WO2023190176A1 - 脾臓組織へ核酸を送達するための脂質ナノ粒子およびこれを用いて脾臓組織へ核酸を送達する方法 - Google Patents

脾臓組織へ核酸を送達するための脂質ナノ粒子およびこれを用いて脾臓組織へ核酸を送達する方法 Download PDF

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WO2023190176A1
WO2023190176A1 PCT/JP2023/011850 JP2023011850W WO2023190176A1 WO 2023190176 A1 WO2023190176 A1 WO 2023190176A1 JP 2023011850 W JP2023011850 W JP 2023011850W WO 2023190176 A1 WO2023190176 A1 WO 2023190176A1
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group
glycero
carbon atoms
formula
lipid
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French (fr)
Japanese (ja)
Inventor
悠太 中井
耕太 丹下
遊 櫻井
英万 秋田
浩揮 田中
瑞歩 堀
昌樹 五味
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Tohoku University NUC
Chiba University NUC
NOF Corp
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Tohoku University NUC
Chiba University NUC
NOF Corp
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Priority to JP2024512364A priority Critical patent/JPWO2023190176A1/ja
Priority to CN202380031620.9A priority patent/CN118973614A/zh
Priority to EP23780186.5A priority patent/EP4501360A4/en
Priority to CA3247039A priority patent/CA3247039A1/en
Priority to KR1020247035352A priority patent/KR20240163154A/ko
Publication of WO2023190176A1 publication Critical patent/WO2023190176A1/ja
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    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the present invention relates to lipid nanoparticles used to deliver nucleic acids to spleen tissue, and methods for delivering nucleic acids to spleen tissue using the same.
  • Viral vectors are nucleic acid delivery carriers with high expression efficiency, but have practical problems from the viewpoint of safety. Therefore, development of non-viral nucleic acid delivery carriers that can be used more safely is underway.
  • lipid nanoparticles which are carriers using ionic lipids, are currently the most commonly used non-viral nucleic acid delivery carriers.
  • Ionic lipids are broadly divided into amine parts and lipid parts.
  • the amine moiety that protonates under acidic conditions interacts electrostatically with nucleic acids, which are polyanions, to form lipid nanoparticles, which promotes uptake into cells and enhances nucleic acids. It is delivered into cells.
  • a known ionic lipid that is generally widely used includes 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP). It is known that by combining such known ionic lipids with phospholipids, cholesterol, and PEG lipids, lipid nanoparticles can be formed and nucleic acids can be delivered into cells (for example, Non-Patent Document 1). reference).
  • DODAP 1,2-dioleoyloxy-3-dimethylaminopropane
  • Patent Document 1 describes an ionic lipid having a structure in which compounds consisting of one or two amine sites and one lipid site are connected to each other by disulfide bonds that exhibit biodegradability.
  • Patent Document 1 shows that the ionic lipid improves in-vivo dynamics such as blood stability and tumor targeting.
  • the pKa of the lipid membrane structure can be adjusted to a value that is advantageous for endosomal escape within cells, and by utilizing the fact that disulfide bonds are broken within cells, It has been shown to have the effect of dissociating nucleic acids from lipid membrane structures.
  • an ionic lipid with an aromatic ring introduced near the lipid site is used to increase the ability to fuse with endosomal membranes and improve the efficiency of nucleic acid delivery to the cytoplasm.
  • a lipid membrane structure with even higher efficiencies is shown.
  • ionic lipids with improved intracellular dynamics have been developed by increasing endosomal escape efficiency and membrane fusion ability, while lipid nanoparticles made of ionic lipids have been used as nucleic acid delivery carriers in vivo. In order to have more practical effects, it is necessary to have directivity to target organs and cells.
  • Dimyristoylglycerol PEG is one of the PEG lipids widely used in lipid nanoparticles.
  • PEG lipids gradually dissociate from the lipid nanoparticles in the blood, and apolipoprotein E (ApoE) present in the blood adheres to the lipid nanoparticles.
  • ApoE apolipoprotein E
  • DMG-PEG which has a hydrophobic group derived from myristic acid as a PEG lipid, but distearoylglycerol PEG (which has a hydrophobic group derived from stearic acid).
  • DSG-PEG Non-Patent Document 3
  • DSG-PEG is difficult to dissociate from lipid nanoparticles in the blood, so it avoids ApoE adhesion in the blood, suppresses its accumulation in the liver, and has high retention in the blood. As a result, the accumulation in tumors increases.
  • Another example of providing directivity to organs other than the liver is an example in which the efficiency of nucleic acid introduction into the spleen was increased by controlling the particle size of lipid nanoparticles to 140 to 230 nm (for example, patented (See Reference 3). It has been shown that lipid nanoparticles with a particle diameter of 200 nm avoid nucleic acid introduction into the liver and increase the efficiency of nucleic acid introduction into the spleen compared to lipid nanoparticles with a particle diameter of 100 nm.
  • the efficiency of nucleic acid introduction can be increased by using lipid nanoparticles with improved intracellular dynamics, and by changing the PEG lipid that is a component of the lipid nanoparticles and adjusting the particle size of the lipid nanoparticles, It is also possible to efficiently deliver nucleic acids to target tissues such as the liver and spleen.
  • the spleen can be a target for nucleic acid vaccine applications because it is an organ with many immune cells, but despite advances in this field, conventional nucleic acid delivery to the spleen using lipid nanoparticles is difficult. Efficiency is not sufficient and there is room for improvement.
  • Non-Patent Document 4 and Patent Document 3 each indicate the use of ionic lipids with different structural characteristics regarding lipid nanoparticles for delivery to the spleen.
  • different phospholipids are used as components of lipid nanoparticles, and the optimal combination with phospholipids differs depending on the structure of the ionic lipid.
  • the present invention aims to provide lipid nanoparticles for delivering nucleic acids to spleen tissue that can improve the efficiency of nucleic acid delivery to spleen tissue cells, and a method for delivering nucleic acids to spleen tissue using the same. purpose.
  • the appropriate combination of phospholipids differs depending on the structure of the ionic lipid, and for efficient delivery of nucleic acids, it is necessary to optimize the combination of ionic lipids and phospholipids. is required.
  • an ionic lipid that has a pKa suitable for endosomal escape and is specifically decomposed in a reducing environment within cells and an anionic phospholipid or a formula (2) It has been found that lipid nanoparticles produced using anionic cholesterol represented by ) can efficiently introduce nucleic acids into spleen tissue.
  • the present invention based on this knowledge is as follows.
  • R 1a and R 1b each independently represent an alkylene group having 1 to 6 carbon atoms
  • X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or 2 to 5 carbon atoms, and represents a cyclic alkylene tertiary amino group having 1 to 2 tertiary amino groups
  • R 2a and R 2b each independently represent an alkylene group or an oxydialkylene group having 8 or less carbon atoms
  • Y a and Y b each independently represent an ester bond, amide bond, carbamate bond, ether bond or urea bond
  • Z a and Z b each independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a hetero atom.
  • n a and n b are each independently 0 or 1
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride.
  • R 9 -O-CO-(CH 2 )a- (4) (In formula (4), R 9 represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, a represents an integer from 2 to 10. ) represents a group represented by ) ionic lipid represented by (B) Anionic phospholipid or formula (2):
  • T represents a divalent aliphatic hydrocarbon group having 1 to 8 carbon atoms.
  • Any two of R 6 , R 7 and R 8 represent a myristoyl group, and the remaining one represents a myristoyl group having 1 to 6 carbon atoms connected via a polyethylene glycol chain having a number average molecular weight of 1,000 to 3,000. Represents an alkyl group.
  • Dimyristoylglycerol PEG represented by Lipid nanoparticles used to deliver nucleic acids to spleen tissue.
  • the anionic phospholipid is 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl -2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1-stearoyl-2-oleoyl-sn-glycero-3 -Phosphoglycerol (SOPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), 1-palmitoyl-2- Oleoyl-sn-glycero-3-phosphoserine (POPS), 1,2-dioleoyl-sn-s
  • the anionic phospholipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), or 1 , 2-dilinoleoyl-sn-glycero-3-phosphoserine (DLoPS).
  • POPG 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol
  • DOPG 1,2-dioleoyl-sn-glycero-3-phosphoglycerol
  • DLoPS 2-dilinoleoyl-sn-glycero-3-phosphoserine
  • the ionic lipid represented by formula (1) has the following formula:
  • lipid nanoparticle according to any one of [1] to [5], which is any ionic lipid represented by:
  • the neutral phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dierkyl- sn-glycero-3-phosphocholine (DEPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 -stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1-palmitoyl-2-oleoyl-sn-
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOPE 1,2-dioleoyl-sn-
  • the ionic lipid is 30 to 70 mol% of the total of the ionic lipid, the anionic phospholipid or the compound represented by formula (2), the neutral phospholipid, and the cholesterol. and the anionic phospholipid or the compound represented by formula (2) is 2.5 to 15 mol%, the neutral phospholipid is 0 to 15 mol%, and the cholesterol is 20 to 60 mol%.
  • a method for delivering a nucleic acid to a spleen tissue which comprises intravenously administering to a living body the lipid nanoparticle according to any one of [1] to [11] encapsulating a nucleic acid.
  • T is as defined in [1].
  • Dimyristoylglycerol PEG represented by Use of lipid nanoparticles comprising: for the manufacture of a medicament used to deliver nucleic acids to spleen tissue.
  • the anionic phospholipid is 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl -2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1-stearoyl-2-oleoyl-sn-glycero-3 -Phosphoglycerol (SOPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), 1-palmitoyl-2- Oleoyl-sn-glycero-3-phosphoserine (POPS), 1,2-dioleoyl-sn-s
  • the anionic phospholipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), or 1 , 2-dilinoleoyl-sn-glycero-3-phosphoserine (DLoPS).
  • POPG 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol
  • DOPG 1,2-dioleoyl-sn-glycero-3-phosphoglycerol
  • DLoPS 2-dilinoleoyl-sn-glycero-3-phosphoserine
  • T in formula (2) is a divalent aliphatic hydrocarbon group having 1 to 3 carbon atoms.
  • the ionic lipid represented by formula (1) has the following formula:
  • lipid nanoparticles may contain a neutral phospholipid as a component.
  • the neutral phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dierkyl- sn-glycero-3-phosphocholine (DEPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 -stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1-palmitoyl-2-oleoyl-sn-
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOPE 1,2-dioleoyl-sn-
  • the ionic lipid is 30 to 70 mol% of the total of the ionic lipid, the anionic phospholipid or the compound represented by formula (2), the neutral phospholipid, and the cholesterol. and the anionic phospholipid or the compound represented by formula (2) is 2.5 to 15 mol%, the neutral phospholipid is 0 to 15 mol%, and the cholesterol is 20 to 60 mol%. , the use according to any one of [19] to [22], wherein the dimyristoylglycerol PEG is 0.5 to 1.5 mol%.
  • the lipid nanoparticles of the present invention are a combination of an ionic lipid and an anionic phospholipid or anionic cholesterol that have a pKa suitable for endosomal escape and are specifically decomposed in a reducing environment within cells, and a lipid nanoparticle that has a pKa suitable for endosomal escape.
  • the optimized composition allows for more efficient delivery of nucleic acids to spleen tissue compared to prior art lipid compositions.
  • FIG. 2 is a diagram showing the gene expression activity in the spleen of the lipid nanoparticles (LNP) of Examples 1 and 2 and the LNP of Comparative Example 1, which is a composition for liver delivery.
  • FIG. 3 is a diagram showing the accumulation of LNP in the spleen of LNP of Example 3 and LNP of Comparative Example 2, which is a composition for liver transport.
  • FIG. 3 is a diagram showing the gene expression activity in the spleen of LNP of Example 3 and LNP of Comparative Example 2, which is a composition for liver transfer.
  • FIG. 2 is a diagram showing gene expression activity in the spleen of LNPs of Examples 1 and 4 to 10 and LNPs of Comparative Example 1, which is a composition for liver transfer.
  • FIG. 3 is a diagram showing the gene expression activity in the spleen of LNPs of Examples 5 and 11 to 24.
  • FIG. 7 is a diagram showing the gene expression activity in the spleen of LNPs of Examples 18 and 25 to 29.
  • FIG. 7 is a diagram showing gene expression activity in the spleen of LNP of Example 30 and LNP of Comparative Example 3, which is a composition for liver delivery.
  • FIG. 3 is a diagram showing CTL activity when LNPs of Examples 18 and 24 encapsulating OVA-mRNA and LNPs of Comparative Example 1, which is a composition for liver delivery, are administered to mice.
  • FIG. 7 is a diagram showing CTL activity when LNP of Example 30 encapsulating OVA-mRNA, LNP of Comparative Example 3 having a composition for liver delivery, and LNP of Comparative Example 4 having a composition for spleen delivery were administered to mice. It is.
  • FIG. 7 is a diagram showing CTL activity when LNP of Example 31 encapsulating OVA-mRNA and LNP of Comparative Example 4, which is a composition for spleen delivery, were administered to mice.
  • FIG. 7 is a diagram showing pathological scores of mice administered with LNP or PBS of Example 32 encapsulating MOG-mRNA or luc-mRNA.
  • FIG. 7 is a diagram showing body weight fluctuation (%) (relative to Day 10) of mice administered with LNP or PBS of Example 32 encapsulating MOG-mRNA or luc-mRNA.
  • the present invention provides an ionic lipid represented by formula (1) (i.e., an ionic lipid having a tertiary amino group, a lipid moiety, and a disulfide bond that is a biodegradable group), an anionic phospholipid, or a formula (2).
  • the present invention relates to lipid nanoparticles containing anionic cholesterol represented by formula (3), cholesterol, and dimyristoylglycerol PEG represented by formula (3), and a method for delivering nucleic acids to spleen cells using the same.
  • Lipid nanoparticles are nanoparticles in which the hydrophilic groups of amphiphilic lipids are directed toward the aqueous phase side of the interface.
  • amphiphilic lipid refers to a lipid having a membrane structure arranged in a diaphragm structure and having a particle diameter of less than 1 ⁇ m.
  • the particle size of the lipid nanoparticles of the present invention is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm.
  • the particle size can be measured using, for example, a particle size distribution measuring device such as Zetasizer Nano (Malvern).
  • the particle size of the lipid nanoparticles can be adjusted as appropriate by the method for producing lipid nanoparticles.
  • particle size means an average particle size (zeta average) measured by a dynamic light scattering method.
  • amphipathic lipids examples include ionic lipids, phospholipids, PEG lipids, and the like.
  • PEG means polyethylene glycol
  • PEG lipid means a lipid modified with PEG
  • Y modified with X for example, X: PEG, Y: lipid
  • PEG lipid means a lipid to which PEG is attached.
  • the lipid nanoparticles of the present invention include an ionic lipid represented by the formula (1), an anionic phospholipid, or an anionic cholesterol represented by the formula (2), a neutral phospholipid, cholesterol, and a lipid nanoparticle represented by the formula (3). It may contain lipids other than the dimyristoylglycerol PEG (hereinafter sometimes referred to as "other lipids"). Examples of other lipids include sterols other than cholesterol and PEG lipids other than dimyristoylglycerol PEG represented by formula (3).
  • the amount of other lipids in the lipid nanoparticles of the present invention is preferably 0 to 50 mol%, more preferably 0 to 30 mol%, and even more preferably 0 to 10 mol%, based on the total amount of lipids in the lipid nanoparticles.
  • the "total amount of lipids in the lipid nanoparticles” means, for example, that the lipid nanoparticles contain ionic lipids represented by formula (1), phospholipids, cholesterol, and dioxins represented by formula (3).
  • the amount of B (mol%) with respect to A means “100 ⁇ the amount of B (mol)/the amount of A (mol)".
  • the amount of other lipids (mol%) relative to the total amount of lipids means “100 x amount of other lipids (mol)/total amount of lipids (mol)”.
  • the lipids constituting the lipid nanoparticles of the present invention are ionic lipids represented by formula (1), anionic phospholipids represented by formula (2), or Most preferably, it consists of anionic cholesterol, neutral phospholipid, cholesterol, and dimyristoylglycerol PEG represented by formula (3).
  • the ionic lipid used in the present invention is an ionic lipid represented by the following formula (1) (herein sometimes abbreviated as "ionic lipid (1)"). Only one type of ionic lipid (1) may be used, or two or more types may be used in combination.
  • R 1a and R 1b each independently represent an alkylene group having 1 to 6 carbon atoms
  • X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or 2 to 5 carbon atoms, and represents a cyclic alkylene tertiary amino group having 1 to 2 tertiary amino groups
  • R 2a and R 2b each independently represent an alkylene group or an oxydialkylene group having 8 or less carbon atoms
  • Y a and Y b each independently represent an ester bond, amide bond, carbamate bond, ether bond or urea bond
  • Z a and Z b each independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a hetero atom.
  • n a and n b are each independently 0 or 1
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride.
  • R 9 -O-CO-(CH 2 )a- (4) (In formula (4), R 9 represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, a represents an integer from 2 to 10. ) represents a group represented by )
  • R 1a and R 1b each independently represent an alkylene group having 1 to 6 carbon atoms, and may be linear or branched, but are preferably linear.
  • the alkylene group preferably has 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms.
  • Specific examples of the alkylene group having 1 to 6 carbon atoms include methylene group, ethylene group, trimethylene group, isopropylene group, tetramethylene group, isobutylene group, pentamethylene group, and neopentylene group.
  • R 1a and R 1b are preferably each independently a methylene group, ethylene group, trimethylene group, isopropylene group or tetramethylene group, most preferably each is an ethylene group.
  • R 1a may be the same as or different from R 1b , but preferably R 1a is the same group as R 1b .
  • X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or 2 to 5 carbon atoms, and Represents a cyclic alkylene tertiary amino group having 1 to 2 tertiary amino groups, preferably each independently a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups. is an alkylene tertiary amino group.
  • the alkyl group having 1 to 6 carbon atoms in the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group may be linear or branched. It may also be circular.
  • the alkyl group preferably has 1 to 3 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentyl group, and isopentyl group.
  • neopentyl group, tert-pentyl group 1,2-dimethylpropyl group, 2-methylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 2,2-dimethylbutyl group, 2,3-dimethylbutyl group
  • Examples include a cyclohexyl group, preferably a methyl group, an ethyl group, a propyl group or an isopropyl group, and most preferably a methyl group.
  • a preferred specific structure of the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group is represented by X 1 .
  • R 5 in X 1 represents an alkyl group having 1 to 6 carbon atoms, and may be linear, branched, or cyclic.
  • the alkyl group preferably has 1 to 3 carbon atoms.
  • Examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentyl group, and isopentyl group.
  • neopentyl group, tert-pentyl group 1,2-dimethylpropyl group, 2-methylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 2,2-dimethylbutyl group, 2,3-dimethylbutyl group
  • Examples include a cyclohexyl group, preferably a methyl group, an ethyl group, a propyl group or an isopropyl group, and most preferably a methyl group.
  • the number of carbon atoms in the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups is preferably 4 to 5 carbon atoms.
  • Examples of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups include aziridylene group, azetidylene group, pyrrolidylene group, piperidylene group, imidazolidylene group, It is a piperadylene group, preferably a pyrrolidylene group, a piperidylene group, or a piperadylene group, and most preferably a piperidylene group.
  • a preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 tertiary amino group is represented by X 2 .
  • p in X 2 is 1 or 2.
  • X 2 is a pyrrolidylene group
  • X 2 is a piperidylene group.
  • p is 2.
  • a preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 2 tertiary amino groups is represented by X 3 .
  • w in X 3 is 1 or 2.
  • X 3 is an imidazolidylene group
  • X 3 is a piperazylene group.
  • X a may be the same as or different from X b , preferably X a is the same group as X b .
  • R 2a and R 2b each independently represent an alkylene group or oxydialkylene group having 8 or less carbon atoms, and preferably each independently represents an alkylene group having 8 or less carbon atoms.
  • the alkylene group having 8 or less carbon atoms may be linear or branched, but is preferably linear.
  • the number of carbon atoms contained in the alkylene group is preferably 6 or less, most preferably 4 or less.
  • Examples of the alkylene group having 8 or less carbon atoms include methylene group, ethylene group, trimethylene group, isopropylene group, tetramethylene group, isobutylene group, pentamethylene group, hexamethylene group, heptamethylene group, octamethylene group, etc. Among them, methylene group, ethylene group, trimethylene group, and tetramethylene group are preferable, and ethylene group is most preferable.
  • oxydialkylene group having 8 or less carbon atoms refers to an alkylene group via an ether bond (alkylene-O-alkylene, in other words, “alkyleneoxyalkylene group”), of which there are two It means a group in which the total number of carbon atoms in the alkylene group is 8 or less.
  • alkylene-O-alkylene in other words, "alkyleneoxyalkylene group”
  • Examples of the oxydialkylene group having 8 or less carbon atoms include an oxydimethylene group, an oxydiethylene group, an oxydi(trimethylene) group (i.e., a trimethyleneoxytrimethylene group), an oxydi(tetramethylene) group (i.e., tetramethyleneoxytetramethylene group) and the like.
  • Preferred are an oxydimethylene group, an oxydiethylene group, and an oxydi(trimethylene) group, and most preferred is an oxydiethylene group.
  • R 2a may be the same as or different from R 2b , but preferably R 2a is the same group as R 2b .
  • Y a and Y b are each independently an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, preferably each independently an ester bond, an amide bond, or a carbamate bond, and more preferably each Independently, it is an ester bond or an amide bond, most preferably each an ester bond.
  • the direction of the bond between Y a and Y b is not limited, but when Y a and Y b are ester bonds, preferably -Z a -CO-O-R 2a - and -Z b -CO-O-R 2b It exhibits a structure of -.
  • Y a may be the same as or different from Y b , but preferably Y a is the same group as Y b .
  • Z a and Z b each independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a hetero atom. represents.
  • the number of carbon atoms contained in the aromatic compound is preferably 6 to 12, most preferably 6 to 7.
  • the number of aromatic rings contained in the aromatic compound is preferably one.
  • aromatic rings contained in aromatic compounds having 3 to 16 carbon atoms include benzene rings, naphthalene rings, anthracene rings for aromatic hydrocarbon rings, imidazole rings, pyrazole rings, oxazole rings for aromatic heterocycles, Isoxazole ring, thiazole ring, isothiazole ring, triazine ring, pyrrole ring, furanthiophene ring, pyrimidine ring, pyridazine ring, pyrazine ring, pyridine ring, purine ring, pteridine ring, benzimidazole ring, indole ring, benzofuran ring, quinazoline ring, phthalazine ring, quinoline ring, isoquinoline ring, coumarin ring, chromone ring, benzodiazepine ring, phenoxazine ring, phenothiazine ring, acrid
  • the aromatic ring may have a substituent, and examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, and a carbamoyl group having 2 to 4 carbon atoms.
  • acyloxy group acylamino group having 2 to 4 carbon atoms, alkoxycarbonylamino group having 2 to 4 carbon atoms, fluorine atom, chlorine atom, bromine atom, iodine atom, alkylsulfanyl group having 1 to 4 carbon atoms, 1 carbon number ⁇ 4 alkylsulfonyl group, arylsulfonyl group with 6 to 10 carbon atoms, nitro group, trifluoromethyl group, cyano group, alkyl group with 1 to 4 carbon atoms, ureido group with 1 to 4 carbon atoms, 1 to 4 carbon atoms 4 alkoxy group, aryl group having 6 to 10 carbon atoms, aryloxy group having 6 to 10 carbon atoms, and preferable examples include acetyl group, methoxycarbonyl group, methylcarbamoyl group, acetoxy group, acetamido group, Methoxycarbonylamino group,
  • a preferred specific structure of Z a and Z b includes Z 1 .
  • s represents an integer of 0 to 3
  • t represents an integer of 0 to 3
  • u represents an integer of 0 to 4
  • u R 4 each independently represent a substituent.
  • s in Z 1 is preferably an integer of 0 to 1, more preferably 0.
  • t in Z 1 is preferably an integer of 0 to 2, more preferably 1.
  • u in Z 1 is preferably an integer of 0 to 2, more preferably an integer of 0 to 1.
  • R 4 in Z 1 is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms, which does not inhibit the reaction in the synthesis process of the ionic lipid.
  • substituents include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, and an acylamino group having 2 to 4 carbon atoms.
  • Arylsulfonyl group, nitro group, trifluoromethyl group, cyano group, alkyl group having 1 to 4 carbon atoms, ureido group having 1 to 4 carbon atoms, alkoxy group having 1 to 4 carbon atoms, aryl group having 6 to 10 carbon atoms , an aryloxy group having 6 to 10 carbon atoms, and preferable examples include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamido group, a methoxycarbonylamino group, a fluorine atom,
  • each R 4 may be the same or different.
  • Z a may be the same as or different from Z b , but preferably Z a is the same group as Z b .
  • n a and n b are each independently 0 or 1.
  • na may be the same as or different from nb , preferably na is the same as nb .
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride.
  • ) represents a group represented by, preferably each independently, a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or a carbonized aliphatic group having 12 to 22 carbon atoms.
  • a hydrogen group most preferably each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms.
  • a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride means that the hydroxyl group of the fat-soluble vitamin having a hydroxyl group is *-O-CO-CH 2 -CH 2 - or * -O-CO-CH 2 -CH 2 -CH 2 - represents a group having a structure replaced with -. * represents the bonding position with fat-soluble vitamins.
  • a residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride means that the hydroxyl group of the sterol derivative having a hydroxyl group is *-O-CO-CH 2 -CH 2 - or *-O -CO-CH 2 -CH 2 -CH 2 - represents a group having a structure replaced with -CO-CH 2 -CH 2 -CH 2 -. * represents the bonding position with the sterol derivative.
  • fat-soluble vitamins having a hydroxyl group examples include retinol, ergosterol, 7-dehydrocholesterol, calciferol, colcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, and tocotrienol.
  • the fat-soluble vitamin having a hydroxyl group is preferably tocopherol.
  • sterol derivatives having a hydroxyl group examples include cholesterol, cholestanol, stigmasterol, ⁇ -sitosterol, lanosterol, and ergosterol, with cholesterol or cholestanol being preferred.
  • the aliphatic hydrocarbon group having 1 to 40 carbon atoms may be linear or branched.
  • the aliphatic hydrocarbon group may be saturated or unsaturated.
  • the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, preferably 1 to 3, more preferably 1 to 2.
  • Unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, with carbon-carbon double bonds being preferred.
  • the number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 12 to 22, more preferably 13 to 19, and most preferably 13 to 17.
  • the aliphatic hydrocarbon group includes an alkyl group, an alkenyl group, an alkynyl group, etc., and preferably an alkyl group or an alkenyl group.
  • Examples of aliphatic hydrocarbon groups having 1 to 40 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, and pentyl group.
  • the aliphatic hydrocarbon group having 1 to 40 carbon atoms is preferably a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, and particularly preferably a tridecyl group, They are a heptadecyl group, a heptadecenyl group, and a heptadecadienyl group.
  • the aliphatic hydrocarbon groups having 1 to 40 carbon atoms (preferably 12 to 22 carbon atoms) represented by R 3a and R 3b are derived from fatty acids.
  • the carbonyl carbon derived from the fatty acid is included in -CO-O- in formula (1).
  • Specific examples of the aliphatic hydrocarbon group include a heptadecadienyl group when linoleic acid is used as the fatty acid, and a heptadecenyl group when oleic acid is used as the fatty acid.
  • the alkyl group having 3 to 40 carbon atoms having a cyclopropane ring in R 3a and R 3b means an alkyl group having 3 to 40 carbon atoms having at least one cyclopropane ring in the alkyl chain.
  • the number of carbon atoms in the alkyl group from 3 to 40 does not include the number of carbon atoms in the cyclopropane ring.
  • the number of cyclopropane rings that the alkyl group has is preferably one.
  • the alkyl group having 3 to 40 carbon atoms having a cyclopropane ring in R 3a and R 3b preferably has the formula (5):
  • b and c are each independently integers, and the sum of b and c is 2 to 39.
  • b is an integer from 1 to 20 and c is an integer from 1 to 19.
  • b is more preferably an integer of 2 to 18, still more preferably an integer of 3 to 17, even more preferably an integer of 4 to 12.
  • c is more preferably an integer of 3 to 15, still more preferably an integer of 3 to 11, even more preferably an integer of 3 to 9.
  • Examples of the group represented by formula (5) include 7-(2-octylcyclopropyl)heptyl.
  • the aliphatic hydrocarbon group having 2 to 20 carbon atoms represented by R 9 may be linear or branched.
  • the aliphatic hydrocarbon group may be saturated or unsaturated.
  • the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, preferably 1 to 3, more preferably 1 to 2.
  • Unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, with carbon-carbon double bonds being preferred.
  • the number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 8 to 20, more preferably 9 to 19, still more preferably 13 to 19, and most preferably 13 to 17.
  • the aliphatic hydrocarbon group includes an alkyl group, an alkenyl group, an alkynyl group, etc., preferably an alkyl group or an alkenyl group, and more preferably an alkyl group.
  • Examples of aliphatic hydrocarbon groups having 2 to 20 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, and pentyl group.
  • the aliphatic hydrocarbon group having 2 to 20 carbon atoms is preferably a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, and particularly preferably a tridecyl group, They are a heptadecyl group, a heptadecenyl group, and a heptadecadienyl group.
  • a is preferably an integer of 3 to 9, more preferably an integer of 3 to 7, even more preferably an integer of 5 to 7, and most preferably 5 or 7.
  • R 3a may be the same as or different from R 3b , but preferably R 3a is the same group as R 3b .
  • R 1a is the same as R 1b
  • X a is the same as X b
  • R 2a is the same as R 2b
  • Y a is the same as Y b
  • Z a is the same as Y b
  • Z is the same as b
  • R 3a is the same as R 3b .
  • ionic lipid (1) include the following ionic lipids.
  • R 1a and R 1b are each independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene group, ethylene group);
  • X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and having 1 tertiary amino group (e.g., -N(CH 3 )-); or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups (e.g., piperidylene group);
  • R 2a and R 2b are each independently an alkylene group having 8 or less carbon atoms (e.g., methylene group, ethylene group, trimethylene group);
  • Y a and Y b are each independently an ester bond or an amide bond;
  • a divalent group in which Z a and Z b are each alkylene
  • tocopherol and succinic anhydride or glutaric anhydride, or an aliphatic group having 12 to 22 carbon atoms.
  • is a hydrocarbon group e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group
  • Ionic lipids (1).
  • R 1a and R 1b are each independently an alkylene group having 1 to 4 carbon atoms (e.g., methylene group, ethylene group);
  • X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 3 carbon atoms and having 1 tertiary amino group (e.g., -N(CH 3 )-); or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 tertiary amino group (e.g., piperidylene group);
  • R 2a and R 2b are each independently an alkylene group having 6 or less carbon atoms (e.g., methylene group, ethylene group, trimethylene group);
  • Y a and Y b are each independently an ester bond or an amide bond;
  • Z a and Z b are each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally
  • tocopherol and succinic anhydride, or an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g. , heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group); Ionic lipids (1).
  • R 1a and R 1b are each independently an alkylene group having 1 to 2 carbon atoms (i.e., a methylene group or an ethylene group);
  • X a and X b are each independently X 1 :
  • R 5 is an alkyl group having 1 to 3 carbon atoms (eg, methyl group).), or X 2 :
  • R 2a and R 2b are each independently an alkylene group having 4 or less carbon atoms (e.g., methylene group, ethylene group, trimethylene group); Y a and Y b are each independently an ester bond or an amide bond; Z a and Z b are each independently Z 1 :
  • s is an integer of 0 to 1
  • t is an integer of 0 to 2
  • u is an integer of 0 to 2 (preferably 0)
  • u R 4 are each independently (represents a substituent)
  • n a and n b are each independently 0 or 1
  • R 3a and R 3b each independently represent a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g. tocopherol) and succinic anhydride, or an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g. , heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group); Ionic lipids (1).
  • the ionic lipid (1) include the following O-Ph-P3C1, O-Ph-P4C1, O-Ph-P4C2, O-Bn-P4C2, E-Ph-P4C2, L-Ph-P4C2, HD -Ph-P4C2, O-Ph-amide-P4C2, O-Ph-C3M, and TS-P4C2.
  • Lipid 1 to Lipid 20 described in WO 2021/195529 A2 can be mentioned, and in particular, the following Lipid 1, Lipid 5, and Lipid 8 can be mentioned.
  • the ionic lipid (1) is preferably an ionic lipid represented by the following formula.
  • the amount of ionic lipid (1) in the lipid nanoparticles of the present invention is determined from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and stability of lipid nanoparticles.
  • Ionic lipid (1) can be produced by a known method (for example, the method described in WO 2019/188867 A1, US 9708628 B2, WO 2021/195529 A2).
  • the lipid nanoparticles of the present invention contain anionic phospholipids as phospholipids.
  • anionic phospholipids may be used, or anionic phospholipids and other neutral phospholipids may be used in combination.
  • anionic phospholipids include 1,2-diacyl-sn-glycero-3-phosphoserine (PS), 1,2-diacyl-sn-glycero-3-phosphoglycerol (PG), and 1,2-diacyl-sn-glycero-3-phosphoglycerol (PG).
  • PS 1,2-diacyl-sn-glycero-3-phosphoserine
  • PG 1,2-diacyl-sn-glycero-3-phosphoglycerol
  • PG 1,2-diacyl-sn-glycero-3-phosphoglycerol
  • PA -sn-glycero-3-phosphatidic acid
  • 1,2-diacyl-sn-glycero-3-phosphoserine include: 1,2-didecanoyl-sn-glycero-3-phosphoserine (DDPS), 1,2-dilauroyl-sn-glycero-3-phosphoserine (DLPS), 1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dilinoleoyl-sn-glycero-3-phosphoserine (DLoPS), 1,2-diercyl-sn-glycero-3-phosphoserine (DEPS), 1-myristoyl-2-palmitoyl-sn-glycero-3-
  • phospholipids may be described by their abbreviations.
  • 1,2-diacyl-sn-glycero-3-phosphoserine is sometimes described as PS
  • 1,2-didecanoyl-sn-glycero-3-phosphoserine is sometimes described as DDPS.
  • 1,2-diacyl-sn-glycero-3-phosphoglycerol include: 1,2-didecanoyl-sn-glycero-3-phosphoglycerol (DDPG), 1,2-dilauroyl-sn-glycero-3-phosphoglycerol (DLPG), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dilinoleoyl-sn-glycero-3-phosphoglycerol (DLoPG), 1,2-diercyl-sn-glycero-3-phosphoglycerol (DEPG), 1-myristoyl-2-pal, 1,2-pal
  • 1,2-diacyl-sn-glycero-3-phosphatidic acid include: 1,2-didecanoyl-sn-glycero-3-phosphatidic acid (DDPA), 1,2-dilauroyl-sn-glycero-3-phosphatidic acid (DLPA), 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), 1,2-distearoyl-sn-glycero-3-phosphatidic acid (DSPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), 1,2-dilinoleoyl-sn-glycero-3-phosphatidic acid (DLoPA), 1,2-diercyl-sn-glycero-3-phosphatidic acid (DEPA), 1-myristoyl-2-palmitoyl-sn-glycero-3-
  • the anionic phospholipid is preferably 1,2-diacyl-sn-glycero-3-phosphoglycerol, more preferably at least one selected from the group consisting of DMPG, DPPG, POPG, DOPG, SOPG and DSPG. , more preferably POPG or DOPG. Since these phospholipids have similar acyl chain structures, they are thought to have similar effects.
  • anionic phospholipid are preferably 1,2-diacyl-sn-glycero-3-phosphoserine, more preferably at least one selected from the group consisting of DPPS, POPS, DOPS, DLoPS, SOPS and DSPS.
  • DPPS 1,2-diacyl-sn-glycero-3-phosphoserine
  • DOPS DOPS
  • DLoPS DOPS
  • DLoPS DLoPS
  • Examples of other neutral phospholipids include 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE), Examples include the body.
  • PC 1,2-diacyl-sn-glycero-3-phosphocholine
  • PE 1,2-diacyl-sn-glycero-3-phosphoethanolamine
  • 1,2-diacyl-sn-glycero-3-phosphocholine include: 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC), 1,2-Dialachidoyl-sn-glycero-3-phosphocholine (DAPC) 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEi)
  • 1,2-diacyl-sn-glycero-3-phosphoethanolamine examples include: 1,2-didecanoyl-sn-glycero-3-phosphoethanolamine (DDPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), 1,2-diercyl-sn-glycero-3-phosphoethanolamine (DEPE), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine (DDPE), 1,
  • neutral phospholipids are not particularly limited, but are preferably at least one selected from the group consisting of DPPC, DOPC, DSPC, DEPC, POPC, SOPC, POPE, and DOPE, and more preferably DSPC, DOPC, and DOPE. At least one selected from the group consisting of DEPC, most preferably DEPC.
  • the ratio of anionic phospholipids to neutral phospholipids is preferably 100:0-25:75 mol%, more preferably 75:25-25:75 mol%.
  • the ratio is 25:75 mol%, more preferably 50:50 mol%.
  • the total amount of anionic phospholipids in the lipid nanoparticles of the present invention is determined from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability. It is preferably 1 to 20 mol%, more preferably 2.5 to 15 mol%, and even more preferably It is 2.5 to 10 mol%.
  • the total amount of neutral phospholipids in the lipid nanoparticles of the present invention is determined from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability. It is preferably 0 to 15 mol%, more preferably 2 to 10 mol%, even more preferably 2. It is 5 to 7.5 mol%.
  • the lipid nanoparticles of the present invention may contain the anionic cholesterol of formula (2) (herein sometimes abbreviated as "anionic cholesterol (2)”) instead of the anionic phospholipid.
  • T represents a divalent aliphatic hydrocarbon group having 1 to 8 carbon atoms.
  • the divalent aliphatic hydrocarbon group having 1 to 8 carbon atoms represented by T may be linear, branched, or contain unsaturation.
  • T is preferably a straight chain divalent saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms, more preferably a straight chain divalent saturated aliphatic hydrocarbon group having 1 to 3 carbon atoms.
  • a specific example of the anionic cholesterol (2) is cholesteryl hemisuccinate.
  • the total amount of anionic cholesterol (2) in the lipid nanoparticles of the present invention is determined from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and stability of lipid nanoparticles. It is preferably 1 to 20 mol%, more preferably 2.5 to 15 mol%, and even more preferably 2. .5 to 10 mol%.
  • the lipid nanoparticles of the present invention contain cholesterol.
  • the amount of cholesterol in the lipid nanoparticles of the present invention is determined from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability.
  • the amount is preferably 20 to 60 mol%, more preferably 25 to 50 mol%, and even more preferably 30 to 40 mol%, based on the total of anionic cholesterol (2), neutral phospholipid, and cholesterol.
  • Dimyristoylglycerol PEG The lipid nanoparticles of the present invention have the formula (3): CH 2 (OR 6 )-CH (OR 7 )-CH 2 (OR 8 ) (3) (In formula (3), Any two of R 6 , R 7 and R 8 represent a myristoyl group, and the remaining one represents the number of carbon atoms connected via a polyethylene glycol chain (PEG chain) with a number average molecular weight of 1,000 to 3,000. Represents 1 to 6 alkyl groups. ) It includes dimyristoylglycerol PEG (sometimes abbreviated as "dimyristoylglycerol PEG (3)" herein) represented by:
  • the number average molecular weight of the PEG chain in formula (3) is 1,000 to 3,000, preferably 1,500 to 2,500.
  • the number average molecular weight of the PEG used to form this PEG chain can be determined by gel permeation chromatography (GPC).
  • the alkyl group having 1 to 6 carbon atoms may be linear, branched, or cyclic.
  • the alkyl group preferably has 1 to 3 carbon atoms.
  • Examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentyl group, and isopentyl group.
  • Examples include cyclohexyl group. Preferably it is a methyl group.
  • the amount of dimyristoylglycerol PEG (3) in the lipid nanoparticles of the present invention is determined based on the ionic lipid (1) from the viewpoint of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and stability of lipid nanoparticles. , anionic phospholipid or anionic cholesterol (2), neutral phospholipid, and cholesterol, preferably 0.5 to 1.5 mol%, more preferably 1.0 mol%.
  • Optimal Composition The optimal molar ratio of ionic lipid (1): anionic phospholipid or anionic cholesterol (2): neutral phospholipid: cholesterol: dimyristoylglycerol PEG (3) in the lipid nanoparticles of the present invention is 55 :5:5:35:1.0.
  • the lipid nanoparticles of the present invention are produced by preparing lipid raw materials containing an ionic lipid (1), an anionic phospholipid or anionic cholesterol (2), cholesterol, and dimyristoylglycerol PEG (3) in an appropriate manner. It can be produced by dispersing it in a dispersion medium (for example, an aqueous dispersion medium, an alcoholic dispersion medium), and performing an operation to induce organization as necessary.
  • a dispersion medium for example, an aqueous dispersion medium, an alcoholic dispersion medium
  • Examples of the "organization-inducing operation" for producing the lipid nanoparticles of the present invention include an alcohol (e.g., ethanol, tert-butanol, etc.) dilution method using a microchannel or vortex, and a simple hydration method. , ultrasonication, heating, vortexing, ether injection method, French press method, cholic acid method, Ca 2+ fusion method, freeze-thaw method, reverse phase evaporation method, etc., and preferably microflow method.
  • the method is an alcohol dilution method using a channel or a vortex, and more preferably an alcohol dilution method using a microchannel.
  • a dispersion containing lipid nanoparticles is prepared by mixing an acidic buffer containing a nucleic acid with an ethanol solution of a lipid. liquid can be produced.
  • the dispersion produced by this method contains lipid nanoparticles and a dispersion medium (acidic buffer and alcohol), but the dispersion medium (especially alcohol) can be removed by operations such as ultrafiltration, dialysis, and dilution. (especially buffer solutions) can be exchanged.
  • the present invention also provides a method for delivering nucleic acids to spleen cells, which comprises administering to a subject the lipid nanoparticles of the present invention encapsulating nucleic acids.
  • the lipid nanoparticles are preferably administered intravenously to the subject.
  • nucleic acid examples include, but are not limited to, DNA, RNA, chimeric nucleic acids of RNA, hybrids of DNA/RNA, and the like. Further, the nucleic acid can be used in any one- to three-stranded form, but is preferably single-stranded or double-stranded. Nucleic acids are, for example, nucleotides having N-glycosides of purine or pyrimidine bases, oligomers having non-nucleotide backbones (such as commercially available peptide nucleic acids (PNA), etc.), or oligomers having special bonds (provided that the oligomers are not DNA). or containing nucleotides having a configuration that allows base pairing or base attachment as found in RNA).
  • PNA commercially available peptide nucleic acids
  • nucleic acids may include, for example, nucleic acids with known modifications, nucleic acids with labels known in the art, capped nucleic acids, methylated nucleic acids, substitution of one or more natural nucleotides with analogs.
  • Nucleic acids with side groups such as proteins (e.g., nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) or sugars (e.g., monosaccharides, etc.)
  • Nucleic acids containing intercurrent compounds e.g., acridine, psoralen, etc.
  • nucleic acids containing chelating compounds e.g., metals, radioactive metals, boron, oxidizing metals, etc.
  • alkylating agents e.g., metals, radioactive metals, boron, oxidizing metals, etc.
  • It may be a nucleic acid having a modified bond (for example, an ⁇ -anomer type nucleic acid, etc.).
  • the type of DNA that can be used in the present invention is not particularly limited, and can be appropriately selected depending on the purpose of use.
  • the DNA include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligo, etc., preferably plasmid DNA, cDNA, and antisense DNA, and more preferably plasmid DNA.
  • Circular DNA such as plasmid DNA can be digested with restriction enzymes as appropriate and used as linear DNA.
  • RNA that can be used in the present invention is not particularly limited, and can be appropriately selected depending on the purpose of use.
  • examples of RNA include siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc., and preferably, siRNA, miRNA, shRNA, mRNA, antisense RNA, and RNA replicon.
  • nucleic acids used in the present invention are preferably purified by methods commonly used by those skilled in the art.
  • the nucleic acids used in the present invention preferably have prophylactic and/or therapeutic activity against a certain disease (preventive/therapeutic nucleic acids).
  • examples of such nucleic acids include nucleic acids used in so-called gene therapy.
  • the particle size of the lipid nanoparticles encapsulating nucleic acid is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm.
  • the particle size can be measured using, for example, a particle size distribution measuring device such as Zetasizer Nano (Malvern).
  • Zetasizer Nano Zetasizer Nano
  • the particle size of lipid nanoparticles encapsulating nucleic acids can be adjusted as appropriate depending on the manufacturing method.
  • the surface potential (zeta potential) of lipid nanoparticles encapsulating nucleic acids for introducing nucleic acids into spleen cells is preferably -30 to +10 mV, more preferably -25 to 0 mV.
  • particles with positively charged surfaces have been mainly used. This is useful as a method for promoting electrostatic interaction with negatively charged heparin sulfate on the cell surface and promoting its uptake into cells.
  • a positive surface potential may result in (a) inhibition of the release of nucleic acid from the carrier due to interaction with the delivered nucleic acid within the cell, and (b) inhibition of protein synthesis due to the interaction of mRNA with the delivered nucleic acid.
  • the surface potential (zeta potential) can be measured using, for example, a zeta potential measuring device such as Zetasizer Nano.
  • the surface potential (zeta potential) of lipid nanoparticles can be adjusted by the composition of the constituent components of the lipid nanoparticles.
  • the lipid nanoparticles of the present invention By administering the lipid nanoparticles of the present invention encapsulating a nucleic acid to a living body, the lipid nanoparticles reach and contact the spleen tissue, and the nucleic acid encapsulated in the lipid nanoparticles is delivered to the spleen tissue in the living body.
  • the subjects to which the lipid nanoparticles can be administered are not particularly limited, and include, for example, mammals (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), birds (e.g., chickens, ostriches, etc.), amphibians (e.g. , frog, etc.), fish (e.g., zebrafish, medaka, etc.), and the like.
  • the subject into which the lipid nanoparticles are introduced is preferably a human or other mammalian cell.
  • the method of administering the lipid nanoparticles encapsulating a nucleic acid to a subject is not particularly limited as long as the lipid nanoparticles can deliver the nucleic acid to spleen cells, and can be administered by any known method (for example, oral administration, parenteral administration).
  • any known method for example, oral administration, parenteral administration.
  • intravenous administration is preferred.
  • the dosage of the lipid nanoparticles can be appropriately selected in consideration of the type of recipient, administration method, and the like.
  • the lipid nanoparticles of the present invention can be used as they are or mixed with a pharmaceutically acceptable carrier to form oral preparations (e.g., tablets, capsules, etc.) or parenteral preparations (e.g., nasal preparations, injections, inhalation preparations, etc.). It can be produced as a parenteral agent (more preferably a nasal agent).
  • pharmaceutically acceptable carriers those commonly used as pharmaceutical ingredients are used; for example, excipients, lubricants, binders, disintegrants, etc. are used in solid preparations, and in liquid preparations, A solvent, a solubilizing agent, a suspending agent, an isotonizing agent, a buffering agent, an analgesic agent, and the like are used. Further, if necessary, formulation additives such as preservatives, antioxidants, colorants, and sweeteners can also be used. For example, in the case of nasal preparations, they are used in the form of nasal drops or sprays.
  • ionic lipid (1) is shown by the name listed in the table above. Furthermore, the meanings of the abbreviations used in the following examples are as follows.
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
  • DSPC 1,2-distearoyl-sn -Glycero-3-phosphocholine
  • SOPC 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • DEPC 1,2-diercoyl-sn-glycero-3-phosphocholine
  • POPE 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOPG 1,2-
  • the acidic buffer solution of nucleic acid is to prepare mRNA (luc-mRNA) encoding the luciferase gene at 0.0067 ⁇ g/ ⁇ L in 20 mM acidic malate buffer containing 30 mM NaCl ( pH 3.0).
  • the transferred LNP solution was subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, about 10 minutes) and concentrated to about 500 ⁇ L.
  • the obtained concentrate was diluted to 4 mL using PBS, and ultrafiltrated again under centrifugal conditions (25° C., 1000 g, about 10 minutes) to concentrate to about 200 ⁇ L.
  • the solution was diluted with PBS to a nucleic acid concentration of 10 ⁇ g/mL.
  • Spleen gene expression activity evaluation 1 (combination evaluation with anionic phospholipid or anionic cholesterol)
  • IV administration to mice Luc-mRNA-encapsulated LNP prepared according to the method described in Production Example 1 was diluted with PBS to an mRNA concentration of 5.0 ⁇ g/mL.
  • the diluted mRNA-encapsulated LNPs were intravenously administered to 6-week-old female BALB/c mice at a dose of 200 ⁇ L per mouse (mRNA dose: 1.0 ⁇ g per mouse).
  • mRNA dose 1.0 ⁇ g per mouse
  • the cryopreserved tube was taken out, and 800 ⁇ L of Lysis buffer (77 mM Tris, 2 mM EDTA 2Na, MQ pH 7.8 containing 0.1% Triton X-100) was added. Homogenization was repeated twice at -2° C. and 4500 rpm for 30 seconds using Micro Smash (TOMY). 500 ⁇ L of the supernatant was transferred to another tube, centrifuged (4° C., 13000 rpm, 10 minutes), and 300 ⁇ L of the supernatant was collected.
  • Lysis buffer 77 mM Tris, 2 mM EDTA 2Na, MQ pH 7.8 containing 0.1% Triton X-100
  • the gene expression activity is shown in FIG. 1 as a relative value to Comparative Example 1.
  • the LNPs of Examples 1 and 2 had higher gene expression activity in the spleen than the LNPs of Comparative Example 1, which had a composition for liver transport. Since gene expression activity depends on the efficiency of nucleic acid delivery into target cells, this shows that the LNP can efficiently deliver nucleic acids to the spleen.
  • the diluted mRNA-encapsulated LNPs were intravenously administered to 6-week-old female BALB/c mice at a dose of 200 ⁇ L per mouse (mRNA dose: 1.0 ⁇ g per mouse).
  • FIG. 2 is a diagram showing the fluorescence intensity of each LNP, and shows the amount of fluorescently labeled LNP accumulated in the spleen.
  • FIG. 3 is a diagram showing gene expression activity in the spleen and shows nucleic acid delivery efficiency. It was confirmed that the LNP of Example 3 has higher LNP accumulation in the spleen and higher nucleic acid delivery efficiency than the LNP of Comparative Example 2, which is LNP with a composition for liver transport.
  • Test Example 4 Evaluation of gene expression activity in spleen 3 (combination evaluation with neutral phospholipid) (1) IV administration to mice Luc-mRNA-encapsulated LNPs prepared according to the method described in Production Example 1 were intravenously administered to mice under the same conditions as Test Example 1 (mRNA dose: 1.0 ⁇ g per mouse). ). (2) Measurement of gene expression activity Measurement was performed by the method described in Test Example 2, and gene expression activity was calculated as a relative value to Comparative Example 1.
  • CTL OVA-specific cytotoxic T cell activity.
  • the following composition was used as a culture medium for splenocytes: 500 mL of RPMI1640, 50 mL of FBS, 5 mL of 100 mM sodium pyruvate, 5 mL of 1M HEPES, 500 ⁇ L of 55 mM 2-mercaptoethanol, and 5 mL of penicillin/streptomycin (hereinafter referred to as the medium). Untreated mice were euthanized by cervical dislocation, and the spleens were harvested.
  • the spleen was cut from the center, and spleen cells were taken out from the cut surface using tweezers and suspended in a medium.
  • the cell suspension medium was collected through a 40 ⁇ m cell strainer into a 50 mL tube and centrifuged (4° C., 500 g, 5 minutes). The supernatant was discarded, 1 mL of Red Blood Cell Lysis Buffer (SIGMA) was added per animal, and the mixture was allowed to stand for 5 minutes. After diluting the mixture 5 times with a medium, it was centrifuged (4°C, 500g, 5 minutes). Furthermore, it was suspended in 10 mL of medium and centrifuged (4° C., 500 g, 5 minutes).
  • SIGMA Red Blood Cell Lysis Buffer
  • the cells were suspended in 30 mL of medium and the number of cells was counted. Thereafter, the mixture was passed through a 40 ⁇ m cell strainer and divided into two 50 mL tubes in equal amounts and centrifuged (4° C., 500 g, 5 minutes). The cells were suspended in a medium to a concentration of 1.0 ⁇ 10 7 cells/mL.
  • a 2 mM OVA epitope (consisting of the amino acid sequence of positions 257-264 of OVA) solution was added to one of the cell suspensions in an amount of 1/400 of the cell suspension, and the suspension was left standing in a culture incubator for 1 hour. This epitope pulse group was centrifuged (4°C, 500g, 5 minutes).
  • mice 100 ⁇ L of “target cells” and 100 ⁇ L of “control cells” were mixed and administered to immunized mice. Both can be distinguished from the intensity of CFSE fluorescence. Twenty hours after administration of the cell mixture, the spleen was collected from the mouse and subjected to flow cytometry analysis. Epitope-specific CTL activity was quantified by correcting the abundance of "target cells” with "control cells.”
  • the evaluation results are shown in Figures 8 and 9.
  • the LNPs with the compositions of Examples 18 and 24 exhibited approximately three times as much CTL activity as the LNPs with the composition of Comparative Example 1, which is the LNP composition for liver delivery.
  • the LNP having the composition of Example 30 showed higher CTL activity than the LNP of Comparative Example 3, which is LNP having a composition for liver delivery, and the LNP of Comparative Example 4, which has LNP having a composition for spleen delivery.
  • CTL activity is an index for evaluating whether acquired immunity can be obtained, and the higher the CTL activity, the more acquired immunity can be obtained. Therefore, it was shown that the LNPs of Examples 18, 24, and 30 were able to enhance acquired immunity more than the comparative example.
  • mice Ten-week-old female C57BL/6J mice were preliminarily bred for one week to acclimate them to the breeding environment.
  • Myelin oligodendrocyte glycoprotein hereinafter referred to as MOG
  • EAE experimental autoimmune encephalomyelitis
  • a mixed emulsion of an autoantigen peptide consisting of the amino acid sequence of positions 35-55 of MOG
  • complete Freund's adjuvant was subcutaneously administered to the neck and lumbar region of the mouse in an amount of 100 ⁇ L.
  • the pertussis toxin stock included in the kit product was diluted with PBS to 165 ng/100 ⁇ L.
  • the diluted pertussis toxin was intraperitoneally administered at 100 ⁇ L per animal twice, on the day of administration of the mixed emulsion and the next day.
  • MOG-mRNA IV administration to mice mRNA encoding luc-mRNA-encapsulated LNP or MOG peptide (consisting of the amino acid sequence at positions 27-63 of MOG) prepared according to the method described in Production Example 1 (hereinafter referred to as MOG-mRNA)
  • MOG-mRNA MOG-mRNA
  • the LNPs encapsulating LNPs were diluted with PBS to an mRNA concentration of 1.0 ⁇ g/200 ⁇ L.
  • the diluted mRNA-encapsulated LNPs were intravenously administered to EAE mice in an amount of 200 ⁇ L per mouse three times on days 7, 10, and 13 from the day of EAE induction.
  • the lipid nanoparticles of the present invention are useful for delivering nucleic acids to spleen cells.

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