WO2024248146A1 - ｐＨ感受性カチオン性脂質及び脂質ナノ粒子 - Google Patents

ｐＨ感受性カチオン性脂質及び脂質ナノ粒子 Download PDF

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WO2024248146A1
WO2024248146A1 PCT/JP2024/020084 JP2024020084W WO2024248146A1 WO 2024248146 A1 WO2024248146 A1 WO 2024248146A1 JP 2024020084 W JP2024020084 W JP 2024020084W WO 2024248146 A1 WO2024248146 A1 WO 2024248146A1
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carbon atoms
lipid
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悠介 佐藤
秀吉 原島
里奈 伊藤
裕一 鈴木
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Hokkaido University NUC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/145—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/15—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/60—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D211/62—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals attached in position 4

Definitions

  • the present invention relates to a pH-sensitive cationic lipid having three hydrophobic chains, and to lipid nanoparticles containing the pH-sensitive cationic lipid.
  • Lipid nanoparticles are used as carriers for encapsulating and delivering nucleic acids such as lipophilic drugs, siRNA (short interfering RNA), and mRNA to target cells.
  • nucleic acids such as lipophilic drugs, siRNA (short interfering RNA), and mRNA
  • lipid nanoparticles containing pH-sensitive cationic lipids as constituent lipids that are electrically neutral at physiological pH and change to cationic in a weakly acidic pH environment such as an endosome have been reported as lipid nanoparticles that serve as carriers for efficiently delivering nucleic acids such as siRNA into target cells (Patent Document 1).
  • Lipid nanoparticles that are positively charged in blood interact nonspecifically with plasma proteins and are rapidly eliminated from the blood by the reticuloendothelial system.
  • lipid nanoparticles containing pH-sensitive cationic lipids as constituent lipids are uncharged under neutral conditions in blood, but are positively charged under the weakly acidic conditions in endo
  • Non-Patent Document 1 As an example of a pH-sensitive cationic lipid, Jayaraman et al. developed DLin-MC3-DMA, which achieved an ED 50 of 0.005 mg siRNA/kg in factor 7 (F7) knockdown in mouse liver (Non-Patent Document 1). The present inventors have also developed their own pH-sensitive cationic lipids YSK05 and YSK13-C3, which achieved ED 50 of 0.06 and 0.015 mg siRNA/kg, respectively, in F7 knockdown (Non-Patent Documents 2 to 4).
  • the present inventors have also developed a compound having two hydrocarbon chains and one hydrophilic group, such as 7-(4-(diisopropylamino)butyl)-7-hydroxytridecane-1,13-diyl dioleate (CL4H6), as a pH-sensitive cationic lipid (Patent Document 1).
  • lipid nanoparticles containing nucleic acids, etc. based on the alcohol dilution method using a flow channel.
  • lipid nanoparticles with a diameter of about 30 nm can be produced with good reproducibility by using a microflow channel with a built-in three-dimensional micromixer that can achieve instantaneous mixing of two liquids (Non-Patent Document 5).
  • a nano-sized lipid particle formation system with higher particle size controllability can be formed by using a simple two-dimensional flow channel structure in which baffles (baffle plates) of a certain width relative to the flow channel width are arranged alternately on both sides of a micro-sized flow channel through which a raw material solution flows (Patent Document 2).
  • baffles baffle plates
  • These methods for producing nanoparticle preparations have been mainly adopted in recent years for the production of lipid nanoparticles (LNPs) loaded with fat-soluble drugs and nucleic acids such as siRNA (short interfering RNA) or mRNA.
  • the present invention aims to provide a pH-sensitive cationic lipid that can be synthesized more easily with fewer steps without using organometallic reactions or relatively expensive raw materials, and a lipid nanoparticle containing the pH-sensitive cationic lipid.
  • Tris tris(hydroxymethyl)aminomethane
  • R 1 is represented by the following general formulae (T-1) to (T-3):
  • R 21 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 22 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 23 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 24 is a hydrogen atom or a hydrocarbon group having 1 to 22 carbon atoms
  • R 25 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 26 is a hydrogen atom or a hydrocarbon group having 1 to 22 carbon atoms.
  • the sum of the carbon numbers of R 21 and R 22 is 8 or more
  • the sum of the carbon numbers of R 23 and R 24 is 6 or more
  • the sum of the carbon numbers of R 25 and R 26 is 6 or more.
  • R 1 which is three in one molecule, may be the same or different;
  • Z 1 is an alkylene group having 1 to 6 carbon atoms which may be substituted with one hydroxyalkyl group having 1 to 3 carbon atoms, or a single bond;
  • X is -N(R 2 )(R 3 ) or a 5- to 7-membered non-aromatic heterocyclic group (wherein the group is bonded to Z 1 via a carbon atom and the ring may be substituted with one or two hydrocarbon groups or amide groups having 1 to 4 carbon atoms);
  • R 2 and R 3 are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms in which one or two hydrogen atoms may be substituted with a phenyl group, a pyrrolyl group, a pyrimidyl group, or an indolyl group, but R 2 and R 3 may be bonded to each other to form a 5- to 7-membered non-ar
  • a pH-sensitive cationic lipid comprising a compound represented by the formula: [2]
  • -Z 1 -X is represented by the following formulae (H-1) to (H-8) and (H-16) to (H-26):
  • the pH-sensitive cationic lipid according to [1] above selected from the following: [3] The pH-sensitive cationic lipid according to [1] or [2] above, wherein all three R 1s in one molecule are the same group. [4] The pH-sensitive cationic lipid according to any one of [1] to [3] above, which is represented by the general formula (I') and wherein the s is 2 or 3.
  • the R 1 is represented by the following formula (A8), (A8 ⁇ ), (A10), (A12), (A12 ⁇ ), (A14), (A16), (A16 ⁇ ), (A16b), (A20), (A24), (E12), (E14), (A14 ⁇ ), (A24 ⁇ ), (A10L), (A12L), or (A14L):
  • the black circle represents a bond.
  • a method for producing a pH-sensitive cationic lipid comprising condensing a compound represented by the following general formula (A), a compound represented by the following general formula (B), and tris(hydroxymethyl)aminomethane to produce a pH-sensitive cationic lipid consisting of a compound represented by the following general formula (I):
  • R 1 represents the following general formulae (T-1) to (T-3):
  • R 21 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 22 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 23 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 24 is a hydrogen atom or a hydrocarbon group having 1 to 22 carbon atoms
  • R 25 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 26 is a hydrogen atom or a hydrocarbon group having 1 to 22 carbon atoms.
  • the sum of the carbon numbers of R 21 and R 22 is 8 or more
  • the sum of the carbon numbers of R 23 and R 24 is 6 or more
  • the sum of the carbon numbers of R 25 and R 26 is 6 or more.
  • Z 2 is a linear alkylene group having 4 to 6 carbon atoms, or a cycloalkylene group
  • Z 1 is an alkylene group having 1 to 6 carbon atoms which may be substituted with one hydroxyalkyl group having 1 to 3 carbon atoms, or a single bond
  • X is -N(R 2 )(R 3 ), or a 5- to 7-membered non-aromatic heterocyclic group (wherein the group is bonded to Z 1 via a carbon atom, and the ring may be substituted with one or two hydrocarbon groups or amide groups having 1 to 4 carbon atoms)
  • R 2 and R 3 are each independently a hydrogen atom, or a hydrocarbon group having 1 to 4 carbon atoms in which one or two hydrogen atoms may be substituted with a phenyl group, a pyrrolyl group, a pyrimidyl group, or an indolyl group, but R 2 and R 3 may be bonded to each other to form
  • a method for producing a pH-sensitive cationic lipid comprising: producing a compound represented by the following general formula (A') by condensing a compound represented by the following general formula (A-0) with a compound represented by the following general formula (D); and producing a pH-sensitive cationic lipid consisting of a compound represented by the following general formula (I') by condensing the compound represented by the general formula (A'), a compound represented by the following general formula (B), and tris(hydroxymethyl)aminomethane.
  • R 1 represents the following general formulae (T-1) to (T-3):
  • R 21 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 22 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 23 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 24 is a hydrogen atom or a hydrocarbon group having 1 to 22 carbon atoms
  • R 25 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 26 is a hydrogen atom or a hydrocarbon group having 1 to 22 carbon atoms.
  • the sum of the carbon numbers of R 21 and R 22 is 8 or more
  • the sum of the carbon numbers of R 23 and R 24 is 6 or more
  • the sum of the carbon numbers of R 25 and R 26 is 6 or more.
  • the black circles represent bonds.) or a single bond;
  • X is -N(R 2 )(R 3 ) or a 5-7 membered non-aromatic heterocyclic group (wherein the group is bonded to Z 1 via a carbon atom and the ring may be substituted with 1 or 2 hydrocarbon groups or amide groups having 1 to 4 carbon atoms);
  • R 2 and R 3 are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms in which 1 or 2 hydrogen atoms may be substituted with a phenyl group, a pyrrolyl group, a pyrimidyl group, or an indolyl group, but R 2 and R 3 may be bonded to each other to form a 5- to 7-membered non-aromatic heterocycle (a hydrogen atom in the ring may be substituted with a hydrocarbon group having 1 to 4 carbon atoms, an amide group, or a nitrogen-containing cyclic group; one carbon atom in the
  • R2 and R3 are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms in which one or two hydrogen atoms may be substituted by a phenyl group, a pyrrolyl group, a pyrimidyl group, or an indolyl group, and R2 and R3 may be bonded to each other to form a 5- to 7-membered non-aromatic heterocycle (in which a hydrogen atom may be substituted by a hydrocarbon group having 1 to 4 carbon atoms, an amide group, or a nitrogen-containing cyclic group; one carbon atom of the ring may be a carbon atom constituting another nitrogen-containing cyclic group)] After adding N-[tris(hydroxymethyl)methyl]acrylamide to a compound represented by the following general formula (A) or general formula (A'):
  • R 1 is represented by the following general formulae (T-1) to (T-3):
  • R 21 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 22 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 23 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 24 is a hydrogen atom or a hydrocarbon group having 1 to 22 carbon atoms
  • R 25 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 26 is a hydrogen atom or a hydrocarbon group having 1 to 22 carbon atoms.
  • the sum of the carbon numbers of R 21 and R 22 is 8 or more
  • the sum of the carbon numbers of R 23 and R 24 is 6 or more
  • the sum of the carbon numbers of R 25 and R 26 is 6 or more.
  • R 1 , R 2 and R 3 are the same as defined above; the three R 1 s in one molecule may be the same or different groups;
  • Z2 is the same as defined above.
  • formula (I'-a) s is the same as defined above.
  • a method for producing a pH-sensitive cationic lipid comprising producing a pH-sensitive cationic lipid comprising a compound represented by the formula:
  • a lipid nanoparticle comprising the pH-sensitive cationic lipid according to any one of [1] to [7] above.
  • the lipid nanoparticle of [11] further comprising a sterol and a polyalkylene glycol-modified lipid.
  • the lipid nanoparticle according to [14], wherein the nucleic acid is siRNA, mRNA, gRNA, or plasmid DNA.
  • a pharmaceutical composition comprising, as an active ingredient, lipid nanoparticles containing the pH-sensitive cationic lipid according to any one of [1] to [7] above.
  • a pharmaceutical composition comprising, as an active ingredient, lipid nanoparticles containing the pH-sensitive cationic lipid according to any one of [1] to [7] above and a nucleic acid.
  • a method for expressing an exogenous gene comprising administering to a subject animal the lipid nanoparticles according to [14] above, which encapsulate an exogenous gene to be expressed in a target cell, and expressing the exogenous gene in the target cell of the subject animal.
  • a method for expressing an exogenous gene comprising administering the lipid nanoparticles according to [14] above, which encapsulate an exogenous gene to be expressed in a target cell, to a subject animal (excluding humans), thereby expressing the exogenous gene in the target cell of the subject animal.
  • the pH-sensitive cationic lipid according to the present invention is relatively easy to synthesize and is suitable for mass synthesis. Therefore, the pH-sensitive cationic lipid is useful as a constituent lipid of lipid nanoparticles used as an active ingredient in pharmaceuticals.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the liver, spleen, kidney, lung, heart, brain, and muscle of mice administered Fluc mRNA-loaded lipid nanoparticles in Example 22.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the liver of mice administered Fluc mRNA-loaded lipid nanoparticles in Example 23.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the spleens of mice administered Fluc mRNA-loaded lipid nanoparticles in Example 23.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the kidneys of mice administered Fluc mRNA-loaded lipid nanoparticles in Example 23.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the lungs of mice administered Fluc mRNA-loaded lipid nanoparticles in Example 23.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the hearts of mice administered Fluc mRNA-loaded lipid nanoparticles in Example 23.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the liver of mice administered each Fluc mRNA-loaded lipid nanoparticle in Example 24.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the spleens of mice administered each Fluc mRNA-loaded lipid nanoparticle in Example 24.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the kidneys of mice administered each Fluc mRNA-loaded lipid nanoparticle in Example 24.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the lungs of mice administered each Fluc mRNA-loaded lipid nanoparticle in Example 24.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the hearts of mice administered each Fluc mRNA-loaded lipid nanoparticle in Example 24.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the liver of mice administered each Fluc mRNA-loaded lipid nanoparticle in Example 25.
  • Example 26 these are microscopic images of liver sections from mice administered with each EGFP mRNA-loaded lipid nanoparticle (upper row: EGFP, lower row: vascular endothelial cells).
  • Example 27 these are microscopic images of liver sections from mice administered with each EGFP mRNA-loaded lipid nanoparticle (upper row: EGFP, lower row: vascular endothelial cells).
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the liver of mice administered Fluc mRNA-loaded lipid nanoparticles in Example 28.
  • Example 23 shows the results of measuring the expression level of human erythropoietin (hEPO activity) in the serum of mice administered with hEPO mRNA-loaded lipid nanoparticles in Example 29.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the liver of mice administered Fluc mRNA-loaded lipid nanoparticles in Example 30.
  • This figure shows the results of measuring Fluc activity (RLU/mg protein) in the liver, spleen, and lungs of mice administered Fluc mRNA-loaded lipid nanoparticles in Example 31.
  • This figure shows the results of measuring the amount of TTR ( ⁇ g/mL) in the serum of mice administered lipid nanoparticles for TTR-targeted genome editing carrying Cas9-mRNA and TTR-gRNA in Example 32 and the TTR knockdown efficiency (%).
  • This figure shows the results of measuring the amount of TTR ( ⁇ g/mL) in the serum of mice administered lipid nanoparticles for TTR-targeted genome editing carrying Cas9-mRNA and TTR-gRNA in Example 33.
  • Example 34 the figure shows the results of measuring the amount of TTR (ng / mL) in the serum of mice administered lipid nanoparticles for PCSK9-targeted genome editing carrying Cas9-mRNA and PCSK9-gRNA.
  • X1 to X2 (X1 and X2 are real numbers satisfying X1 ⁇ X2)
  • X1 or more and X2 or less means "X1 or more and X2 or less”.
  • C X3-X4 (X3 and X4 are integers satisfying X3 ⁇ X4)” means "the number of carbon atoms is X3 or more and X4 or less”.
  • the pH-sensitive cationic lipid of the present invention is a compound in which a hydrocarbon chain is linked to each of the three oxygen atoms of the tris skeleton via a linking group serving as a spacer, and a hydrophilic group is bonded to one nitrogen atom of the tris skeleton.
  • tris skeleton refers to a structure in which one hydrogen atom is removed from each of the three hydroxyl groups and amino group of tris(hydroxymethyl)aminomethane.
  • the pH-sensitive cationic lipid of the present invention is a compound having three hydrocarbon chains and a hydrophilic portion, and due to the similarity of its structure, it has high affinity with lipids such as phospholipids, and is suitable as a constituent lipid of lipid nanoparticles.
  • the pH-sensitive cationic lipid of the present invention is a compound represented by the following general formula (I) or (I'):
  • R1 is a group represented by any one of the following general formulas (T-1) to (T-3): In the formulas, a black circle represents a bond.
  • R 21 is each independently a hydrocarbon group having 1 to 22 carbon atoms (C 1-22 hydrocarbon group)
  • R 22 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 23 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 24 is a hydrogen atom or a hydrocarbon group having 1 to 22 carbon atoms
  • R 25 is a hydrocarbon group having 1 to 22 carbon atoms
  • R 26 is a hydrogen atom or a hydrocarbon group having 1 to 22 carbon atoms.
  • the hydrocarbon group in R 21 to R 26 may be an alkyl group or an alkenyl group.
  • the alkenyl group may be a group having one unsaturated bond, a group having two unsaturated bonds, or a group having three unsaturated bonds.
  • the C 1-22 hydrocarbon group may be linear or branched.
  • R 21 to R 26 are each a C 1-22 alkyl group
  • the alkyl group is, for example, A methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group; n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1-ethylpropyl group, 1,1-dimethylpropyl group, 2,2-dimethylpropyl group; n-hexyl group, 1-methylpentyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 1-ethylbutyl group, 1,1-dimethylbutyl group, 2,2-dimethylbutyl group, 3,3-dimethylbutyl group, 1,2-dimethylbutyl group, 1-methyl-2,
  • R 21 to R 26 are each a C 2-22 alkenyl group
  • examples of the alkenyl group include groups in which 1 to 3 of the saturated bonds between carbon molecules in the groups exemplified as the C 2-22 alkyl groups are replaced with unsaturated bonds.
  • the three R 1s in general formula (I) and (I') are regions that interact with the hydrophobic portion of other lipid molecules that constitute the lipid nanoparticles when the pH-sensitive cationic lipid represented by general formula (I) or (I') is used as a constituent lipid of the lipid nanoparticle.
  • the hydrocarbon group contained in R 1 is a group consisting of a sufficient number of carbon atoms
  • the hydrophobic interaction between the pH-sensitive cationic lipid represented by general formula (I) or (I') and other lipid molecules increases, and lipid nanoparticles that can more stably hold the encapsulated substances such as nucleic acids can be obtained.
  • the hydrocarbon groups contained in R 1 are R 21 and R 22 in general formula (T-1), R 23 and R 24 in general formula (T-2), and R 25 and R 26 in general formula (T-3).
  • the sum of the carbon numbers of R 21 and R 22 is 8 or more, preferably 10 or more, and more preferably 12 or more. In general formula (T-1), the sum of the carbon numbers of R 21 and R 22 is preferably 28 or less, more preferably 26 or less, and even more preferably 24 or less.
  • the sum of the carbon numbers of R 23 and R 24 is 6 or more, preferably 8 or more, and more preferably 10 or more. In general formula (T-2), the sum of the carbon numbers of R 23 and R 24 is preferably 26 or less, more preferably 24 or less, and even more preferably 22 or less.
  • the sum of the carbon numbers of R 25 and R 26 is 6 or more, preferably 8 or more, and more preferably 10 or more. In general formula (T-3), the sum of the carbon numbers of R 25 and R 26 is preferably 26 or less, more preferably 24 or less, and even more preferably 22 or less.
  • Z2 is a linear alkylene group having 4 to 6 carbon atoms (linear C4-6 alkylene group) or a cycloalkylene group.
  • the cycloalkylene group preferably has 3 to 7 carbon atoms, more preferably 4 to 6 carbon atoms.
  • Z2 is preferably a linear C4-6 alkylene group, and particularly preferably a linear alkylene group having 4 or 5 carbon atoms (n-butylene group or n-pentylene group).
  • R1 which is a hydrocarbon chain, is linked to the tris skeleton via a spacer having a -CO- Z2 -CO- structure, so that the hydrocarbon chain R1 has sufficient freedom.
  • s is 2, 3, or 4, and preferably 2 or 3. In one embodiment, s is 2. In another embodiment, s is 3. In yet another embodiment, s is 4.
  • the hydrocarbon chain R 1 is linked to the tris backbone via a spacer having the structure -CO-(CH 2 ) s -CO-, so that the hydrocarbon chain R 1 has sufficient freedom.
  • the three R 1s in one molecule may be the same or different groups. In terms of ease of synthesis and quality stability, it is preferable that the three R 1s in one molecule of the pH-sensitive cationic lipid according to the present invention are all the same group.
  • the three Z2 in one molecule may be the same or different groups. In terms of ease of synthesis and quality stability, it is preferable that the three Z2 in one molecule are all the same group.
  • the three s's in one molecule may be the same or different. From the standpoint of ease of synthesis and quality stability, it is preferable that the three s's in one molecule of the pH-sensitive cationic lipid according to the present invention all be the same.
  • R1 is preferably any of the groups represented by the following formulae (A8), (A8 ⁇ ), (A10), (A12), (A12 ⁇ ), (A14), (A16), (A16 ⁇ ), (A16b), (A20), (A24), (E12), (E14), (A14 ⁇ ), (A24 ⁇ ), (A10L), (A12L), or (A14L).
  • R 1 is preferably any of the groups represented by the following formulae (A8), (A8 ⁇ ), (A10), (A12), (A12 ⁇ ), (A14), (A16), (A16 ⁇ ), (A16b), (A20), (A24), (E12), or (E14).
  • Z1 is a C1-6 alkylene group which may be substituted with one hydroxy group, or a single bond.
  • the C1-6 alkylene group may be linear or branched.
  • Examples of the C1-6 alkylene group include a methylene group, an ethylene group, a 1-methylmethylene group, a 1-hydroxymethylmethylene group, a n-propylene group, a 1-methylethylene group, a 1-hydroxymethylethylene group, a 2-methylethylene group, a n-butylene group, a 1-methylpropylene group, a 1-hydroxymethylpropylene group, a 2-methylpropylene group, a 3-methylpropylene group, a 1-ethylethylene group, a 2-ethylethylene group, a n-pentylene group, a 1-methylbutylene group, a 2-methylbutylene group, a 3-methylbutylene group, a 4-methylbutylene group, a 1-ethylpropylene group, a
  • a compound in which Z 1 is a methylene group, an ethylene group, an n-propylene group, a 1-hydroxymethylmethylene group, a 1-methylmethylene group, a 1-methylethylene group, a 1-hydroxymethylethylene group, or a 2-methylethylene group is preferred, and a compound in which Z 1 is a methylene group, an ethylene group, an n-propylene group, or a 1-hydroxymethylmethylene group is more preferred.
  • X is -N( R2 )( R3 ) or a 5- to 7-membered non-aromatic heterocyclic group.
  • the 5- to 7-membered non-aromatic heterocyclic group represented by X is bonded to Z1 via a carbon atom.
  • R2 and R3 are each independently a hydrogen atom or a C1-4 hydrocarbon group in which one or two hydrogen atoms may be substituted with a phenyl group, a pyrrolyl group, a pyrimidyl group, or an indolyl group.
  • the C1-4 hydrocarbon group may be a linear or branched C1-4 alkyl group or a linear or branched C2-4 alkenyl group.
  • R2 and R3 are C1-4 alkyl groups
  • examples of the C1-4 alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc.
  • examples of the C2-4 alkenyl groups include vinyl, 1-propenyl, 2-propenyl, 1-methylvinyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, etc.
  • pH-sensitive cationic lipid in general formulas (I) and (I'), when X is -N( R2 )( R3 ), a compound in which R2 and R3 are each independently a hydrogen atom or a linear or branched C1-4 alkyl group is preferred, a compound in which R2 and R3 are each independently a hydrogen atom or a linear C1-4 alkyl group is more preferred, and a compound in which R2 and R3 are each independently a hydrogen atom or a linear C1-4 alkyl group is even more preferred, and a compound in which R2 and R3 are each independently a hydrogen atom, a methyl group, an ethyl group, or an n-propyl group is even more preferred.
  • R2 and R3 may be bonded to each other to form a 5- to 7-membered non-aromatic heterocycle.
  • the 5- to 7-membered non-aromatic heterocycle formed by bonding R2 and R3 to each other include a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, and a 1-piperazinyl group.
  • one or more hydrogen atoms, preferably one or two hydrogen atoms, in the 5- to 7-membered non-aromatic heterocycle may be substituted with a C1-4 hydrocarbon group, an amide group, or a nitrogen-containing cyclic group.
  • the C1-4 hydrocarbon group is preferably a C1-4 alkyl group or a C2-4 alkenyl group.
  • two hydrogen atoms in the ring are substituted with a C1-4 alkyl group or a C2-4 alkenyl group, they may be substituted with the same group or different groups.
  • a group derived from a ring in which a carbonyl group is adjacent to a nitrogen atom is preferable, for example, benzo[d]oxazol-2(3H)-one, 1,3-dihydro-2H-benzo[d]imidazol-2-one, indolin-2-one, 1,3-dihydro-2H-pyrrol-2-one, 1,3-dihydro-2H-imidazol-2-one, oxazol-2(3H)-one, oxazolidin-2-one, imidazolidin-2-one, pyrrolidin-2-one, 1,4-dihydroisoquinolin-3(2H)-one, 3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one,
  • groups include groups derived from a nitrogen-containing ring, such as 3,4-dihydroquinazolin-2(1H)-one, 3,4-d
  • one of the carbon atoms constituting the 5- to 7-membered non-aromatic heterocycle may be a carbon atom constituting another nitrogen-containing cyclic group.
  • the other nitrogen-containing cyclic group a group derived from a ring in which a carbonyl group is adjacent to a nitrogen atom is preferable, and specific examples thereof include the same groups as those mentioned above.
  • heteroatom contained in the heterocyclic group examples include a nitrogen atom, an oxygen atom, and a sulfur atom.
  • the heteroatom constituting the heterocycle in the heterocyclic group may be one, or may be two or more heteroatoms that are the same or different.
  • the heterocycle in the heterocyclic group may be a saturated heterocycle and may contain one or more double bonds, but the heterocycle cannot be an aromatic ring.
  • the heterocyclic group is bonded to Z1 via a carbon atom, and the ring may be substituted with one or two C1-4 hydrocarbon groups or amide groups.
  • the C1-4 hydrocarbon group may be a linear or branched C1-4 alkyl group, or a linear or branched C2-4 alkenyl group.
  • the linear or branched C1-4 alkyl group and the linear or branched C2-4 alkenyl group may be those listed above.
  • -Z 1 -X is preferably any one of the groups of the following formulae (H-1) to (H-8) and (H-16) to (H-26):
  • a black circle represents a bond.
  • a compound in which in general formula (I), -Z 1 -X is any group selected from the group consisting of formulas (H-1) to (H-8) and (H-16) to (H-26) is preferred;
  • a compound in which in general formula (I), R More preferred are compounds in which R 1 is a group represented by the following formula (A8), (A8 ⁇ ),
  • R 1 is a group represented by the following formula (A8), (A8 ⁇ ), (A10), (A12), (A12 ⁇ ), (A14), (A16), (A16 ⁇ ), (A16b), (A20), (A24), (E12), or (E14), and -Z 1 -X is any group of the formulas (H-1) to (H-8) and (H-16) to (H-26), is more preferred; in the general formula (I), R 1 is a group represented by the following formula (A8), (A8 ⁇ ), (A10), (A12), (A12 ⁇ ), (A14), (A16), (A16 ⁇ ), (A16b), (A20), (A24), (E12), or (E14), Z 2 is a linear C 4-6 alkylene group, and -Z 1 More preferred is a compound in which -X is any group selected from the formulae (H-1) to (H-8) and (H-16) to (H-26); a compound in which -X is any group selected from the
  • a compound in which in general formula (I'), -Z 1 -X is any group selected from the formulae (H-1) to (H-8) and (H-16) to (H-26) is preferred;
  • a compound in which in general formula (I'), R More preferred are compounds in which R 1 is a group represented by the following formula (A8), (A8 ⁇ ), (A10), (
  • R 1 is a group represented by the following formula (A8), (A8 ⁇ ), (A10), (A12), (A12 ⁇ ), (A14), (A16), (A16 ⁇ ), (A16b), (A20), (A24), (E12), or (E14), and -Z 1 -X is any group of the formulas (H-1) to (H-8) and (H-16) to (H-26) is more preferred; in the general formula (I'), R 1 is a group represented by the following formula (A8), (A8 ⁇ ), (A10), (A12), (A12 ⁇ ), (A14), (A16), (A16 ⁇ ), (A16b), (A20), (A24), (E12), or (E14), s is 2 or 3, and -Z 1 More preferred is a compound in which -X is any one of the groups of the formulae (H-1) to (H-8) and (H-16) to (H-26); a compound in which in general formula (A8), (A8 ⁇ ), (A
  • the pH-sensitive cationic lipid according to the present invention has a hydrocarbon chain consisting of three R 1 extending from one Tris skeleton via a spacer, and the Tris skeleton is further linked to a hydrophilic group consisting of X via Z 1. Therefore, the pH-sensitive cationic lipid according to the present invention can be oriented such that the hydrophilic group consisting of X is sterically shielded by the three hydrocarbon chains consisting of R 1.
  • the pH-sensitive cationic lipid described in Patent Document 1 such as CL4H6 has two hydrophobic chains and a hydrophilic group, and is in a positional relationship in which the hydrophilic group and the hydrophobic chain are separated from each other.
  • the pH-sensitive cationic lipid according to the present invention can be expected to have characteristics different from those of conventional pH-sensitive cationic lipids such as CL4H6 when used as a constituent lipid of lipid nanoparticles.
  • the pKa of the pH-sensitive cationic lipid represented by general formula (I) or (I') is not particularly limited, but can be selected from, for example, about 4.0 to 9.0, preferably about 4.5 to 8.5, more preferably about 5.0 to 8.5, and it is preferable to select the type of each substituent so as to give a pKa in this range.
  • the pKa of the pH-sensitive cationic lipid is particularly influenced by -Z 1 -X in general formulas (I) and (I').
  • the pH-sensitive cationic lipid according to the present invention can be produced, for example, by condensing a compound represented by the following general formula (A) (hereinafter sometimes referred to as "compound (A)”), a compound represented by the following general formula (B) (hereinafter sometimes referred to as “compound (B)”), and tris(hydroxymethyl)aminomethane.
  • R1 and Z2 in general formula (A) are the same as R1 and Z2 in general formula (I), respectively.
  • Z1 and X in general formula (B) are the same as Z1 and X in general formula (I), respectively.
  • the pH-sensitive cationic lipid represented by general formula (I) is synthesized by dehydrating and condensing the carboxylic acid group of compound (A) with the hydroxyl group of Tris to form an ester bond, and then dehydrating and condensing the carboxylic acid group of compound (B) with the amino group of Tris to form an amide bond.
  • the dehydrating and condensing reaction between the carboxylic acid group of compound (A) and the hydroxyl group of Tris can be generally carried out under the same reaction conditions as the dehydrating and condensing reaction when forming an ester of a carboxylic acid and an alcohol.
  • the dehydrating and condensing reaction between the carboxylic acid group of compound (B) and the amino group of Tris can be generally carried out under the same reaction conditions as the dehydrating and condensing reaction when forming an amide of a carboxylic acid and an amine.
  • the pH-sensitive cationic lipid represented by general formula (I) can be relatively easily synthesized by a general dehydrating and condensing reaction by using Tris as a linker.
  • the pH-sensitive cationic lipid represented by general formula (I) can be synthesized by reacting Tris with compound (A) to carry out a dehydration condensation reaction with the hydroxyl group of Tris, and then reacting the resulting esterified product with compound (B) to carry out a dehydration condensation reaction with the amino group derived from Tris.
  • the pH-sensitive cationic lipid represented by general formula (I) can also be synthesized by reacting Tris with compound (B) to carry out a dehydration condensation reaction with the amino group of Tris, and then reacting the resulting amidated product with compound (A) to carry out a dehydration condensation reaction with the hydroxyl group derived from Tris.
  • a compound in which Z2 in compound (A) is a linear C2-4 alkylene group i.e., a compound represented by the following general formula (A') (hereinafter sometimes referred to as "compound (A')"), can be produced by ring-opening condensation of a compound represented by the following general formula (A-0) with a dicarboxylic acid anhydride represented by the following general formula (D) (hereinafter sometimes referred to as "compound (D)").
  • R1 is the same as in general formula (A).
  • s represents 2, 3, or 4.
  • s is the same as s in general formula (A').
  • the ring-opening condensation reaction of compound (A-0) and compound (D) can generally be carried out under reaction conditions similar to those of the dehydration condensation reaction when forming an amide compound between an acid anhydride and a primary or secondary amine, or the dehydration condensation reaction when forming an ester compound between an acid anhydride and an alcohol.
  • the carboxylic acid group of the produced compound (A') and the hydroxyl group of Tris are dehydrated and condensed to form an ester bond, and the carboxylic acid group of compound (B) and the amino group of Tris are dehydrated and condensed to form an amide bond, thereby synthesizing the pH-sensitive cationic lipid represented by general formula (I').
  • the compound in which Z1 is an ethylene group and X is -N( R2 )( R3 ) can be synthesized by a simpler synthesis method using, for example, N-[tris(hydroxymethyl)methyl]acrylamide. Specifically, it can be produced by adding N-[tris(hydroxymethyl)methyl]acrylamide to the compound represented by general formula (C), and then condensing the resulting compound represented by general formula (E) with the compound represented by general formula (A) or general formula (A').
  • R 2 and R 3 in general formula (C) are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms in which one or two hydrogen atoms may be substituted with a phenyl group, a pyrrolyl group, a pyrimidyl group, or an indolyl group, but R 2 and R 3 may be bonded to each other to form a 5- to 7-membered non-aromatic heterocycle (a hydrogen atom in the ring may be substituted with a hydrocarbon group having 1 to 4 carbon atoms, an amide group, or a nitrogen-containing cyclic group; one carbon atom in the ring may be a carbon atom constituting another nitrogen-containing cyclic group).
  • R 2 and R 3 in general formula (E) are the same as R 2 and R 3 in general formula (C), respectively.
  • R1 and Z2 in formula (A) are the same as R1 and Z2 in formula (I), respectively.
  • R1 and s in formula (A') are the same as R1 and s in formula (I'), respectively.
  • R1 and Z2 are the same as R1 and Z2 in general formula (I), and R2 and R3 are the same as R2 and R3 in general formula (C).
  • R1 and s in general formula (I'-a) are the same as R1 and s in general formula (I'), and R2 and R3 are the same as R2 and R3 in general formula (C).
  • the addition reaction of the compound represented by general formula (C) to the unsaturated bond in N-[tris(hydroxymethyl)methyl]acrylamide can generally be carried out under the same reaction conditions as the addition reaction when adding a secondary amine to an alkene.
  • the dehydration condensation reaction between the carboxylic acid group of the compound represented by general formula (A) or general formula (A') and the hydroxyl group of the compound represented by general formula (E) can generally be carried out under the same reaction conditions as the dehydration condensation reaction when forming an ester of a carboxylic acid and an alcohol.
  • the pH-sensitive cationic lipid represented by general formula (I-a) or general formula (I'-a) can be synthesized relatively easily by general addition reaction and dehydration condensation reaction using N-[tris(hydroxymethyl)methyl]acrylamide.
  • the pH-sensitive cationic lipid represented by general formula (I) or (I') can be easily produced, for example, by the method specifically shown in the Examples of this specification. By referring to this production method and appropriately selecting the raw material compounds, reagents, reaction conditions, etc., a person skilled in the art can easily produce any lipid falling within the scope of general formula (I) or (I').
  • the lipid nanoparticles according to the present invention have the pH-sensitive cationic lipid according to the present invention as a constituent lipid.
  • the pH-sensitive cationic lipid according to the present invention constituting the lipid nanoparticles according to the present invention may be only one type, or may be two or more types.
  • the amount of the pH-sensitive cationic lipid according to the present invention means the total amount of lipid molecules corresponding to the pH-sensitive cationic lipid according to the present invention among the lipid molecules constituting the lipid nanoparticles.
  • the ratio of the pH-sensitive cationic lipid according to the present invention to the lipid molecules constituting the lipid nanoparticles is not particularly limited.
  • the ratio of the amount of the pH-sensitive cationic lipid according to the present invention to the total amount of lipids constituting the lipid nanoparticles ([amount of pH-sensitive cationic lipid according to the present invention (mol)]/([amount of total lipids constituting the lipid nanoparticles (mol)]) ⁇ 100%) is preferably 5 mol% or more, more preferably 10 mol% or more, and even more preferably 20 mol% or more.
  • the ratio of the amount of the pH-sensitive cationic lipid according to the present invention to the total amount of lipids constituting the lipid nanoparticles in the lipid nanoparticles according to the present invention is preferably 90 mol% or less, more preferably 80 mol% or less, and even more preferably 70 mol% or less.
  • lipids other than the pH-sensitive cationic lipid of the present invention can be lipids that are generally used when forming liposomes.
  • examples of such lipids include phospholipids, sterols, glycolipids, and saturated or unsaturated fatty acids. These can be used alone or in combination of two or more kinds.
  • phospholipids examples include glycerophospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, cardiolipin, plasmalogen, ceramide phosphorylglycerol phosphate, and phosphatidic acid; sphingophospholipids such as sphingomyelin, ceramide phosphorylglycerol, and ceramide phosphorylethanolamine; and the like. Phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin can also be used.
  • glycerophospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, cardiolipin, plasmalogen, ceramide phosphorylglycerol phosphate, and
  • the fatty acid residues in the glycerophospholipids and sphingophospholipids are not particularly limited, but examples thereof include saturated or unsaturated fatty acid residues having 12 to 24 carbon atoms, with saturated or unsaturated fatty acid residues having 14 to 20 carbon atoms being preferred. Specific examples include acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, and lignoceric acid. When these glycerolipids or sphingolipids have two or more fatty acid residues, all of the fatty acid residues may be the same group or may be different groups.
  • Sterols include, for example, animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol, and brassicasterol; and microorganism-derived sterols such as zymosterol and ergosterol.
  • animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol
  • plant-derived sterols such as stigmasterol, sitosterol, campesterol, and brassicasterol
  • microorganism-derived sterols such as zymosterol and ergosterol.
  • Glycolipids include, for example, glyceroglycolipids such as sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, and glycosyldiglyceride; sphingoglycolipids such as galactosylcerebroside, lactosylcerebroside, and ganglioside; and the like.
  • Saturated or unsaturated fatty acids include, for example, saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.
  • the lipids constituting the lipid nanoparticles according to the present invention preferably contain a neutral lipid in addition to the pH-sensitive cationic lipid according to the present invention, more preferably a phospholipid or a sterol, even more preferably a sterol, and even more preferably cholesterol.
  • the lipid nanoparticles according to the present invention preferably contain a polyalkylene glycol-modified lipid as a lipid component.
  • Polyalkylene glycol is a hydrophilic polymer, and by constructing lipid nanoparticles using a polyalkylene glycol-modified lipid as a lipid membrane constituent lipid, the surface of the lipid nanoparticles can be modified with polyalkylene glycol. By modifying the surface with polyalkylene glycol, it may be possible to increase the stability of the lipid nanoparticles, such as their retention in blood.
  • polyethylene glycol for example, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol, etc.
  • the molecular weight of the polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably about 1,000 to 5,000.
  • stearylated polyethylene glycol such as PEG 45 stearate (STR-PEG45)
  • PEG 45 stearate can be used to modify lipids with polyethylene glycol.
  • Other examples include N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, n-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol Polyethylene glycol derivatives such as N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene
  • the ratio of the polyalkylene glycol-modified lipid to the total amount of lipids constituting the lipid nanoparticles of the present invention is not particularly limited as long as it is an amount that does not impair the selectivity for target tissues by the pH-sensitive cationic lipid of the present invention, specifically, the target tissue-specific gene expression activity when the lipid nanoparticles of the present invention are used as gene expression carriers.
  • the ratio of the polyalkylene glycol-modified lipid to the total amount of lipids constituting the lipid nanoparticles is preferably 0.5 to 3 mol %.
  • the lipid nanoparticles according to the present invention can be subjected to appropriate surface modification, etc., as necessary.
  • the lipid nanoparticles according to the present invention can have their blood retention improved by modifying their surfaces with hydrophilic polymers, etc.
  • surface modification can be performed by using lipids modified with these modifying groups as constituent lipids of the lipid nanoparticles.
  • lipid nanoparticles for example, glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerin phospholipid derivatives, etc. can be used as lipid derivatives to enhance blood retention.
  • hydrophilic polymers to enhance blood retention include dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan, etc. can also be used for surface modification.
  • the lipid nanoparticles can be surface-modified with an oligosaccharide compound having three or more sugars.
  • the type of oligosaccharide compound having three or more sugars is not particularly limited, but for example, an oligosaccharide compound having about 3 to 10 sugar units bonded thereto can be used, and preferably an oligosaccharide compound having about 3 to 6 sugar units bonded thereto can be used.
  • an oligosaccharide compound having a glucose trimer or hexamer can be preferably used, and more preferably an oligosaccharide compound having a glucose trimer or tetramer can be used. More specifically, isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose, or maltohexaose can be preferably used, and among these, maltotriose, maltotetraose, maltopentaose, or maltohexaose in which glucose is bonded by ⁇ 1-4 bonding are more preferable.
  • Maltotriose or maltotetraose is particularly preferable, and maltotriose is most preferable.
  • the amount of surface modification of lipid nanoparticles with oligosaccharide compounds is not particularly limited, but is, for example, about 1 to 30 mol% of the total lipid amount, preferably about 2 to 20 mol%, and more preferably about 5 to 10 mol%.
  • the method for surface-modifying lipid nanoparticles with oligosaccharide compounds is not particularly limited.
  • liposomes in which the surface of lipid nanoparticles is modified with monosaccharides such as galactose or mannose (WO 2007/102481) are known, and the surface modification method described in this publication can be adopted.
  • the entire disclosure of the above publication is incorporated herein by reference.
  • the lipid nanoparticles according to the present invention can be given one or more functions, such as a temperature change sensitivity function, a membrane permeability function, a gene expression function, and a pH sensitivity function.
  • functions such as a temperature change sensitivity function, a membrane permeability function, a gene expression function, and a pH sensitivity function.
  • the lipid nanoparticles according to the present invention may contain one or more substances selected from the group consisting of antioxidants such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, charged substances, and membrane polypeptides.
  • antioxidants such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene
  • charged substances include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine
  • examples of charged substances that impart a negative charge include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid.
  • membrane polypeptides include membrane surface polypeptides and membrane integral polypeptides. The amounts of these substances to be added are not particularly limited and can be appropriately selected depending on the purpose.
  • the size of the lipid nanoparticles according to the present invention is preferably an average particle diameter of 450 nm or less, more preferably an average particle diameter of 300 nm or less, even more preferably an average particle diameter of 200 nm or less, and even more preferably an average particle diameter of 160 nm or less, since this tends to provide high delivery efficiency to target cells in the body.
  • the average particle diameter of the lipid nanoparticles means the number-average particle diameter measured by dynamic light scattering (DLS). Measurement by dynamic light scattering can be performed by standard methods using a commercially available DLS device, etc.
  • the polydispersity index (PDI) of the lipid nanoparticles according to the present invention is about 0.01 to 0.7, preferably about 0.01 to 0.6, more preferably about 0.01 to 0.45, and even more preferably about 0.01 to 0.3.
  • the zeta ( ⁇ ) potential can be in the range of -50 mV to 5 mV, preferably in the range of -40 mV to 5 mV, more preferably in the range of -20 mV to 5 mV, and even more preferably in the range of -12 mV to 5 mV.
  • the form of the lipid nanoparticles according to the present invention is not particularly limited, but examples of forms dispersed in an aqueous solvent include unilamellar liposomes, multilamellar liposomes, spherical micelles, and amorphous layered structures.
  • the lipid nanoparticles according to the present invention are preferably unilamellar liposomes or multilamellar liposomes.
  • the lipid nanoparticles according to the present invention preferably encapsulate a desired component to be delivered into a target cell inside the particle covered with a lipid membrane.
  • the component encapsulated inside the lipid nanoparticles according to the present invention is not particularly limited as long as it is of a size that allows it to be encapsulated.
  • the lipid nanoparticles according to the present invention can encapsulate any substance, such as nucleic acids, sugars, peptides, low molecular weight compounds, and metal compounds.
  • the component to be encapsulated in the lipid nanoparticles according to the present invention is preferably a nucleic acid.
  • the nucleic acid may be DNA, RNA, or an analog or derivative thereof (e.g., peptide nucleic acid (PNA) or phosphorothioate DNA).
  • PNA peptide nucleic acid
  • the nucleic acid to be encapsulated in the lipid nanoparticles according to the present invention may be a single-stranded nucleic acid, a double-stranded nucleic acid, and may be linear or circular.
  • the nucleic acid to be encapsulated in the lipid nanoparticles according to the present invention preferably contains a foreign gene for expression in a target cell, and more preferably is a nucleic acid that functions to express a foreign gene in the cell by being incorporated into the cell.
  • the foreign gene may be a gene that is originally contained in the genomic DNA of the target cell, or a gene that is not contained in the genomic DNA.
  • Examples of such nucleic acids include gene expression vectors that contain a nucleic acid consisting of a base sequence that codes for the target gene to be expressed.
  • the gene expression vector may exist as an extrachromosomal gene in the cell into which it is introduced, or may be incorporated into the genomic DNA by homologous recombination.
  • the gene expression vector to be encapsulated in the lipid nanoparticles according to the present invention is not particularly limited, and vectors generally used in gene therapy and the like can be used.
  • the gene expression vector to be encapsulated in the lipid nanoparticles according to the present invention is preferably a nucleic acid vector such as a plasmid vector.
  • the plasmid vector may remain circular, or may be encapsulated in the lipid nanoparticles according to the present invention after having been cut into a linear form in advance.
  • the gene expression vector can be designed in a standard manner using commonly used molecular biology tools based on the base sequence information of the gene to be expressed, and can be manufactured by various known methods.
  • the nucleic acid to be encapsulated in the lipid nanoparticles according to the present invention is a functional nucleic acid that controls the expression of a target gene present in a target cell.
  • the functional nucleic acid include antisense oligonucleotides, antisense DNA, antisense RNA, siRNA, microRNA, mRNA, gRNA, and the like.
  • the functional nucleic acid may also be a plasmid DNA (pDNA) that serves as an expression vector such as an siRNA expression vector that expresses siRNA in cells.
  • the expression vector can be prepared from a commercially available one and appropriately modified.
  • an siRNA expression vector can be prepared from a commercially available siRNA expression vector, which may also be appropriately modified.
  • the nucleic acid to be encapsulated in the lipid nanoparticles according to the present invention is preferably mRNA or pDNA, since they have particularly good selectivity for tissues in the body.
  • the method for producing lipid nanoparticles according to the present invention is not particularly limited, and any method available to a person skilled in the art can be used.
  • all lipid components are dissolved in an organic solvent such as chloroform, and a lipid membrane is formed by drying under reduced pressure using an evaporator or spray drying using a spray dryer.
  • an aqueous solvent containing a component to be encapsulated in the lipid nanoparticles, such as nucleic acids is added to the dried mixture, and the lipid nanoparticles are emulsified using an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure jet emulsifier.
  • an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure jet emulsifier.
  • Liposomes can also be produced by a method well known for producing liposomes, such as reverse phase evaporation. If it is desired to control the size of lipid nanoparticles, extrusion (extrusion filtration) can be performed under high pressure using a membrane filter with a uniform pore size.
  • the composition of the aqueous solvent is not particularly limited, but examples include buffer solutions such as phosphate buffer, citrate buffer, and phosphate buffered saline, physiological saline, and cell culture media.
  • aqueous solvents can stably disperse lipid nanoparticles, but they may also contain sugars (aqueous solutions) such as monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose sugars, disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose, trisaccharides such as raffinose and melezinose, polysaccharides such as cyclodextrin, sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol, and polyhydric alcohols (
  • lipid nanoparticles dispersed in this aqueous solvent it is desirable to eliminate electrolytes in the aqueous solvent as much as possible from the perspective of physical stability such as suppressing aggregation. Also, from the perspective of the chemical stability of lipids, it is desirable to set the pH of the aqueous solvent to a weak acidic to neutral range (pH 3.0 to 8.0) and/or remove dissolved oxygen by nitrogen bubbling or the like.
  • the lipid nanoparticles according to the present invention can also be produced by an alcohol dilution method using a flow path.
  • a solution in which a lipid component is dissolved in an alcohol solvent and a solution in which a water-soluble component to be included in the lipid nanoparticles is dissolved in an aqueous solvent are introduced from separate flow paths and merged to produce lipid nanoparticles.
  • lipid nanoparticles with a diameter of about 30 nm can be produced with good reproducibility (Non-Patent Document 5).
  • baffles baffle plates
  • a simple two-dimensional flow path structure in which baffles (baffle plates) of a certain width are arranged alternately on both sides of a microflow path through which a raw material solution flows, as described in Patent Document 2, because this can form a nano-sized lipid particle formation system with high particle size controllability.
  • aqueous solvent used in the alcohol dilution method the above-mentioned one can be used.
  • the stability may be improved by using a sugar (aqueous solution) such as monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose; trisaccharides such as raffinose and melezinose; polysaccharides such as cyclodextrin; and sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol.
  • a sugar aqueous solution
  • monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose
  • disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose
  • the stability may be improved by using a polyhydric alcohol (aqueous solution) such as the above-mentioned sugars, glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, and 1,3-butylene glycol.
  • a polyhydric alcohol aqueous solution
  • the lipid nanoparticles according to the present invention function as a gene expression carrier for target tissue.
  • a foreign gene to be expressed in a target cell in the lipid nanoparticles according to the present invention By encapsulating a foreign gene to be expressed in a target cell in the lipid nanoparticles according to the present invention and then administering the lipid nanoparticles to a test animal, the foreign gene is expressed in the target tissue of the test animal.
  • the lipid nanoparticles according to the present invention can also encapsulate medicinal ingredients other than nucleic acids and introduce the medicinal ingredients into the target tissue, making them useful as drug transport carriers. For this reason, the lipid nanoparticles according to the present invention are useful as active ingredients in pharmaceutical compositions used in various treatments, including gene therapy.
  • Lipid nanoparticles with high liver selectivity can be produced by using, as the constituent lipids, a compound with high tissue specificity for the liver from among the pH-sensitive cationic lipids of the present invention, and selecting and using lipids that are easily taken up by the liver from among the other constituent lipids.
  • lipid nanoparticles with high spleen selectivity can be produced by using, as the constituent lipids, a compound with high tissue specificity for the spleen from among the pH-sensitive cationic lipids of the present invention, and selecting and using lipids that are easily taken up by the spleen from among the other constituent lipids.
  • lipid nanoparticles with high liver selectivity or lipid nanoparticles with high spleen selectivity that have a gene expression vector encapsulated therein are administered to an animal, the gene expression vector encapsulated in the lipid nanoparticles is selectively expressed in the liver or spleen rather than in other organs.
  • the siRNA expression vector encapsulated in the lipid nanoparticles is selectively expressed in the liver or spleen rather than in other organs, and the expression of the gene targeted by the expression vector is suppressed.
  • Lipid nanoparticles with high liver selectivity include those in which the content of phosphatidylcholine in the total lipid composition is 10 mol% or less, preferably 2-9 mol%, more preferably 2-5 mol%, and even more preferably 2-4 mol%.
  • the animals to which the lipid nanoparticles of the present invention are administered are not particularly limited, and may be humans or non-human animals.
  • Non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs, and birds such as chickens, quails, and ducks.
  • the route of administration of the lipid nanoparticles of the present invention to animals is not particularly limited, but is preferably parenteral administration such as intravenous administration, enteral administration, intramuscular administration, subcutaneous administration, transdermal administration, nasal administration, or pulmonary administration.
  • the lipid nanoparticles of the present invention are lipid nanoparticles containing a pH-sensitive cationic lipid as described herein for use as a medicament. In another aspect, the lipid nanoparticles of the present invention are lipid nanoparticles containing a pH-sensitive cationic lipid as described herein and a nucleic acid for use as a medicament.
  • the lipid nanoparticles of the present invention are lipid nanoparticles containing a pH-sensitive cationic lipid as described herein for use in gene therapy. In another embodiment, the lipid nanoparticles of the present invention are lipid nanoparticles containing a pH-sensitive cationic lipid and a nucleic acid as described herein for use in gene therapy.
  • the lipid nanoparticles according to the present invention are lipid nanoparticles that contain a pH-sensitive cationic lipid as described herein and encapsulate a foreign gene to be expressed in a target cell, and a method for expressing the foreign gene in a target cell of a subject animal is provided, the method comprising administering the lipid nanoparticles to the subject animal.
  • the lipid nanoparticles according to the present invention are lipid nanoparticles that contain a pH-sensitive cationic lipid as described herein and encapsulate a nucleic acid capable of suppressing the function of a target gene, and a method for suppressing the function of the target gene is provided, comprising administering the lipid nanoparticles to a subject animal.
  • the lipid nanoparticles according to the present invention are lipid nanoparticles containing a pH-sensitive cationic lipid and a nucleic acid as described herein, and the lipid nanoparticles can be used in a method for treating or preventing a disease associated with a genetic abnormality.
  • a method for treating or preventing a disease associated with a genetic abnormality comprises administering to a subject in need of such treatment or prevention a lipid nanoparticle containing a pH-sensitive cationic lipid and a nucleic acid as described herein, wherein the nucleic acid suppresses or replaces the function of the gene.
  • the lipid nanoparticles according to the present invention may be used in the manufacture of a medicament for gene therapy.
  • lipid nanoparticles containing a pH-sensitive cationic lipid as described herein in the manufacture of a medicament for gene therapy.
  • lipid nanoparticles containing a pH-sensitive cationic lipid and a nucleic acid as described herein in the manufacture of a medicament for gene therapy.
  • TLC Thin Layer Chromatography
  • SM-102 was manufactured by Cayman Chemical
  • ALC-0315 was manufactured by Ambeed
  • DLin-MC3-DMA (MC3) was manufactured by Selleck Biotech
  • cholesterol (Chol) was manufactured by SIGMA Aldrich.
  • 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and methoxyethylene glycol 2000-modified 2-dimyristoyl-rac-glycerol (PEG-DMG) were manufactured by NOF Corporation.
  • mRNA encoding firefly luciferase (Fluc) (CleanCap FLuc mRNA, 5 moU)
  • mRNA encoding green fluorescent protein EGFP EGFP mRNA, 5 moU
  • hEPO mRNA, 5 moU mRNA encoding human erythropoietin hEPO
  • lipid nanoparticles were prepared by the alcohol dilution method using a flow channel.
  • the flow channel used was a micro flow channel iLiNP (manufactured by Lilac Pharma) with a built-in baffle mixer.
  • RNA solution 13 mg/mL mRNA solution was diluted with 50 mM citrate buffer (pH 4.0) to make a total volume of 250 ⁇ L, and this solution was dispensed into a 1 mL syringe (HAMILTON) as the RNA solution.
  • 50 mM citrate buffer pH 4.0
  • HAMILTON 1 mL syringe
  • pH-responsive cationic lipid/chol/DSPC/PEG-DSG 50/35/15/1.5 (mol%)
  • 200 ⁇ L of 10 mM pH-responsive cationic lipid/ethanol solution, 140 ⁇ L of 10 mM cholesterol/ethanol solution, 60 ⁇ L of 10 mM DSPC/ethanol solution, 60 ⁇ L of 1 mM PEG-DSG/ethanol solution, and 40 ⁇ L of ethanol were mixed to make a total volume of 500 ⁇ L (final lipid concentration 8 mM, 100% ethanol) and dispensed into a 1 mL syringe (HAMILTON) as the lipid solution.
  • HAMILTON 1 mL syringe
  • the lipid nanoparticle solution was placed in a dialysis membrane (MWCO 12,000-14,000, Spectrum Laboratories) and dialyzed at 4°C for more than 2 hours against PBS (-) (pH 7.4) as the external aqueous phase to remove ethanol and exchange the buffer, after which the lipid nanoparticle solution was collected from the dialysis membrane.
  • ⁇ Measurement of mean particle size and zeta potential of lipid nanoparticles The mean particle size (number average value), ⁇ -mean particle size, and polydispersity index (PdI) of the lipid nanoparticles in PBS(-) (pH 7.4), as well as the zeta potential in 10 mM HEPES buffer (pH 7.4), were measured using a dynamic light scattering analyzer, Zetasizer Nano ZS ZEN3600 (Malvern).
  • encapsulation efficiency of lipid nanoparticles was determined by measuring the amount of encapsulated nucleic acid (mRNA) using Ribogreen (Life Technologies), an RNA intercalator. The amount of mRNA was quantified based on a calibration curve created from a dilution series of mRNA with known concentrations.
  • TNS p-Toluenesulfonic acid
  • the highest and lowest values of the measured values were set to 100% and 0% charge rate, respectively, and the pH showing a 50% charge rate was calculated as the apparent pKa.
  • the percentage of cationically charged pH-sensitive cationic lipid was calculated using the Henderson-Hasselbalch equation.
  • Lipid nanoparticles were administered to mice via tail vein injection using a 27 G needle. Unless otherwise specified, BALB/c mice (4-5 weeks old, female) were used.
  • lipid nanoparticles encapsulating Fluc mRNA were intravenously injected into BALB/c mice (female, 4 weeks old) at a dose of 0.1 mg mRNA/kg. 6 hours, 24 hours, or 48 hours after intravenous injection, the mice were euthanized, and each tissue was collected, frozen in liquid nitrogen, and stored at -80°C. The tissues were homogenized in passive lysis buffer (Promega) using a bead cell disrupter (Micro Smash MS-100R, TOMY Seiko). Tissue debris was removed by centrifugation, and the supernatant was collected.
  • Fluc activity in the supernatant was measured using a luciferase assay system (E1500, Promega) according to the manufacturer's protocol. Luminescence was measured using a luminometer (Luminescencer-PSN, ATTO). Protein concentration was determined using a commercially available measurement kit (BCA Protein Assay Kit, Pierce). Fluc activity was expressed as relative light units (RLU) per mg of protein.
  • TxT-5-5A10 (194.5 mg, 38.1% yield) was obtained as a yellow oil by reacting with TxT-5 head and purifying it in the same manner as in the synthesis of compound (TxT-5-5A8), except that compound (5A10) (447.8 mg, 1.65 mmol) was used instead of compound (5A8).
  • the compound (TxT-5-5A20) was obtained as a yellow oil (448.6 mg, 62.9% yield) by reacting with TxT-5 head and purifying in the same manner as in the synthesis of compound (TxT-5-4A20), except that compound (5A20) (679.3 mg, 1.65 mmol) was used instead of compound (4A20).
  • the compound (TxT-5-5A24) (634.1 mg, 79.5% yield) was obtained as a yellow oil by reacting with TxT-5 head and purifying it in the same manner as in the synthesis of compound (TxT-5-4A20), except that compound (5A24) (771.8 mg, 1.65 mmol) was used instead of compound (4A20).
  • TxT-5-5E12 is obtained as a yellow oil by reacting with TxT-5 head and purifying it in the same manner as in the synthesis of compound (TxT-5-5E14), except that compound (5E12) is used instead of compound (5E14).
  • the compound (TxT-5-5A24 ⁇ ) (548.2 mg, yield 68.1%) was obtained as a yellow oil by reacting with TxT-5 head and purifying it in the same manner as in the synthesis of compound (TxT-5-4A20), except that compound (5A24 ⁇ ) (884 mg, 1.65 mmol) was used instead of compound (4A20).
  • the compound (TxT-5-5A10L) (410.6 mg, 80.5% yield) was obtained as a white solid by reacting with TxT-5 head and purifying it in the same manner as in the synthesis of compound (TxT-5-4(6)A14), except that compound (5A10L) (447.8 mg, 1.65 mmol) was used instead of compound (4(6)A14).
  • the compound (TxT-5-5A12L) (220.1 mg, yield 39.8%) was obtained as a white powder by reacting with TxT-5 head and purifying it in the same manner as in the synthesis of compound (TxT-5-4(6)A14), except that compound (5A12L) (494.1 mg, 1.65 mmol) was used instead of compound (4(6)A14).
  • the reaction was monitored by TLC, and TEA (809.5 mg, 8.0 mmol) was added to the resulting reaction mixture, the solids were filtered, and DMF and THF were evaporated in vacuum. The resulting residue was loaded onto a normal phase column (Sfaer Amino D, Biotage) and purified by flash chromatography using a gradient mobile phase of DCM and methanol. This resulted in the TxT-19 head (508.2 mg, 51.4% yield) being obtained as a yellow powder.
  • Lipid nanoparticles were prepared using TxT lipids TxT-5-5A8, TxT-5-5A8 ⁇ , TxT-5-5A10, TxT-5-5A12, TxT-5-5A12 ⁇ , TxT-5-5A14, and TxT-5-5A16 as constituent lipids.
  • lipids other than TxT lipid phosphatidylcholine (PC), cholesterol, and PEG-DMG were used.
  • PC DSPC was used.
  • the ⁇ -average particle size (nm), number-average particle size (nm), polydispersity index (PdI), ⁇ -potential (mV), mRNA encapsulation rate (%), mRNA concentration ( ⁇ g/ ⁇ L), and pKa of each Fluc mRNA-loaded lipid nanoparticle are shown in Table 1. In Table 1, "nd" means not measured.
  • lipid nanoparticles encapsulating mRNA could be produced using any of the TxT lipids. Furthermore, the physical properties of these lipid nanoparticles were such that they could be used as gene carriers.
  • lipid nanoparticles loaded with Fluc mRNA produced using TxT-5-5A10, TxT-5-5A12, TxT-5-5A12 ⁇ , TxT-5-5A14, and TxT-5-5A16 were administered to mice, and the luciferase expression level (Fluc activity) in each tissue was examined.
  • lipid nanoparticles containing TxT lipid as a constituent lipid are useful as gene carriers targeting various organs.
  • these lipid nanoparticles loaded with Fluc mRNA were confirmed to have high Fluc activity, particularly in the liver and spleen.
  • lipid nanoparticles containing TxT-5-5A12 and TxT-5-5A12 ⁇ as constituent lipids had the highest Fluc activity in the spleen, making them promising carriers targeting the spleen.
  • Example 23 Using TxT-5-5A16, which had the highest Fluc activity in the liver in Example 22, lipid nanoparticles with varying DSPC content were prepared, and the luciferase expression level (Fluc activity) in each tissue was examined.
  • the ⁇ -average particle size (nm), number-average particle size (nm), polydispersity index (PdI), ⁇ -potential (mV), mRNA encapsulation rate (%), mRNA concentration ( ⁇ g/ ⁇ L), and pKa of each lipid nanoparticle loaded with Fluc mRNA are shown in Table 2.
  • TxT-5-5A12, TxT-5-5A12 ⁇ , TxT-5-5A14, TxT-5-5A16, TxT-5-5A16b, TxT-5-5A16 ⁇ , TxT-5-5A20, TxT-5-5A24, TxT-5-5E14, and TxT-5-4A20 were used as constituent lipids to prepare lipid nanoparticles containing 3 mol% DSPC.
  • the ⁇ -average particle size (nm), number-average particle size (nm), polydispersity index (PdI), ⁇ -potential (mV), mRNA encapsulation rate (%), mRNA concentration ( ⁇ g/ ⁇ L), and pKa of each lipid nanoparticle loaded with Fluc mRNA are shown in Table 3.
  • the results are shown in Figures 3A to 3E.
  • Fluc activity was observed in the liver (Figure 3A) that was 10 times higher than that in the spleen ( Figure 3B), kidney ( Figure 3C), lung ( Figure 3D), and heart ( Figure 3E).
  • lipid nanoparticles containing TxT-5-5A14 as a constituent lipid were expected to be a carrier that targets the liver.
  • Example 25 The liver uptake efficiency of lipid nanoparticles containing TxT-5-5A14 as the constituent lipid was compared with that of Fluc mRNA-loaded lipid nanoparticles using SM-102, ALC-0315, or MC3 as the cationic lipid.
  • SM-102, ALC-0315, or MC3 was used as the cationic lipid
  • Table 4 shows the ⁇ -average particle size (nm), number-average particle size (nm), polydispersity index (PdI), ⁇ -potential (mV), mRNA encapsulation rate (%), mRNA concentration ( ⁇ g/ ⁇ L), and pKa of each Fluc mRNA-loaded lipid nanoparticle.
  • the results are shown in Figure 4.
  • mice administered lipid nanoparticles containing TxT-5-5A14 as the constituent lipid a clearly higher Fluc activity was observed than in mice administered lipid nanoparticles containing other cationic lipids as the constituent lipid, suggesting that lipid nanoparticles containing TxT-5-5A14 as the constituent lipid may be a superior gene carrier to existing gene carriers.
  • Example 26 The uptake of lipid nanoparticles containing TxT-5-5A14 as the constituent lipid in each cell of the liver was compared with that of Fluc mRNA-loaded lipid nanoparticles using SM-102, ALC-0315, or MC3 as the cationic lipid.
  • each EGFP mRNA-loaded lipid nanoparticle was administered to Balb/c mice at a dose of 0.2 mg mRNA/kg, and 23 hours and 50 minutes after administration, fluorescently labeled lectin (DyLight-649 Lycopersicon (Tomato) Lectin) was administered at 40 ⁇ g/mouse into the tail vein to fluorescently label vascular endothelial cells. 24 hours after lipid nanoparticle administration, the mice were euthanized, the livers were collected, and tissue sections were prepared.
  • fluorescently labeled lectin DyLight-649 Lycopersicon (Tomato) Lectin
  • tissue sections were immersed in a nuclear staining solution (a solution of 1 ⁇ g/mL Hoechst 33342 dissolved in PBS (-)), cooled on ice, and incubated in the dark for more than 30 minutes.
  • the stained tissue sections were observed using a confocal laser scanning microscope.
  • FIG. 5 Microscopic images of liver tissue sections from each mouse are shown in Figure 5.
  • the upper row is a fluorescent image of EGFP
  • the lower row is a fluorescent image of vascular endothelial cells (fluorescently labeled lectin).
  • vascular endothelial cells fluorescently labeled lectin
  • Example 27 Lipid nanoparticles loaded with EGFP mRNA, which consisted of various TxT lipids as constituent lipids, were administered to mice to examine the expression efficiency in liver cells.
  • TxT lipids TxT-5-5A12, TxT-5-5A12 ⁇ , TxT-5-5A14, TxT-5-5A16, TxT-5-5A16b, TxT-5-5A16 ⁇ , TxT-5-5A20, TxT-5-5A24, TxT-5-5E14, and TxT-5-4A20 were used.
  • Table 5 shows the ⁇ -average particle size (nm), number-average particle size (nm), polydispersity index (PdI), ⁇ -potential (mV), mRNA encapsulation rate (%), and mRNA concentration ( ⁇ g/ ⁇ L) of each EGFP mRNA-loaded lipid nanoparticle. As shown in Table 5, uniform particles could be prepared with all TxT lipids.
  • each EGFP mRNA-loaded lipid nanoparticle was administered to a Balb/c mouse, and 23 hours and 50 minutes after administration, fluorescently labeled lectin was administered.
  • 24 hours after lipid nanoparticle administration the mouse was euthanized, the liver was collected, and tissue sections were prepared. Each tissue section was observed using a confocal laser scanning microscope. Microscopic images of liver tissue sections from each mouse are shown in Figure 6. In the figure, the upper row is a fluorescent image of EGFP, and the lower row is a fluorescent image of vascular endothelial cells (fluorescently labeled lectin).
  • EGFP fluorescence was observed in the liver tissue of mice administered EGFP mRNA-loaded lipid nanoparticles containing TxT-5-5A12, TxT-5-5A12 ⁇ , TxT-5-5A14, TxT-5-5A16b, TxT-5-5A16 ⁇ , TxT-5-5A24, TxT-5-5E14, and TxT-5-5A20.
  • very high gene expression was observed in the hepatocytes of mice administered EGFP mRNA-loaded lipid nanoparticles containing TxT-5-5A12 ⁇ , TxT-5-5A14, and TxT-5-5A24.
  • Example 28 The time course of gene expression in the liver of lipid nanoparticles containing TxT-5-5A12 ⁇ and TxT-5-5A14 as constituent lipids was evaluated.
  • the ⁇ -average particle size (nm), number-average particle size (nm), polydispersity index (PdI), ⁇ -potential (mV), mRNA encapsulation rate (%), and mRNA concentration ( ⁇ g/ ⁇ L) of each lipid nanoparticle loaded with Fluc mRNA are shown in Table 6.
  • the results are shown in Figure 7.
  • the lipid nanoparticles containing TxT-5-5A12 ⁇ as the constituent lipid and the lipid nanoparticles containing TxT-5-5A14 as the constituent lipid showed the same gene expression intensity at all times.
  • Example 29 The time course of hEPO expression efficiency in lipid nanoparticles containing TxT-5-5A12 ⁇ and TxT-5-5A14 as constituent lipids was compared with that of hEPO mRNA-loaded lipid nanoparticles using SM-102.
  • SM-102 was used as the cationic lipid
  • ⁇ -average particle size (nm), number-average particle size (nm), polydispersity index (PdI), ⁇ -potential (mV), mRNA encapsulation rate (%), and mRNA concentration ( ⁇ g/ ⁇ L) of each hEPO mRNA-loaded lipid nanoparticle are shown in Table 7.
  • the blood was left to stand at room temperature for 90 minutes, centrifuged at 25°C and 1,000 x g for 15 minutes, the supernatant was collected, and serum was obtained.
  • hEPO activity in serum was measured using a commercially available ELISA kit (Human EPO ELISA Kit, Invitrogen). Standard curve samples were prepared by serial dilution from 100 mIU/mL to prepare 450 ⁇ L each of solutions at 50, 25, 12.5, 6.3, and 3.1 ng/mL. For serum samples, serum was diluted 10 to 10,000 times to fall within the standard curve range.
  • mice administered lipid nanoparticles with TxT-5-5A12 ⁇ as the constituent lipid and mice administered lipid nanoparticles with TxT-5-5A14 as the constituent lipid showed significantly higher hEPO concentrations than mice administered lipid nanoparticles with SM-102 as the constituent lipid, suggesting that lipid nanoparticles with TxT-5-5A12 ⁇ or TxT-5-5A14 as the constituent lipid may be superior gene carriers to existing gene carriers.
  • TxT-5-5A12, TxT-5-5A12 ⁇ , TxT-5-5A14, TxT-5-5A14 ⁇ , TxT-5-5A16, TxT-5-5A16b, TxT-5-5A16 ⁇ , TxT-5-5A20, TxT-5-5A24, TxT-5-5A24 ⁇ , TxT-5-5E14, and TxT-5-4A20 were used as constituent lipids to prepare lipid nanoparticles (Fluc mRNA-loaded lipid nanoparticles) containing 3 mol% DSPC, and the amount of luciferase expression in the liver (Fluc activity) was examined.
  • TxT-5-4(6)A14 was used as the constituent lipid to prepare lipid nanoparticles containing 3 mol% DSPC (Fluc mRNA-loaded lipid nanoparticles), and the luciferase expression level (Fluc activity) in each tissue was examined.
  • FIG. 10 shows the results of measuring Fluc activity (RLU/mg protein) in the liver, spleen, and lungs of mice administered Fluc mRNA-loaded lipid nanoparticles. Fluc activity was observed in the liver, spleen, and lungs, with particularly high activity confirmed in the liver.
  • Example 32 Lipid nanoparticles containing various TxT lipids as constituent lipids were used as DDS carriers for genome editing.
  • TxT lipids TxT-5-5A12 ⁇ , TxT-5-5A14, and TxT-5-5A24, which were observed to have high gene expression in Example 27, were used.
  • Cas9-mRNA TriLink, 5 moU modified
  • TTR gRNA targeting transthyretin
  • lipid nanoparticles lipid nanoparticles for TTR-targeted genome editing
  • TxT/DSPC/chol/PEG-DMG 50/3/47/1.5 (mol%) and loaded with Cas9-mRNA and TTR-gRNA (SEQ ID NO: 1) were prepared in the same manner as in Example 27.
  • ⁇ -average particle size (nm), number-average particle size (nm), polydispersity index (PdI), ⁇ -potential (mV), mRNA encapsulation rate (%), and mRNA concentration ( ⁇ g/ ⁇ L) of each lipid nanoparticle for TTR-targeted genome editing are shown in Table 8.
  • lipid nanoparticles for TTR-targeted genome editing encapsulating Cas9 mRNA tended to have a smaller average particle size than lipid nanoparticles encapsulating Fluc mRNA (Example 24) and lipid nanoparticles encapsulating EGFP mRNA (Example 27), even when the lipid composition was the same.
  • mice 4-week-old female mice
  • tail vein 1.5 mg Cas9 mRNA/kg
  • the blood was left to stand at room temperature for 1 hour, centrifuged at 25°C and 1,000 x g for 15 minutes, the supernatant was collected, and serum was obtained.
  • the TTR concentration in serum was measured using a commercially available ELISA kit (Prealbumin ELISA Kit Mouse, Aviva Systems Biology).
  • ⁇ L of solutions of 250, 125, 62.5, 31.25, and 15.63 ng/mL were prepared by serial dilution from 500 ng/mL.
  • serum samples serum was diluted 10,000 times. 100 ⁇ L of the calibration curve samples and serum samples were applied to the plate and incubated at 25°C for 30 minutes. The wells were washed four times, and 100 ⁇ L of Enzyme-Antibody Conjugate was applied to each well. The plate was protected from light with aluminum foil and then incubated at 25°C for 20 minutes. The wells were washed four times again, and 100 ⁇ L of TMB Substrate Solution (Aviva Systems Biology) was applied to each well.
  • TMB Substrate Solution Aviva Systems Biology
  • the plate was protected from light with aluminum foil and then incubated at 25°C for 10 minutes. After that, 100 ⁇ L of Stop Solution (Aviva Systems Biology) was applied, and the absorbance at 450 nm was measured using a spectrofluorometer (VarioskanLux, Thermo Fisher Scientific).
  • FIG 11 shows the results of measuring the amount of TTR in serum ( ⁇ g/mL) and TTR knockdown efficiency (%).
  • NT indicates the measurement result of serum from mice that were not administered lipid nanoparticles.
  • Lipid nanoparticles for TTR-targeted genome editing containing TxT-5-5A12 ⁇ and lipid nanoparticles for TTR-targeted genome editing containing TxT-5-5A14 showed extremely high TTR knockout activity of over 85%.
  • Lipid nanoparticles containing TxT-5-5A14 as a constituent lipid were used as DDS carriers for genome editing.
  • Cas9-mRNA TriLink, 5 moU modified
  • highly modified gRNA SEQ ID NO: 2
  • ⁇ -average particle size (nm), number-average particle size (nm), polydispersity index (PdI), ⁇ -potential (mV), mRNA encapsulation rate (%), and mRNA concentration ( ⁇ g/ ⁇ L) of each TTR-targeted genome editing lipid nanoparticle are shown in Table 9.
  • the measurement results are shown in Figure 12.
  • "NT" indicates the measurement results of serum from mice that were not administered lipid nanoparticles.
  • a dosage-dependent increase in TTR knockout efficiency was observed, with a TTR knockout activity of 60% or more at 0.3 mg RNA/kg.
  • Lipid nanoparticles containing TxT-5-5A14 as a constituent lipid were used as DDS carriers for genome editing.
  • Cas9-mRNA TriLink, 5 moU modified
  • gRNA SEQ ID NO: 3
  • ⁇ -average particle size (nm), number-average particle size (nm), polydispersity index (PdI), ⁇ -potential (mV), mRNA encapsulation rate (%), and mRNA concentration ( ⁇ g/ ⁇ L) of the PCSK9-targeted genome editing lipid nanoparticles are shown in Table 10.
  • mice The prepared lipid nanoparticles for PCSK9-targeted genome editing were then administered to Balb/c mice (4-week-old female) via the tail vein (0.3, 1.0, 2.0 mg RNA/kg).
  • the serum PCSK9 concentration was measured using a commercially available ELISA kit (Mouse/Rat PCSK9 ELISA Kit, CircuLex).
  • the wells were washed four times again, and 100 ⁇ L of Substrate Reagent (CircuLex) was applied to each well.
  • the plate was protected from light with aluminum foil and incubated at 25°C for 10-20 minutes while shaking at 300 rpm. 100 ⁇ L of Stop Solution (CircuLex) was then applied, and the absorbance at 450 nm and 540 nm was measured using a spectrofluorometer (VarioskanLux, Thermo Fisher Scientific).
  • Figure 13 shows the results of measuring the amount of PCSK9 (ng/mL) in serum. A dose-dependent increase in PCSK9 knockout efficiency was observed, with 2.0 mg RNA/kg showing PCSK9 knockout activity of over 90%.
  • gRNA sequences used in the above examples are as follows:
  • mU stands for 2'-O-methyluridine
  • mA stands for 2'-O-methyladenosine
  • mC stands for 2'-O-methylcytidine
  • mG stands for 2'-O-methylguanosine
  • * represents a phosphorothioate bond.
  • mU * represents 2'-O-methyluridine with a 3'-phosphorothioate bond
  • mA * represents 2'-O-methyladenosine with a 3'-phosphorothioate bond
  • mC * represents 2'-O-methylcytidine with a 3'-phosphorothioate bond.

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