for cationic lipid A, 5 to 25 mol% for DOPC, 15 to 85 mol% for sterols, and 0.5 to 5.0 mol% for PEG lipids. It is more preferable that the content ratio is 10 to 60 mol%, the DOPC content ratio is 5 to 20 mol%, the sterol content ratio is 20 to 80 mol%, and the PEG lipid content ratio is 0.5 to 4.0 mol%, and even more preferable that the cationic lipid A content ratio is 20 to 60 mol%, the DOPC content ratio is 10 to 20 mol%, the sterol content ratio is 20 to 75 mol%, and the PEG lipid content ratio is 0.5 to 3.0%, and it is also preferable that the cationic lipid A content ratio is 30 to 60 mol%, the DOPC content ratio is 10 to 20 mol%, the sterol content ratio is 20 to 70 mol%, and the PEG lipid content ratio is 0.5 to 2.0 mol%. When the LNP of the present invention contains cationic lipid A, DOPC, sterol, and PEG lipid, the PEG lipid is preferably DMG-PEG5000, DMG-PEG2000, or ALC-0159, with DMG-PEG2000 being particularly preferred.

[0032] 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)) in the LNP of the present invention is usually 2 to 48, preferably 4 to 32, more preferably 4 to 12, even more preferably 4 to 10, and even more preferably 4 to 8 (e.g., 6), but is not limited to these values.

[0033] In another aspect, the LNP of the present invention may contain components other than nucleic acids and lipids. Such components include, for example, surfactants, hyaluronic acid or its derivatives, etc., but are not limited thereto.

[0034] In one aspect, the LNP of the present invention may contain a surfactant. Surfactants that can be included in the LNP of the present invention include polyoxyethylene sorbitan monooleate (e.g., polysorbate 80, etc.), polyoxyethylene polyoxypropylene glycol (e.g., pluronic F68, etc.), sorbitan fatty acid esters (e.g., sorbitan monolaurate, sorbitan monooleate, etc.), polyoxyethylene derivatives (e.g., polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, etc.), glycerin fatty acid esters or polyethylene glycol alkyl ethers, etc., but are not limited thereto. In a preferred aspect, the surfactant contained in the LNP of the present invention is polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester or polyethylene glycol alkyl ether.

[0035] In one aspect, in addition to nucleic acids and lipids, the LNP of the present invention may further contain hyaluronic acid or its derivatives. Examples of hyaluronic acid derivatives include compounds obtained by dehydrative condensation of hyaluronic acid with the hydroxyl group of fatty acid glyceryl.

[0036] In one aspect, the average particle diameter of the LNP of the present invention can have an average particle diameter 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, about 70 nm to about 80 nm. In a preferred aspect, the average particle diameter of the LNP of the present invention can be about 30 nm to about 200 nm.

[0037] In addition, as used in this specification, the term "about" when applied to a target value refers to a value similar to the recited reference value. The term "about" means a range of values that fall within ±10% of the reference value.

[0038] In one aspect, the lower limit of the polydispersity index (PDI) of the LNP of the present invention can usually be 0.001 or more, but is not limited thereto. The upper limit of the PDI of the LNP of the present invention is preferably 0.50 or less, more preferably 0.35 or less, still more preferably 0.30 or less, even more preferably 0.26 or less, and particularly preferably 0.22 or less.

[0039] In the present invention and this specification, the average particle diameter of the LNP means the volume-based D ５０ particle diameter (nm) measured by the dynamic light scattering method. The PDI of the LNP is calculated from the particle size distribution measured by the dynamic light scattering method.

[0040] <Method for Producing Lipid Nanoparticles> The method for producing lipid nanoparticles of the present embodiment (hereinafter sometimes referred to as "the production method of the present invention") is a method for producing an LNP containing at least a cationic lipid A, DOPC, sterol, and PEG lipid as constituent lipids among the LNPs of the present invention, and includes the following steps. A step of preparing a lipid solution containing a cationic lipid A, DOPC, sterol, and PEG lipid, a step of preparing a nucleic acid solution containing a nucleic acid, and a step of mixing the lipid solution and the nucleic acid solution.

[0041] The cationic lipid A, DOPC, sterol, and PEG lipid used are those to be incorporated into the LNP of the present invention described above.

[0042] In the production method of the present invention, first, a lipid solution containing cationic lipid A, DOPC, sterols, and PEG lipids is prepared. Specifically, cationic lipid A, DOPC, sterols, and PEG lipids are added to an organic solvent in a desired molar ratio. The organic solvent is not particularly limited as long as it is an organic solvent capable of dissolving all of cationic lipid A, DOPC, sterols, and PEG lipids. Examples of such organic solvents include alcoholic solvents such as methanol, ethanol, propanol, isopropanol, and butanol; ester-based solvents such as ethyl acetate and butyl acetate; ketone-based solvents such as acetone, methyl ethyl ketone, and methyl isobutyl ketone; and chlorine-based solvents such as chloroform. Alternatively, a mixed solvent of two or more of these organic solvents, or a mixed solvent of one or more of these organic solvents with water, may also be used. In the production method of the present invention, from the viewpoint of relatively high safety for living organisms, an alcoholic solvent or a mixed solvent of water and an alcoholic solvent is preferred, ethanol or a mixed solvent of ethanol and water is more preferred, and ethanol is particularly preferred.

[0043] Furthermore, 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 with an aqueous solvent capable of dissolving the nucleic acid. The aqueous solvent is not particularly limited as long as it can dissolve the nucleic acid, and any solvent may be used. Examples of aqueous solvents include water and buffer solutions (e.g., citrate buffer solution, acetate buffer solution, etc.), but are not limited to these. In one preferred embodiment, the aqueous solvent is an acetate buffer solution.

[0044] In one embodiment, the pH of the aqueous solvent used in the manufacturing 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.

[0045] 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.

[0046] 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).

[0047] 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.

[0048] 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.

[0049] <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").

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] [Example 1] Cationic lipids include CL4H6, Lipid_23, CL4F8-6, YK-009, Lipid_III-45, cKK-E12, C12-200, OF-02, OC2-K3-E10, 306-O12B, 113-O12B, 113-O16B, 306Oi10, CIN-16645, and Lipid_2,2(8,8)_4C_CH３ Using one of the following, LNPs encapsulating mRNA were prepared using 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), or DSPC (1,2-distearoyl-sn-glycero-3-phosphatidylcholine, Cas number: 816-94-4) as the phospholipid, cholesterol as the sterol, and DMG-PEG5000, DMG-PEG2000, or ALC-0159 as the PEG lipid, and their physical properties and cellular activity were investigated. The mRNA used was the mRNA encoding green fluorescent protein GFP (SEQ ID NO: 1).

[0056] (Preparation of LNP) Cationic lipids, phospholipids, cholesterol, and PEG lipids were mixed in the molar ratios shown in Tables 1 to 7 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.

[0057] (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.

[0058] 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.

[0059] [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 (%)

[0060] (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.

[0061] (Cell assay) AAVpro HEK293T cells pre-cultured using FBS-DMEM (DMEM containing 10% FBS and 1 mM SP) were seeded in a 24-well plate at a density of 80,000 cells / cm ２ and cultured until 70-80% confluent (37 °C, 5% CO ２ ). Twenty-four hours after seeding the cells, the medium was changed (FBS-DMEM). Then, the prepared LNP was added to each well to transfect the AAVpro HEK293T cells, which were further cultured. At the time point 24 hours after transfection, the cells were detached from the wells and collected by adding a trypsin-like enzyme (TrypLE select, manufactured by Thermo Fisher Scientific).

[0062] (Measurement of cell viability (%)) The collected cells were passed through a strainer, and then the cell count and GFP fluorescence intensity were measured using a FACSVerse flow cytometer (manufactured by Becton Dickinson). The measurement results were analyzed using the analysis software "FlowJo" to determine the percentage of eGFP-positive cells ([number of cells expressing GFP] / [total number of cells] × 100%) and the MFI (Mean Fluorescence Intensity: average value of GFP fluorescence intensity). The percentage of eGFP-positive cells (%) was taken as the cell viability (%). LNPs with a cell viability of 1% or more were evaluated as useful carriers, that is, carriers capable of effectively exerting the function of the encapsulated nucleic acid inside the cells.

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] Tables 1-7 show the measurement results for the average particle size (nm), PDI, mRNA inclusion rate (%), and cell activity (%) of each prepared LNP. When comparing the test groups 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, 13-15, 16-18, 19-20, 21-22, 23-25, 26-27, 28-29, 30-31, and 32-34, which all used the same cationic lipid, the cell activity of LNPs using DOPC as the phospholipid was clearly higher in all test groups. These results suggest that when LNPs are used as carriers to deliver nucleic acids for protein expression, such as mRNA, to cells, the constituent lipids of LNPs are DOPC, CL4H6, Lipid_23, CL4F8-6, YK-009, Lipid_III-45, cKK-E12, C12-200, OF-02, OC2-K3-E10, 306-O12B, 113-O12B, 113-O16B, 306Oi10, CIN-16645, and Lipid_2,2(8,8)_4C_CH ３ It was confirmed that combining DOPC with any of the following significantly improves the efficiency of nucleic acid delivery to cells: DOPC with CL4H6, Lipid_23, CL4F8-6, YK-009, Lipid_III-45, cKK-E12, C12-200, OF-02, OC2-K3-E10, 306-O12B, 113-O12B, 113-O16B, 306Oi10, CIN-16645, and Lipid_2,2(8,8)_4C_CH ３ LNPs formed by combining any of the above with other lipids have been shown to be very useful as carriers for introducing nucleic acids for protein expression, such as mRNA, into cells. In particular, LNPs formed by combining DOPC with any of CL4H6, YK-009, cKK-E12, C12-200, OF-02, OC2-K3-E10, 113-O12B, 306Oi10, and CIN-16645 have been shown to have high cellular activity and be very useful as carriers for introducing nucleic acids for protein expression, such as mRNA, into cells.

[0071] [Example 2] Cationic lipids include CL4H6, Lipid_23, CL4F8-6, YK-009, Lipid_III-45, cKK-E12, C12-200, OF-02, OC2-K3-E10, 306-O12B, 113-O12B, 113-O16B, 306Oi10, CIN-16645, and Lipid_2,2(8,8)_4C_CH ３ Using one of the following methods, DOPC was used as the phospholipid, cholesterol (Chol) as the sterol, and DMG-PEG2000 as the PEG lipid, LNPs encapsulating mRNA were prepared, and their physical properties and cellular activity were investigated. The mRNA used was the mRNA encoding the luciferase protein FLuc (SEQ ID NO: 2).

[0072] (Preparation of LNPs) Cationic lipids, phospholipids, cholesterol, and PEG lipids were mixed in the molar ratios shown in Tables 8 to 52 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.

[0073] (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.

[0074] (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.

[0075] (Cell assay) AAVpro 293T cells, pre-cultured using FBS-DMEM (DMEM containing 10% FBS and 1 mM SP), were seeded into 96-well plates at a density of 80,000 cells / cm² and cultured until 70–80% confluence (37°C, 5% CO2). ２ ). Twenty-four hours after seeding, the prepared LNPs were added to each well to transfect AAVpro 293T cells, which were then cultured. Twenty-four hours after transfection, Steady-Glo® Luciferase Assay System (Promega) was added, and the luminescence intensity was analyzed using BioTek Synergy Neo2 (Agilent Technologies).

[0076] (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 Seeds were sown and cultured until 70-80% confluence (at 37°C, 5% CO2). ２). Twenty-four hours after seeding, AAVpro 293T cells were transfected by adding the prepared LNPs to each well and then cultured. Twenty-four hours after transfection, Cell Counting Kit-8 (Dojin Chemical Co., Ltd.) was added, and the cells were cultured for approximately 60 minutes before the absorbance was analyzed using BioTek Synergy Neo2 (Agilent Technologies). As a control, LNPs prepared in the same manner except that nucleic acids were not encapsulated were transfected and the absorbance was analyzed in the same manner. 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. The measurement results for the average particle size (nm), PDI, mRNA encapsulation rate (%), luminescence intensity, and cell viability (%) of each prepared LNP are shown in Tables 8 to 52. Under the experimental conditions of this experiment, the luminescence intensity is preferably 20,000 or higher, more preferably 500,000 or higher, and even more preferably 950,000 or higher. In the PDI measurement results, "Multimodal" means that multiple peaks were observed due to aggregates, etc.

[0077]

[0078]

[0079]

[0080] In test groups 1-20, LNPs using CL4H6 as the cationic lipid all exhibited good luminescence intensity and high cell viability. Although the mRNA inclusion rate was low in LNPs in test groups 9, 17, and 19, they showed good luminescence intensity, indicating good efficiency in introducing nucleic acids for protein expression, such as mRNA, into cells.

[0081]

[0082]

[0083]

[0084] In test groups 21-40, where Lipid_23 was used as the cationic lipid, the LNPs all showed mRNA inclusion rates exceeding 30%, and exhibited good luminescence intensity and cell viability.

[0085]

[0086]

[0087]

[0088] In test groups 41-60, where CL4F8-6 was used as the cationic lipid, the LNPs all exhibited good luminescence intensity and high cell viability.

[0089]

[0090]

[0091]

[0092] In test groups 61-80, where YK-009 was used as the cationic lipid, the LNPs all showed high mRNA inclusion rates exceeding 50%, and also exhibited good luminescence intensity and cell viability.

[0093]

[0094]

[0095]

[0096] In test groups 81-100, where Lipid III-45 was used as the cationic lipid, the LNPs all showed good luminescence intensity and cell viability.

[0097]

[0098]

[0099]

[0100] In test groups 101-120, where cKK-E12 was used as the cationic lipid, the LNPs all showed high mRNA inclusion rates exceeding 70%, and also exhibited good luminescence intensity and cell viability.

[0101]

[0102]

[0103]

[0104] In test groups 121-140, where C12-200 was used as the cationic lipid, the LNPs all showed high mRNA inclusion rates exceeding 70%, and also exhibited good luminescence intensity and cell viability.

[0105]

[0106]

[0107]

[0108] In test groups 141-160, where OF-02 was used as the cationic lipid, the LNPs all showed high mRNA inclusion rates exceeding 50%, and also exhibited good luminescence intensity and cell viability.

[0109]

[0110]

[0111]

[0112] In test plots 161-180, where OC2-K3-E10 was used as the cationic lipid, the LNPs all showed high mRNA inclusion rates exceeding 40%, and exhibited good luminescence intensity and cell viability.

[0113]

[0114]

[0115]

[0116] In test groups 181-200, LNPs using 306-O12B as the cationic lipid all showed good luminescence intensity and cell viability. Although the mRNA inclusion rate of LNPs in test group 185 was low at 8%, the luminescence intensity was good, indicating good efficiency in introducing nucleic acids for protein expression, such as mRNA, into cells.

[0117]

[0118]

[0119]

[0120] In test plots 201-220, where 113-O12B was used as the cationic lipid, the LNPs all showed mRNA inclusion rates of 25% or more, and exhibited good luminescence intensity and cell viability.

[0121]

[0122]

[0123]

[0124] In test plots 221-240, where 113-O16B was used as the cationic lipid, the LNPs all showed good luminescence intensity and cell viability.

[0125]

[0126]

[0127]

[0128] In test plots 221-240, where 306Oi10 was used as the cationic lipid, the LNPs all showed good luminescence intensity and cell viability.

[0129]

[0130]

[0131]

[0132] In test plots 261-280, where CIN-16645 was used as the cationic lipid, the LNPs all showed mRNA inclusion rates exceeding 50%, and exhibited good luminescence intensity and cell viability.

[0133]

[0134]

[0135]

[0136] Cationic lipids include Lipid_2,2(8,8)_4C_CH ３In all of the LNPs in test plots 281-300, the mRNA inclusion rate was over 45%, and the luminescence intensity and cell viability were good.

[0137] In the test groups using cationic lipids Lipid_23, YK-009, CIN-16645, and C12-200, the LNPs exhibited significantly higher luminescence intensity and superior cell viability. In particular, the LNPs in all test groups using CIN-16645 as the cationic lipid had a sufficiently high mRNA inclusion rate of over 50%, and both cell viability and luminescence intensity were also sufficiently high. From the above results, DOPC, CL4H6, Lipid_23, CL4F8-6, YK-009, Lipid_III-45, cKK-E12, C12-200, OF-02, OC2-K3-E10, 306-O12B, 113-O12B, 113-O16B, 306Oi10, CIN-16645, and Lipid_2,2(8,8)_4C_CH ３ LNPs formed by combining one of the cationic lipids with another lipid exhibit high cellular activity and have been shown to be extremely useful as carriers for introducing nucleic acids for protein expression, such as mRNA, into cells.

[0138] The LNPs of the present invention can more effectively exert the function of the encapsulated nucleic acid within the cell. 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.

[0139] 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.

Claims

Lipid nanoparticles, It contains nucleic acids, cationic lipids, and DOPC. The cationic lipids are CL4H6, Lipid_23, CL4F8-6, YK-009, Lipid_III-45, cKK-E12, C12-200, OF-02, OC2-K3-E10, 306-O12B, 113-O12B, 113-O16B, 306Oi10, CIN-16645, and Lipid_2,2(8,8)_4C_CH ３ At least one selected from the group consisting of, Lipid nanoparticles having a molar ratio of cationic lipid to DOPC content of 0.2 to 15.

0. Lipid nanoparticles according to claim 1, wherein the molar ratio of the cationic lipid to the DOPC content is 0.4 to 12.

0. The lipid nanoparticles according to claim 1, wherein the lipid nanoparticles further comprise PEG lipids. 　 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. 　 The lipid nanoparticles according to claim 1, wherein the lipid nanoparticles further comprise sterols. 　 The lipid nanoparticle according to claim 5, wherein the sterol is cholesterol. 　 The lipid nanoparticles further comprise sterols and PEG lipids. Lipid nanoparticles according to claim 1, wherein the content ratio of the lipid nanoparticles relative to the total amount of constituent lipids is 5 to 65 mol% of cationic lipids, 5 to 30 mol% of DOPC, 15 to 50 mol% of sterols, and 0.5 to 3 mol% of PEG lipids. 　 The lipid nanoparticles according to claim 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%. 　 The lipid nanoparticles according to claim 7, wherein the content ratio of DOPC to the total amount of constituent lipids in the lipid nanoparticles is 5 to 25 mol%. 　 The content ratio of the cationic lipid to the total amount of constituent lipids in the lipid nanoparticles is 10 to 60 mol%, The lipid nanoparticles according to claim 7, wherein the content ratio of DOPC to the total amount of constituent lipids in the lipid nanoparticles is 5 to 25 mol%. 　 The lipid nanoparticle according to any one of claims 1 to 10, wherein the nucleic acid is mRNA or siRNA. 　 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. 　 A method for producing lipid nanoparticles according to any one of claims 7 to 10, comprising the following steps: A step of preparing a lipid solution containing the cationic lipid, DOPC, sterol, and PEG lipid, A step of preparing a nucleic acid solution containing nucleic acids, and, A step of mixing the lipid solution and the nucleic acid solution. 　 The method according to claim 13, wherein the solvent in the lipid solution is an alcoholic solvent. 　 The method according to claim 14, wherein the alcoholic solvent is ethanol. 　 The method according to claim 13, wherein the solvent in the nucleic acid solution is an acetate buffer. 　 The method according to claim 13, wherein the pH of the nucleic acid solution is 3.0 to 7.

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

Images