Lipid nanoparticles

Abstract

The present invention provides lipid nanoparticles comprising a polymer represented by formula (I) [a represents an integer of 3-5; b represents 0 or 1; r1 and R2 each independently represent a linear or branched C1-15 alkyl group represented by general formula (A) (R11 and R12 each independently represent a linear or branched C1-15 alkyl group; c represents an integer from 1 to 7; and e represents an integer of 4-12); x represents a compound represented by general formula (B) (d represents an integer of 0-3; and R3 and R4 each independently represent a C1-4 alkyl group or a C2-4 alkenyl group, but R3 and R4 may bind to each other to form a 5-7-membered non-aromatic heterocyclic ring) or a 5-7-membered non-aromatic heterocyclic ring group. Formula (R1) (R2) C (OH)-(CH2) a-(O-CO) b-X (I) (R11) (R12) HC-(CH2) c-(CO-O)-(CH2) e-(A)-(CH2) d-N (R3) (R4) (B).

Description

Technical Field

[0001] The present invention relates to lipid nanoparticles and their constituent lipids that can be used as gene delivery vectors targeting hepatocytes and spleen cells. This application claims priority based on patent application No. 2022-060960 filed in Japan on March 31, 2022, and incorporates its contents herein. Background Art

[0002] Lipid nanoparticles (LNPs) are used as carriers for encapsulating lipophilic drugs, nucleic acids (such as siRNA (short interfering RNA), mRNA), etc.) and delivering them to target cells. For example, lipid nanoparticles containing pH-sensitive cationic lipids as constituent lipids that are electrically neutral at physiological pH and change to cationic properties in a weakly acidic pH environment (such as endosomes) have been reported as carriers for effectively delivering nucleic acids (such as siRNA) to target cells (Patent Document 1).

[0003] For example, Jayaraman et al. developed DLin-MC3-DMA (hereinafter, sometimes referred to as "MC3") as a pH-sensitive cationic lipid and achieved an ED of 0.005 mg siRNA / kg in factor 7 (F7) knockdown in mouse liver. 50 (Non-patent Document 1) The present inventors have developed unique pH-sensitive cationic lipids YSK05 and YSK13-C3 as EDs in F7 knockdown. 50 0.06 and 0.015 mg siRNA / kg were achieved respectively (Non-patent Documents 2-4). In addition, Mailer et al. reportedly developed L319 that imparted biodegradability to MC3-DMA and reported an ED of 0.01 mg siRNA / kg. 50 and high safety (Non-patent Documents 5-7). However, it has been shown that the endosomal escape efficiency of lipid nanoparticles containing these pH-sensitive cationic lipids is still only about a few percent (Non-patent Document 8), and it is necessary to develop a technology that can further improve the bioavailability.

[0004] On the other hand, as the manufacture method of lipid nanoparticles such as nucleic acid, there is the method for principle with the alcohol dilution method that has used flow channel.For example, it is reported: can make the lipid nanoparticle (non-patent literature 9) of diameter about 30nm with good reproducibility by using the microfluidic channel of the built-in three-dimensional micro-mixer that can realize two kinds of liquid instant mixing.And, also report: by using the flow channel structure body of simple two-dimensional structure compared with the flow channel structure body of using traditional three-dimensional mixer, can form the lipid particle formation system (patent documentation 2) with the nanometer size of high particle diameter controllability, the flow channel structure body of described simple two-dimensional structure is on the flow channel of the micro-size of circulation stock solution, and is staggered with the baffle (spoiler) of certain width with respect to flow channel width from two sides.In recent years, the manufacture method of these nanoparticle preparations is mainly used for making the lipid nanoparticle (LNP) that loads with nucleic acid such as fat-soluble drug, siRNA (short interfering RNA) or mRNA. Prior art literature Patent Literature

[0005] Patent Document 1: International Publication No. 2018 / 230710 Patent Document 2: International Publication No. 2018 / 190423 Non-patent literature

[0006] Non-patent document 1: Jayaraman et al., Angewandte ChemIe International Edition, 2012, vol. 51, p. 8529-8533. Non-patent document 2: Watanabe et al., Scientific Reports, 2014, 4: 4750, DOI: 10.1038 / srep04750. Non-patent document 3: Yamamoto et al., Journal of Hepatology, 2016, vol. 64, p. 547-555. Non-patent document 4: Sato et al., Molecular Therapy, 2016, vol. 24, p. 788-795. Non-patent document 5: Mailer et al., Molecular Therapy, 2013, vol. 21(8), p. 1570-1578. Non-patent document 6: WIttrup et al., Nature Biotechnology, 2015, vol. 33(8), p. 870-876. Non-patent document 7: Xu et al., Molecular PharmaceutIcs, 2014, vol.11, p.1424-1434. Non-patent document 8: GIlleron et al., Nature Biotechnology, 2013, vol. 31(7), p. 638-646. Non-patent document 9: Leung et al., Journal of PhysIcal Chemistry C Nanomaterial Interfaces, 2012, vol. 116(34), p. 18440-18450. Summary of the invention Problems to be solved by the invention

[0007] The present invention aims to provide lipid nanoparticles useful as gene delivery vectors targeting hepatocytes and spleen cells. Means used to solve problems

[0008] The present inventors have found that lipid nanoparticles comprising a pH-sensitive cationic lipid having a branched hydrocarbon chain as a constituent lipid can be used as a gene delivery vector to hepatocytes and spleen cells, and have completed the present invention.

[0009] That is, the present invention provides the following lipid nanoparticles and the like. [1] A lipid nanoparticle comprising a pH-sensitive cationic lipid represented by the following formula (I):

[0010] Chemical formula 1 (R 1 )(R 2 )C(OH)-(CH2)a-(O-CO)bX (I)

[0011] In formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R 1 and R 2 Each independently represents a group represented by the following general formula (A),

[0012] Chemical formula 2 (R 11 )(R 12 )HC-(CH2)c-(CO-O)-(CH2)e- (A)

[0013] In formula (A), R 11 and R 12 Each independently represents a linear or branched C 1-15 alkyl; c represents an integer of 1-7; e represents an integer of 4-12; but R11 and R 12 can be connected to each other to form a ring, X represents a group represented by the following general formula (B) or a 5-7 membered non-aromatic heterocyclic group, but the group is bonded to (O-CO)b- through a carbon atom, and one or two hydrogen atoms of the ring can be replaced by C 1-4 Alkyl or C 2-4 Alkenyl substitution,

[0014] Chemical formula 3 -(CH2)dN(R 3 )(R 4 ) (B)

[0015] In formula (B), d represents an integer of 0-3; R 3 and R 4 Each independently represents C 1-4 Alkyl or C 2-4 alkenyl, wherein the C 1-4 Alkyl or C 2-4 In the alkenyl group, one or two hydrogen atoms may be replaced by a phenyl group), but R 3 and R 4 They can combine with each other to form a 5-7 membered non-aromatic heterocyclic ring, wherein one or two hydrogen atoms of the ring can be replaced by C 1-4 Alkyl or C 2-4 alkenyl substitution). [2] According to the lipid nanoparticles described in [1], c is an integer of 4-7. [3] The lipid nanoparticle according to [1] or [2], wherein R 11 and the R 12 The sum of the number of carbon atoms of the group with more carbon atoms and the number of c is 5-17. [4] The lipid nanoparticle according to any one of [1] to [3], further comprising sterol- and polyalkylene glycol-modified lipids. [5] According to any one of [1]-[4], the lipid nanoparticle comprises a nucleic acid. [6] According to the lipid nanoparticles of [5], the nucleic acid is siRNA, mRNA or plasmid DNA. [7] According to any one of the lipid nanoparticles [1]-[5], it contains a DNA nuclease, a guide RNA, and a single-stranded oligonucleotide containing a region that can pair with a portion of the guide RNA. [8] According to the lipid nanoparticle of [7], in the region where the single-stranded oligonucleotide can pair with part of the guide RNA, 30-60% of the bases in the region are complementary to the bases in the guide RNA. [9] According to the lipid nanoparticles of [7] or [8], the DNA nuclease is Cas9 protein, and the guide RNA consists of crRNA and tracrRNA.

[10] A lipid nanoparticle, The method comprises a lipid component, a DNA nuclease, a guide RNA, and a single-stranded oligonucleotide containing a region that can pair with a portion of the guide RNA. In the region where the single-stranded oligonucleotide can pair with a portion of the guide RNA, 30-60% of the bases in the region are complementary to the bases in the guide RNA.

[11] A pharmaceutical composition comprising the lipid nanoparticles described in any one of [1] to

[10] as an active ingredient.

[12] The pharmaceutical composition according to

[11] , which is used for gene therapy.

[13] A method for expressing an exogenous gene, comprising administering a lipid nanoparticle as described in any one of [1] to

[10] , encapsulating a target exogenous gene expressed in hepatocytes or spleen cells, to a test animal, but the test animal does not include humans, and the exogenous gene is expressed in the liver or spleen of the test animal.

[14] A genome editing method, wherein any lipid nanoparticle described in [7]-

[10] is introduced into a cell.

[15] A pH-sensitive cationic lipid represented by the above formula (I).

[16] According to the pH-sensitive cationic lipid according to

[15] , which is 7-(4-(dipropylamino)butyl)-7-hydroxy-13-((3-propylhexanoyl)oxy)tridecyl 3-ethylheptanoic acid, 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-butylheptanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-pentyloctanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-hexylnonanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-heptyldecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-octyl undecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(2-cycloheptyl acetate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(2-cyclododecyl acetate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(2-cyclopentadecanyl acetate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(6-ethyldecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(6-propyl undecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(6-butyl dodecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(6-hexyl dodecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(7-ethyl undecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(8-ethyldodecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(8,10,10-trimethylundecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(7,9,9-trimethyldecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(9,11,11-trimethyldodecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(7,11-dimethyldodecanoate) or 7-(4-(Dipropylamino)butyl)-7-hydroxytridecane-1,13-diyl bis(8,12-dimethyltridecanoate). Effects of the Invention

[0016] The lipid nanoparticles according to the present invention can highly express encapsulated genes in hepatocytes and spleen cells. Thus, the lipid nanoparticles can be used as gene delivery vectors used in gene therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the results of measuring the Fluc activity (RLU / mg protein) in the liver and spleen of mice to which each Fluc mRNA-loaded lipid nanoparticle was administered in Example 1. Figure 2 This is a graph showing the results of measuring Fluc activity (RLU / mg protein) in the liver and spleen of mice to which each Fluc mRNA-loaded lipid nanoparticle was administered in Example 1. Figure 3 This is a graph showing the results of measuring Fluc activity (RLU / mg protein) in the liver and spleen of mice to which each Fluc mRNA-loaded lipid nanoparticle was administered in Example 2. Figure 4 This is a graph showing the results of measuring the amount of lipid in the liver of mice to which each Fluc mRNA-loaded lipid nanoparticle was administered in Example 3 (percentage of the total dose per gram of liver; % / g liver). Figure 5 This is a graph showing the results of measuring TTR activity (μg / mL) in mouse serum 7 days after administration of TTR-RNP-ssODN-loaded lipid nanoparticles in Example 4. Figure 6 This is a graph showing the results of measuring TTR activity (μg / mL) in mouse serum 7 days after administration of TTR-RNP-ssODN-loaded lipid nanoparticles in Example 4. Figure 7 This is a graph showing the results of measuring the in vitro DNA cleavage activity of TTR-RNP-ssODN-loaded lipid nanoparticles in Example 6. Figure 8 This is a graph showing the results of measuring the TTR protein content (μg / mL) in mouse serum 7 days after administration of TTR-RNP-ssODN-loaded lipid nanoparticles in Example 6. Fig. 9 This is a graph showing the results of measuring Fluc activity (RLU / mg protein) in various tissues of mice administered with lipid nanoparticles loaded with each Fluc mRNA in Example 7. Fig.10 This is a graph showing the results of measuring Fluc activity (RLU / mg protein) in various tissues of mice administered with lipid nanoparticles loaded with each Fluc mRNA in Example 7. Fig.11 This is a graph showing the results of measuring Fluc activity (RLU / mg protein) in various tissues of mice to which frozen samples or non-frozen samples were administered in Example 7. Fig.12 This is a graph showing the results of measuring the TTR protein content (μg / mL) in mouse serum 7 days after administration of TTR-RNP-ssODN-loaded lipid nanoparticles in Example 8. Fig.13This is a graph showing the results of measuring the TTR protein content (μg / mL) in mouse serum 7 days after administration of lipid nanoparticles loaded with TTR-RNP-ssODN having different PEG-DMG content ratios and [RNP weight] / [total lipid weight] in Example 8. Fig.14 This figure shows the results of measuring the TTR protein content (μg / mL) in mouse serum 7 days after administration of lipid nanoparticles loaded with TTR-RNP-ssODN having different content ratios of CL4F11-ζ-2 and DSPC in Example 8. Fig.15 The results of the measurement of TTR protein content (μg / mL) in mouse serum 7 days after administration of lipid nanoparticles loaded with TTR-RNP-ssODN containing CL4F11-ζ-2 in Example 8 ( Fig.15 (A)), and the gene knockout efficiency (%) obtained from the results ( Fig.15 (B))Figure. Fig.16 It is a graph showing the results of measuring the TTR protein content (μg / mL) in mouse serum 1, 4 or 12 weeks after administration of lipid nanoparticles containing CL4F11-ζ-2 loaded with TTR-RNP-ssODN or lipid nanoparticles containing CL4F11-ζ-2 loaded with GFP-RNP-ssODN in Example 8. Fig.17 This is a graph showing the results of measuring the TTR protein content (μg / mL) in mouse serum 7 days after administration of TTR-RNP-ssODN-loaded lipid nanoparticles containing CL4F11-ζ-2, SM, ALC, or MC3 in Example 8. Fig.18 This is a graph showing the results of measuring Fluc activity (RLU / mg protein) in the liver or spleen of mice administered with lipid nanoparticles loaded with Fluc mRNA containing CL4F11-ζ-2, SM, ALC, or MC3 in Example 8. Fig.19 This is a graph showing the results of measuring the TTR protein content (μg / mL) in mouse serum 7 days after administration of frozen samples or non-frozen samples of lipid nanoparticles loaded with TTR-RNP-ssODN containing CL4F11-ζ-2, CL4F11-e-3 or CL4F12-e-4 in Example 8. Fig. 20This is a graph showing the measurement results of the DIR fluorescence intensity of each tissue of mice 1 hour after administration of lipid nanoparticles loaded with TTR-RNP-ssODN labeled with DIR or lipid nanoparticles loaded with sITTR labeled with DIR diluted to have the same DIR fluorescence intensity in Example 8. Fig.21 This is a graph showing the fluorescence intensity of the reaction solution containing sgTTR-G211-5FAM and ssODN-80%-3IBFQ, ssODN-40%-3IBFQ, or ssODN-0%-3IBFQ at each temperature from 11 to 70 °C in Example 9 ( Fig.21 part (A)), and the change amount of the fluorescence intensity at each temperature ( Fig.21 part (B)). Fig. 22 This is a graph showing the in vitro measurement results of the DNA cleavage activity of the RNP formed using sgTTR-G211 and ssODN-TTR_3-80%, ssODN-TTR 3-40%, or ssODN-TTR 3-0% at 37 °C ( Fig. 22 part (A)) or 15 °C ( Fig. 22 part (B)) in Example 9. Fig.23 This is a graph showing the measurement results of the TTR protein content (μg / mL) in the serum of mice 7 days after administration of lipid nanoparticles loaded with TTR-RNP-ssODN prepared at 4 °C in Example 9. Detailed Embodiments

[0018] Hereinafter, the embodiments of the present invention will be specifically described. In this specification, "X1-X2 (X1 and X2 are real numbers satisfying X1 < X2)" means "greater than or equal to X1 and less than or equal to X2"

[0019] pH-Sensitive Cationic Lipid The lipid nanoparticles according to the present invention are lipid nanoparticles containing a pH-sensitive cationic lipid represented by the following general formula (I) (hereinafter, sometimes referred to as "the pH-sensitive cationic lipid of the present invention"). By having the pH-sensitive cationic lipid represented by the general formula (I) as a constituent lipid of the lipid nanoparticles, the lipid nanoparticles according to the present invention have good import efficiency and expression efficiency for hepatocytes and splenocytes and can be used as gene carriers.

[0020] Chemical Formula 4 (R 1 )(R 2 )C(OH)-(CH2)a-(O-CO)b-X (I)

[0021] In the general formula (I), a represents an integer of 3-5, but is preferably 4. b represents 0 or 1. When b is 0, it means that there is no -O-CO- group and it is a single bond.

[0022] In the general formula (I), R 1 and R 2 Each independently represents a group represented by the following general formula (A). In the general formula (A), R 11 and R 12 Each independently represents a linear or branched C 1-15 Alkyl group (alkyl group having 1 to 15 carbon atoms).

[0023] Chemical formula 5 (R 11 )(R 12 )HC-(CH2)c-(CO-O)-(CH2)e- (A)

[0024] Straight or branched chain C 1-15 Alkyl groups include: methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, and tert-butyl. 2-4 Alkenyl includes: vinyl, 1-propenyl, 2-propenyl, 1-methylvinyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl; n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1-ethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl; n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-ethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1-methyl-2,2-dimethylbutyl; n-heptyl, 1-methylhexyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 1-ethylpentyl, 1,1-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1-methyl-3,3-dimethylbutyl, 2-methyl-3,3-dimethylbutyl; n-octyl, 1-methylheptyl, 2-methylheptyl, 3-methylheptyl, 4-methylheptyl, 5-methylheptyl, 6-methylheptyl, 1-ethylhexyl, 1,1-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 5,5-dimethylhexyl, 1-methyl-4,4-dimethylpentyl, 2-methyl-4,4-dimethylpentyl, 3-methyl-4,4-dimethylpentyl; n-nonyl, 1-methyloctyl, 2-methyloctyl, 3-methyloctyl, 4-methyloctyl, 5-methyloctyl, 6-methyloctyl, 7-methyloctyl, 1-ethylheptyl, 1,1-dimethylheptyl, 2,2-dimethylheptyl, 3,3-dimethylheptyl, 4,4-dimethylheptyl, 5,5-dimethylheptyl, 6,6-dimethylheptyl, 1-methyl-5,5-dimethylhexyl, 2-methyl-5,5-dimethylhexyl, 3-methyl-5,5-dimethylhexyl, 4-methyl-5,5-dimethylhexyl; n-Decyl, 1-methylnonyl, 2-methylnonyl, 3-methylnonyl, 4-methylnonyl, 5-methylnonyl, 6-methylnonyl, 7-methylnonyl, 8-methylnonyl, 1-ethyloctyl, 1,1-dimethyloctyl, 2,2-dimethyloctyl, 3,3-dimethyloctyl, 4,4-dimethyloctyl, 5,5-dimethyloctyl, 6,6-dimethyloctyl, 7,7-dimethyloctyl, 1-methyl-6,6-dimethylheptyl, 2-methyl-6,6-dimethylheptyl, 3-methyl-6,6-dimethylheptyl, 4-methyl-6,6-dimethylheptyl, 5-methyl-6,6-dimethylheptyl; n-Undecyl, 1-methyldecyl, 2-methyldecyl, 3-methyldecyl, 4-methyldecyl, 5-methyldecyl, 6-methyldecyl, 7-methyldecyl, 8-methyldecyl, 9-methyldecyl, 1-ethylnonyl, 1,1-dimethylnonyl, 2,2-dimethylnonyl, 3,3-dimethylnonyl, 4,4-dimethylnonyl, 5,5-dimethylnonyl, 6,6-dimethylnonyl, 7,7-dimethylnonyl, 8,8-dimethylnonyl, 1-methyl-7,7-dimethyloctyl, 2-methyl-7,7-dimethyloctyl, 3-methyl-7,7-dimethyloctyl, 4-methyl-7,7-dimethyloctyl, 5-methyl-7,7-dimethyloctyl, 6-methyl-7,7-dimethyloctyl; n-dodecyl, 1-methylundecyl, 2-methylundecyl, 3-methylundecyl, 4-methylundecyl, 5-methylundecyl, 6-methylundecyl, 7-methylundecyl, 8-methylundecyl, 9-methylundecyl, 10-methylundecyl, 1-ethyldecyl, 1,1-dimethyldecyl, 2,2-dimethyldecyl, 3,3-dimethyldecyl, 4,4-dimethyldecyl, 5,5-dimethyldecyl, 6,6-dimethyldecyl, 7,7-dimethyldecyl, 8,8-dimethyldecyl, 9,9-dimethyldecyl, 1-methyl-8,8-dimethylnonyl, 2-methyl-8,8-dimethylnonyl, 3-methyl-8,8-dimethylnonyl, 4-methyl-8,8-dimethylnonyl, 5-methyl-8,8-dimethylnonyl, 6-methyl-8,8-dimethylnonyl, 7-methyl-8,8-dimethylnonyl; tridecyl, 1-methyldodecyl, 2-methyldodecyl, 3-methyldodecyl, 4-methyldodecyl, 5-methyldodecyl, 6-methyldodecyl, 7-methyldodecyl, 8-methyldodecyl, 9-methyldodecyl, 10-methyldodecyl, 11-methyldodecyl, 1-ethylundecyl, 1,1-dimethylundecyl, 2,2-dimethylundecyl, 3,3-dimethylundecyl, 4,4-dimethylundecyl, 5,5-dimethylundecyl, 6,6 -dimethylundecyl, 7,7-dimethylundecyl, 8,8-dimethylundecyl, 9,9-dimethylundecyl, 10,10-dimethylundecyl, 1-methyl-9,9-dimethyldecyl, 2-methyl-9,9-dimethyldecyl, 3-methyl-9,9-dimethyldecyl, 4-methyl-9,9-dimethyldecyl, 5-methyl-9,9-dimethyldecyl, 6-methyl-9,9-dimethyldecyl, 7-methyl-9,9-dimethyldecyl, 8-methyl-9,9-dimethyldecyl; n-tetradecyl, 1-methyltridecyl, 2-methyltridecyl, 3-methyltridecyl, 4-methyltridecyl, 5-methyltridecyl, 6-methyltridecyl, 7-methyltridecyl, 8-methyltridecyl, 9-methyltridecyl, 10-methyltridecyl, 11-methyltridecyl, 12-methyltridecyl, 1-ethyldodecyl, 1,1-dimethyldodecyl, 2,2-dimethyldodecyl, 3,3-dimethyldodecyl, 4,4-dimethyldodecyl, 5,5-dimethyldodecyl, 6,6-dimethyldodecyl, 7,7-dimethyldodecyl, 8,8-dimethyldodecyl 10,10-dimethylundecyl, 9,9-dimethyldodecyl, 10,10-dimethyldodecyl, 11,11-dimethyldodecyl, 1-methyl-10,10-dimethylundecyl, 2-methyl-10,10-dimethylundecyl, 3-methyl-10,10-dimethylundecyl, 4-methyl-10,10-dimethylundecyl, 5-methyl-10,10-dimethylundecyl, 6-methyl-10,10-dimethylundecyl, 7-methyl-10,10-dimethylundecyl, 8-methyl-10,10-dimethylundecyl, 9-methyl-10,10-dimethylundecyl; n-pentadecyl, 1-methyltetradecyl, 2-methyltetradecyl, 3-methyltetradecyl, 4-methyltetradecyl, 5-methyltetradecyl, 6-methyltetradecyl, 7-methyltetradecyl, 8-methyltetradecyl, 9-methyltetradecyl, 10-methyltetradecyl, 11-methyltetradecyl, 12-methyltetradecyl, 13-methyltetradecyl, 1-ethyltridecyl, 1,1-dimethyltridecyl, 2,2-dimethyltridecyl, 3,3-dimethyltridecyl, 4,4-dimethyltridecyl, 5,5-dimethyltridecyl, 6,6-dimethyltridecyl, 7,7-dimethyltridecyl, 8,8-dimethyltridecyl, 9,9-dimethyl Tridecyl, 10,10-dimethyltridecyl, 11,11-dimethyltridecyl, 12,12-dimethyltridecyl, 1-methyl-11,11-dimethyldodecyl, 2-methyl-11,11-dimethyldodecyl, 3-methyl-11,11-dimethyldodecyl, 4-methyl-11,11-dimethyldodecyl, 5-methyl-11,11-dimethyldodecyl, 6-methyl-11,11-dimethyldodecyl, 7-methyl-11,11-dimethyldodecyl, 8-methyl-11,11-dimethyldodecyl, 9-methyl-11,11-dimethyldodecyl, 10-methyl-11,11-dimethyldodecyl, and the like.

[0025] In the general formula (A), R 11 and R 12 Each independently preferably is a linear or branched C 1-12 Alkyl (alkyl having 1 to 12 carbon atoms), more preferably a linear or branched C 1-10 Alkyl (alkyl having 1 to 10 carbon atoms), more preferably a linear or branched C 1-8 Alkyl (alkyl having 1 to 8 carbon atoms). In addition, in the pH-sensitive cationic lipid of the present invention, R 1 and R 2 They may be the same group as or different from each other.

[0026] In the general formula (A), R 11 and R 12 The group with more carbon atoms in the main chain is 11 and R 12 The carbon number of the group with more carbon atoms is Nc L The carbon number of the smaller group is Nc S . R 11 and R 12 With the same number of carbon atoms, Nc L and Nc SIn order to achieve higher delivery efficiency, in the general formula (A), Nc L The sum of Nc + c is preferably 5-17, more preferably 5-12, and further preferably 6-11. L The sum of Nc + c is preferably 10-15, and also preferably 13-15. From the viewpoint of delivery efficiency, Nc S Preferably, it is 1-8, more preferably 1-5, and more preferably 1-3. L Preferably, it is 4-8, more preferably 4-6. From the viewpoint of delivery efficiency, Nc L +Nc S The sum of +c is preferably 10-17, more preferably 10-15, and further preferably 11-13.

[0027] In the general formula (A), R 11 and R 12 Can be connected to each other to form a ring. Examples of the ring include cycloalkanes with 5-15 carbon atoms. Specifically, include: cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, cycloundecane, cyclododecane, cyclotridecane, cyclotetradecane, cyclopentadecane. These cycloalkanes may also have alkyl substituents.

[0028] In the general formula (A), c represents an integer of 1 to 7. From the viewpoint of improving biodegradability, the pH-sensitive cationic lipid of the present invention preferably has c as an integer of 4 or more, that is, the chain of the general formula (A) is preferably branched at the ε position or a carbon atom away therefrom.

[0029] In the general formula (A), e represents an integer of 4 to 12. In the pH-sensitive cationic lipid of the present invention, e is preferably an integer of 4 to 10, and more preferably an integer of 6 to 10.

[0030] In the general formula (I), X represents a group represented by the following general formula (B) 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 (O-CO)b- via a carbon atom.

[0031] Chemical formula 6 -(CH2)dN(R 3 )(R 4 ) (B)

[0032] In the formula (B), d represents an integer of 0 to 3. When d is 0, it means that there is no -(CH2)- group and it is a single bond.

[0033] In formula (B), R 3 and R 4 Each independently represents a C1-4 alkyl group (an alkyl group having 1 to 4 carbon atoms) or a C 2-4Alkenyl (alkenyl having 1 to 4 carbon atoms). 3 and R 4 The C 1-4 Alkyl or C 2-4 In the alkenyl group, one or two hydrogen atoms may be replaced by a phenyl group. 3 and R 4 Can be C 1-4 Alkyl or C 2-4 The alkenyl groups may be the same group or different groups.

[0034] C 1-4 Alkyl groups include: methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, and tert-butyl. 2-4 The alkenyl group includes vinyl, 1-propenyl, 2-propenyl, 1-methylvinyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl and 3-butenyl.

[0035] In formula (B), R 3 and R 4 They can combine with each other to form a 5-7 membered non-aromatic heterocyclic ring. 3 Examples of the 5-7 membered non-aromatic heterocyclic ring formed by combining R and R4 with each other include 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl and 1-piperazinyl. 3 and R 4 In the 5-7 membered non-aromatic heterocyclic ring formed by combining with each other, one or two hydrogen atoms in the ring may be replaced by C 1-4 Alkyl or C 2-4 When the two hydrogen atoms in the ring are replaced by C 1-4 Alkyl or C 2-4 When the alkenyl group is substituted, it may be substituted with the same group or with different groups.

[0036] In the general formula (I), when X is a 5-7 membered non-aromatic heterocyclic group, the heteroatom contained in the heterocyclic group may include a nitrogen atom, an oxygen atom or a sulfur atom. The heteroatom constituting the heterocyclic ring in the heterocyclic group may be one, or the same or different heteroatoms may be two or more. The heterocyclic ring in the heterocyclic group may be a saturated heterocyclic ring, or may contain one or more double bonds, but the heterocyclic ring will not become an aromatic ring.

[0037] The pH-sensitive cationic lipid of the present invention is preferably the following compound: In the general formula (I), R 1 and R 2 Each independently is a group (in the general formula (A), R 11 and R 12 Each independently is a linear or branched C 1-12alkyl, c is an integer of 1-7, e is an integer of 6-10), a is an integer of 3-5, b is 1, X is a 5-7 membered non-aromatic heterocyclic group (but is bonded to (O-CO)b- through a carbon atom in the heterocyclic group.), preferably 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl (bonded to (O-CO)b- through a carbon atom in the ring, and one hydrogen atom may be C 1-4 Alkyl or C 2-4 or, in the general formula (I), R 1 and R 2 Each independently is a group (in the general formula (A), R 11 and R 12 Each independently is a linear or branched C 1-12 alkyl, c is an integer of 1-7, e is an integer of 6-10), a is an integer of 3-5, b is 0, when X is the general formula (B), d is 0, R 3 and R 4 Each independently is C 1-4 Alkyl or C 2-4 Alkenyl (in R 3 and R 4 The C 1-4 Alkyl or C 2-4 In the alkenyl group, one or two hydrogen atoms may be substituted by a phenyl group). In particular, the pH-sensitive cationic lipid of the present invention is preferably a compound as follows: In the general formula (I), R 1 and R 2 Each independently is a group (in the general formula (A), R 11 and R 12 Each independently is a linear or branched C 1-8 alkyl, c is an integer of 1-7, e is an integer of 6-10), and a is an integer of 3-5, b is 1, X is 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl (bonded to (O-CO)b- through the carbon atom in the ring, and one hydrogen atom can be C 1-4 Alkyl or C 2-4 or, in the general formula (I), R 1 and R 2 Each independently is a group (in the general formula (A), R 11 and R 12 Each independently is a linear or branched C 1-8 alkyl, c is an integer of 1-7, e is an integer of 6-10), a is an integer of 3-5, b is 0, when X is the general formula (B), d is 0, R 3 and R 4 Each independently is C 1-4 alkyl.

[0038] The pH-sensitive cationic lipid of the present invention is more preferably the following compound: In the general formula (I), R 1 and R 2 Each independently is a group (in the general formula (A), R 11 and R 12 Each independently is a linear or branched C 1-8 alkyl, c is an integer of 4-7, e is an integer of 6-10), and a is an integer of 3-5, b is 1, X is 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl (bonded to (O-CO)b- through the carbon atom in the ring, and one hydrogen atom can be C 1-4 Alkyl or C 2-4 In the general formula (I), R 1 and R 2 Each independently is a group (in the general formula (A), R 11 and R 12 The carbon atoms in the ring are connected to form a cycloalkane having 5 to 15 carbon atoms, c is an integer of 4 to 7, e is an integer of 6 to 10), a is an integer of 3 to 5, b is 1, and X is 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl (bonded to (O-CO)b- through the carbon atom in the ring, and one hydrogen atom can be C 1-4 Alkyl or C 2-4 In the general formula (I), R 1 and R 2 Each independently is a group (in the general formula (A), R 11 and R 12 Each independently is a linear or branched C 1-8 alkyl, c is an integer of 4-7, e is an integer of 6-10), a is an integer of 3-5, b is 0, when X is the general formula (B), d is 0, R 3 and R 4 Each independently is C 1-4 Alkyl; in the general formula (I), R 1 and R 2 Each independently is a group (in the general formula (A), R 11 and R 12 connected to each other to form a cycloalkane having 5 to 15 carbon atoms, c is an integer of 4 to 7, e is an integer of 6 to 10), a is an integer of 3 to 5, b is 0, when X is the general formula (B), d is 0, R 3 and R 4 Each independently is C 1-4 alkyl.

[0039] The pH-sensitive cationic lipid of the present invention is particularly preferably the following compound: In the general formula (I), R 1 and R 2are the same group (and in the general formula (A), R 11 and R 12 Each independently is a linear or branched C 1-8 alkyl, c is an integer of 4-7, e is an integer of 6-10), a is an integer of 3-5, b is 1, X is 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl (bonded to (O-CO)b- through the carbon atom in the ring, and one hydrogen atom can be C 1-4 Alkyl or C 2-4 In the general formula (I), R 1 and R 2 are the same group (and in the general formula (A), R 11 and R 12 The carbon atoms in the ring are connected to form a cycloalkane having 5 to 15 carbon atoms, c is an integer of 4 to 7, e is an integer of 6 to 10), a is an integer of 3 to 5, b is 1, and X is 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl (bonded to (O-CO)b- through the carbon atom in the ring, and one hydrogen atom can be C 1-4 Alkyl or C 2-4 In the general formula (I), R 1 and R 2 are the same group (and in the general formula (A), R 11 and R 12 Each independently is a linear or branched C 1-8 alkyl, c is an integer of 4-7, e is an integer of 6-10), a is an integer of 3-5, b is 0, when X is the general formula (B), d is 0, R 3 and R 4 Each independently is C 1-4 Alkyl; in the general formula (I), R 1 and R 2 are the same group (and in the general formula (A), R 11 and R 12 connected to each other to form a cycloalkane having 5 to 15 carbon atoms, c is an integer of 4 to 7, e is an integer of 6 to 10), a is an integer of 3 to 5, b is 0, when X is the general formula (B), d is 0, R 3 and R 4 Each independently is C 1-4 alkyl.

[0040] The pH-sensitive cationic lipid of the present invention is particularly preferably the following compound: In the general formula (I), R 1 and R 2 are the same group (and in the general formula (A), R 11 and R 12 Each independently is a linear or branched C 1-8Alkyl, c is an integer from 4 to 7, Nc L +c is 8-12, Nc S is 1-6, e is an integer of 6-10) and a is an integer of 3-5, b is 0, when X is the general formula (B), d is 0, R 3 and R 4 Each independently is C 1-4 Alkyl; more preferably the following compounds: In the general formula (I), R 1 and R 2 are the same group (and in the general formula (A), R 11 and R 12 Each independently is a linear or branched C 1-8 Alkyl, c is an integer from 4 to 7, Nc L +c is 9-11, Nc S is 1-3, e is an integer of 6-10) and a is an integer of 3-5, b is 0, when X is the general formula (B), d is 0, R 3 and R 4 Each independently is C 1-4 Alkyl; further preferably the following compounds: In the general formula (I), R 1 and R 2 are the same group (and in the general formula (A), R 11 and R 12 Each independently is a linear or branched C 1-8 Alkyl, c is an integer of 4-6, Nc L +c is 9-11, Nc S is 1-3, e is an integer of 6-10) and a is an integer of 3-5, b is 0, when X is the general formula (B), d is 0, R 3 and R 4 Each independently is C 1-4 alkyl.

[0041] The pKa of the pH-sensitive cationic lipid represented by the general formula (I) is not particularly limited, and can be selected, for example, from about 4.0 to 9.0; preferably from about 4.5 to 8.5, and the type of each substituent is preferably selected to provide a pKa within this range.

[0042] The pH-sensitive cationic lipid represented by the general formula (I) can be easily produced by the method specifically shown in the examples of this specification, for example. With reference to this production method, by appropriately selecting raw material compounds, reagents and reaction conditions, a person skilled in the art can easily produce any lipid falling within the scope of the general formula (I).

[0043] The group represented by the general formula (A) is a group having two hydrocarbon chains (R 11 and R 12) is a branched structure group connected to a -CO-O- group. That is, the pH-sensitive cationic lipid of the present invention has two branched hydrocarbon chains (R 1 and R 2 ), these hydrocarbon chains become a hydrophobic scaffold embedded in the lipid membrane of the lipid nanoparticle. The lipid nanoparticle according to the present invention is characterized in that the pH-sensitive cationic lipid of the present invention having a hydrophobic scaffold composed of a branched structure is used as a lipid component, and has high selectivity for the liver.

[0044] Lipid Nanoparticles The lipid nanoparticles according to the present invention are composed of the pH-sensitive cationic lipids of the present invention as the constituent lipids, i.e., two hydrocarbon chains (R 11 and R 12 ) has a constituent lipid connected to a carbon atom located at the β position or away from the -CO-O- group. Thus, the lipid nanoparticles according to the present invention also have high selectivity for the spleen.

[0045] The type of pH-sensitive cationic lipid of the present invention constituting lipid nanoparticles according to the present invention may be only one, or may be two or more. When the pH-sensitive cationic lipid of the present invention constituting lipid nanoparticles according to the present invention is two or more, the amount of pH-sensitive cationic lipid of the present invention represents the total amount of lipid molecules equivalent to the pH-sensitive cationic lipid of the present invention in the lipid molecules constituting lipid nanoparticles.

[0046] The higher the percentage of the pH-sensitive cationic lipid of the present invention in the lipid molecules constituting the lipid nanoparticles, the higher the efficiency of the lipid nanoparticles in being taken up by the target cells. Thus, in the lipid nanoparticles according to the present invention, the percentage of the amount of the pH-sensitive cationic lipid of the present invention to the total amount of lipids constituting the lipid nanoparticles ([the amount of the pH-sensitive cationic lipid of the present invention (mol)] / ([the total amount of lipids constituting the lipid nanoparticles (mol)])×100%) is preferably 20 mol% or more. On the other hand, if the percentage of the pH-sensitive cationic lipid in the lipid molecules constituting the lipid nanoparticles is too high, it may be difficult to sufficiently reduce the particle size. From the perspective of the lipid nanoparticles having sufficient uptake efficiency for the target cells and obtaining lipid nanoparticles with a sufficiently small particle size, in the lipid nanoparticles according to the present invention, the amount of the pH-sensitive cationic lipid of the present invention to the total amount of lipids constituting the lipid nanoparticles is more preferably 30 mol% or more, further preferably 30-70 mol%, and further preferably 40-60 mol%.

[0047] Among the constituent lipids of the lipid nanoparticles according to the present invention, lipids other than the pH-sensitive cationic lipids of the present invention can use lipids commonly used when forming liposomes. Examples of such lipids include: phospholipids, sterols, glycolipids, or saturated or unsaturated fatty acids, etc. These can be used alone or in combination of two or more.

[0048] As phospholipids, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, cardiolipin, plasmalogen, ceramide phosphoglycerophosphate, phosphatidic acid and other glycerophospholipids can be listed; and sphingolipids such as sphingomyelin, ceramide phosphoglycerol, ceramide phosphoethanolamine, etc. can be listed. In addition, phospholipids of natural products, such as egg yolk phospholipids and soybean phospholipids, can also be used. The fatty acid residues in glycerophospholipids and sphingolipids are not particularly limited. For example, saturated or unsaturated fatty acid residues with 12-24 carbon atoms can be listed, preferably saturated or unsaturated fatty acid residues with 14-20 carbon atoms. Specifically, 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 can be listed. When these glycerolipids or sphingolipids have two or more fatty acid residues, all the fatty acid residues may be the same group or different groups.

[0049] Examples of sterols include animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, streptosterol, and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol, and brassicasterol; and microbial-derived sterols such as thymosterol and ergosterol. Examples of glycolipids include glyceroglycolipids such as sulforibosylglycerol, disaccharide diglycerides, digalactosyl diglycerides, galactosyl diglycerides, and glycosyl diglycerides; and sphingolipids such as galactosyl cerebroside, lactosyl cerebroside, and ganglioside. Examples of saturated or unsaturated fatty acids include saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.

[0050] The constituent lipids of the lipid nanoparticles according to the present invention preferably contain a neutral lipid, more preferably a phospholipid or a sterol, further preferably a sterol, and even more preferably cholesterol, in addition to the pH-sensitive cationic lipid of the present invention.

[0051] The lipid nanoparticles according to the present invention preferably contain polyalkylene glycol modified lipids as lipid components. Polyalkylene glycol is a hydrophilic polymer. By using polyalkylene glycol modified lipids as lipid membrane constituent lipids to construct lipid nanoparticles, the surface of the lipid nanoparticles can be modified with polyalkylene glycol. Surface modification with polyalkylene glycol can improve the stability of the retention of lipid nanoparticles in the blood.

[0052] As the polyalkylene glycol, polyethylene glycol, polypropylene glycol, polybutylene glycol, polyethylene glycol, etc. can be used. The molecular weight of the polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably 1,000 to 5,000.

[0053] For example, stearoyl polyethylene glycol (e.g., stearic acid PEG45 (STR-PEG45) etc.) can be used to modify lipids with polyethylene glycol. In addition, N-[carbonyl-methoxy polyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, n-[carbonyl-methoxy polyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine can also be used. Polyethylene glycol derivatives such as methoxypolyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG) are not limited to these.

[0054] The percentage of the polyalkylene glycol-modified lipid relative to the total amount of lipids constituting the lipid nanoparticles according to the present invention is not particularly limited, as long as it does not impair the liver selectivity caused by the pH-sensitive cationic lipid of the present invention, specifically, the liver-specific gene expression activity when the lipid nanoparticles according to the present invention are used as gene carriers. For example, the percentage of the polyalkylene glycol-modified lipid relative to the total amount of lipids constituting the lipid nanoparticles is preferably 0.5-3 mol%.

[0055] The lipid nanoparticles according to the present invention can be subjected to appropriate surface modification etc. as needed. The lipid nanoparticles according to the present invention can improve the retention in blood by modifying the surface with a hydrophilic polymer etc. The surface modification can be performed by using lipids modified with these modifying groups as the constituent lipids of the lipid nanoparticles.

[0056] In the manufacture of lipid nanoparticles according to the present invention, as lipid derivatives for improving the retention in blood, glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerol phospholipid derivatives, etc. can also be used. In addition to polyalkylene glycol, dextran, pullulan, polysucrose, 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 as hydrophilic polymers for surface modification to improve the retention in blood.

[0057] In addition, in order to promote the intranuclear migration of the lipid nanoparticles according to the present invention, for example, the lipid nanoparticles can be surface modified with an oligosaccharide compound having more than three sugars. The type of oligosaccharide compound having more than three sugars is not particularly limited, for example, an oligosaccharide compound having about 3-10 sugar units can be used, and preferably an oligosaccharide compound having about 3-6 sugar units can be used. Among them, it is preferred that a trimer or even a hexamer of glucose can be used, and it is further preferred that a trimer or tetramer of glucose can be used. More specifically, isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose or maltohexaose can be appropriately used, among which, maltotriose, maltotetraose, maltopentaose or maltohexaose in which glucose is combined with α1-4 are further preferred. Particularly preferred is maltotriose or maltotetraose, and most preferred is maltotriose. The amount of the oligosaccharide compound used to modify the surface of the lipid nanoparticles is not particularly limited, and for example, it is about 1-30 mol %, preferably about 2-20 mol %, and more preferably about 5-10 mol % based on the total lipid amount.

[0058] The method for surface modification of lipid nanoparticles with oligosaccharide compounds is not particularly limited, for example, since liposomes (International Publication No. 2007 / 102481) in which the surface of lipid nanoparticles is modified with monosaccharides such as galactose or mannose are known, the surface modification method described in the publication can be adopted. The entire disclosure of the above publication is incorporated herein by reference.

[0059] Furthermore, the lipid nanoparticles according to the present invention can be endowed with any one or more functions such as temperature change sensitivity function, membrane permeation function, gene expression function and pH sensitivity function. By appropriately adding these functions, the retention of lipid nanoparticles in the blood can be improved, and the lipid nanoparticles can be effectively escaped from the endosome after endocytosis in the target cells, so that the encapsulated nucleic acid can be more effectively expressed in the hepatocytes.

[0060] Lipid nanoparticles according to the present invention can comprise one or more materials selected from the group consisting of antioxidants such as tocopherol, propyl gallate, ascorbyl palmitate or butylated hydroxytoluene, charged substances, membrane polypeptides, etc. As charged substances that give positive charge, saturated or unsaturated fatty amines such as stearylamine and oleylamine can be enumerated, and as charged substances that give negative charge, 26-hexaphosphate ester, cholesterol hemisuccinate, phosphatidylserine, phosphatidylinositol, phosphatidic acid, etc. can be enumerated. As membrane polypeptides, membrane surface polypeptides or membrane intrinsic polypeptides, etc. can be enumerated. The preparation amount of these materials is not particularly limited and can be appropriately selected according to purpose.

[0061] For the size of the lipid nanoparticles according to the present invention, from the perspective of easily obtaining a high delivery efficiency to liver cells in vivo, the average particle size is preferably below 400nm, the average particle size is more preferably below 300nm, the average particle size is further preferably below 200nm, and further preferably below 150nm. In addition, the average particle size of lipid nanoparticles refers to the number average particle size measured by dynamic light scattering (Dynamic light scattering: DLS). The determination by dynamic light scattering can be carried out using a commercially available DLS device, etc., and according to a conventional method.

[0062] The polydispersity index (PDI) of the lipid nanoparticles according to the present invention is about 0.01-0.7, preferably about 0.01-0.6, and more preferably about 0.01-0.3. The zeta potential may range from -50 mV to 5 mV, preferably from -10 mV to 5 mV.

[0063] The morphology of the lipid nanoparticles according to the present invention is not particularly limited, and for example, as the morphology dispersed in an aqueous solvent, there can be listed unilamellar liposomes, multilamellar liposomes, spherical micelles or amorphous lamellar structures, etc. The lipid nanoparticles according to the present invention are preferably unilamellar liposomes or multilamellar liposomes.

[0064] The lipid nanoparticles according to the present invention preferably contain the target component delivered to the target cell inside the particle covered by the lipid membrane. The components contained in the lipid nanoparticles according to the present invention are not particularly limited as long as they are of a size that can be contained. Any substance such as nucleic acid, sugar, peptide, low molecular weight compound, metal compound, etc. can be encapsulated in the lipid nanoparticles according to the present invention.

[0065] As the component contained in the lipid nanoparticle according to the present invention, nucleic acid is preferably used. The nucleic acid can be DNA, RNA, or analogs or derivatives thereof (e.g., peptide nucleic acid (PNA), thionucleic acid DNA, etc.). The nucleic acid contained in the lipid nanoparticle according to the present invention can be single-stranded nucleic acid, double-stranded nucleic acid, linear, or cyclic.

[0066] The nucleic acid contained in the lipid nanoparticle of the present invention preferably comprises the foreign gene for expressing in the target cell, more preferably the nucleic acid for expressing the function of the foreign gene in the cell by being incorporated into the cell. The foreign gene can be the gene originally contained in the genomic DNA of the target cell (preferably hepatocyte), or the gene not contained in the genomic DNA. This type of nucleic acid comprises the gene expression vector of the nucleic acid consisting of the base sequence of the target gene to be expressed by encoding. The gene expression vector can exist as the extrachromosomal gene in the imported cell, or it can be incorporated into the genomic DNA by homologous recombination.

[0067] The gene expression vector contained in the lipid nanoparticle according to the present invention is not particularly limited, and the vector commonly used in gene therapy, etc. can be used. The gene expression vector contained in the lipid nanoparticle according to the present invention is preferably a nucleic acid vector such as a plasmid vector. The plasmid vector can remain circular, or it can be encapsulated in the lipid nanoparticle according to the present invention in a linear state after being cut in advance. The gene expression vector can be designed by conventional methods using commonly used molecular biology tools based on the base sequence information of the target gene to be expressed, and can be manufactured by various known methods.

[0068] According to the functional nucleic acid of the target gene expression that is present in the target cell that is contained in the lipid nanoparticle of the present invention, it is also preferably controlled.This functional nucleic acid can include antisense oligonucleotide, antisense DNA, antisense RNA, siRNA, mIcroRNA, mRNA etc.And, it can also be the plasmid DNA (pDNA) of the siRNA expression vector used as expressing siRNA in the cell.The siRNA expression vector can be prepared by commercially available siRNA expression vector, and, it is also possible to appropriately modify these.According to the functional nucleic acid of the lipid nanoparticle of the present invention, it is preferably mRNA or pDNA, because its selectivity to liver is particularly good.

[0069] The manufacture method of lipid nanoparticles according to the present invention is not particularly limited, and any method that can be utilized by a person skilled in the art can be used. For example, all lipid components are dissolved in organic solvents such as chloroform, and after forming a lipid film by using an evaporator to carry out reduced pressure drying or using a spray dryer to carry out spray drying, the composition (for example, nucleic acid, etc.) aqueous solvent encapsulated in the lipid nanoparticle is added to the above-mentioned dry mixture, and further, it can be manufactured by emulsifying using an emulsifier such as a homogenizer, an ultrasonic emulsifier or a high-pressure jet emulsifier, etc. In addition, liposomes can also be manufactured by known methods such as reverse phase evaporation. If you want to control the size of lipid nanoparticles, you can use a membrane filter with an aperture to extrude (extrusion filtration) under high pressure.

[0070] The composition of the aqueous solvent (dispersion medium) is not particularly limited, and may include, for example, buffers such as phosphate buffer, citrate buffer, phosphate buffered saline, physiological saline, cell culture medium, etc. These aqueous solvents (dispersion mediums) can be stably dispersed in the lipid nanoparticles, but may further include sugars (aqueous solutions) such as monosaccharides of glucose, galactose, mannose, fructose, inositol, ribose, and xylose, disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose, trisaccharides such as raffinose and raffinose, polysaccharides such as cyclodextrin, sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol, or polyols (aqueous solutions) such as glycerol, diglycerol, polyglycerol, 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. In order to stably preserve the lipid nanoparticles dispersed in the aqueous solvent for a long time, it is preferred to remove electrolytes in the aqueous solvent as much as possible from the viewpoint of physical stability such as suppressing aggregation. In addition, from the viewpoint of chemical stability of lipids, it is preferred to set the pH of the aqueous solvent to a weakly acidic to neutral range (pH is about 3.0-8.0), and / or remove dissolved oxygen by nitrogen bubbling or the like.

[0071] The lipid nanoparticles according to the present invention can also be manufactured by using the alcohol dilution method of the flow channel. The method is to import a solution dissolving the lipid component in an alcohol solvent and a solution dissolving the water-soluble component contained in the lipid nanoparticles in an aqueous solvent from different flow channels, and to manufacture the method of lipid nanoparticles by confluence of these. By using a microfluidic channel with a built-in three-dimensional micromixer that can achieve instant mixing of two liquids, lipid nanoparticles with a diameter of about 30nm can be manufactured with good reproducibility (non-patent literature 9). As the flow channel used during manufacturing, since a nano-sized lipid particle formation system with high particle size controllability can be formed, as described in patent document 2, it is preferred to use a flow channel structure of a simple two-dimensional structure, and the flow channel structure of the simple two-dimensional structure is arranged on the micro-sized flow channel of the circulating raw material solution, and a baffle (spoiler) with a certain width relative to the flow channel width is staggered from both sides. As the aqueous solvent used in the alcohol dilution method, the above-mentioned aqueous solvent can be used.

[0072] When the aqueous dispersion of the obtained lipid nanoparticles is freeze-dried or spray-dried, for example, glucose, galactose, mannose, fructose, inositol, ribose, monosaccharides of xylose, lactose, sucrose, cellobiose, trehalose, disaccharides such as maltose, trisaccharides such as raffinose, polysaccharides such as cyclodextrin, erythritol, xylitol, sorbitol, mannitol, sugar alcohols such as maltitol (aqueous solution) can be used to improve stability. And, when the above-mentioned aqueous dispersion is frozen, for example, the sugar or glycerol, diglycerol, polyglycerol, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol and other polyols (aqueous solution) can be used to improve stability.

[0073] When the lipid nanoparticle according to the present invention encapsulated with a gene expression vector is given to an individual animal, the gene expression vector encapsulated in the lipid nanoparticle is selectively expressed in the liver or spleen rather than in other organs. For example, when the lipid nanoparticle according to the present invention encapsulated with a target exogenous gene to be expressed in a hepatocyte or a spleen cell is given to a subject animal, the exogenous gene can be expressed in the liver or spleen of the subject animal. Similarly, when the lipid nanoparticle according to the present invention encapsulated with a siRNA expression vector is given to an individual animal, the siRNA expression vector encapsulated in the lipid nanoparticle 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.

[0074] Due to the high selectivity gene expression activity for this liver or spleen, lipid nanoparticle according to the present invention plays the effect of the gene expression vector of targeting liver or spleen.By the target exogenous gene to be expressed in hepatocyte or spleen cell is encapsulated in lipid nanoparticle according to the present invention and given to the test animal, this exogenous gene can be expressed in the liver or spleen of this test animal.Thus, lipid nanoparticle according to the present invention is useful as the effective ingredient for gene therapy pharmaceutical composition, particularly useful as the effective ingredient for gene therapy pharmaceutical composition for targeting organ with liver or spleen.

[0075] The animals to which the lipid nanoparticles according to the present invention are administered are not particularly limited, and may be humans or non-human animals. Non-human animals include mammals such as cattle, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, guinea pigs, or birds such as chickens, quails, and ducks. Furthermore, the administration route when the lipid nanoparticles according to the present invention are administered to animals is not particularly limited, but parenteral administration such as intravenous administration, enteral administration, intramuscular administration, subcutaneous administration, transdermal administration, nasal administration, and intrapulmonary administration is preferred.

[0076] Gene vectors for genome editing By including the nucleic acid etc. required for genome editing in the lipid nanoparticles according to the present invention, it can also be used as a gene vector for gene therapy using genome editing. For example, RNP is used in the genome editing performed using the CRISPR / Cas9 system, and the RNP is a complex of crRNA, tracrRNA and Cas9 protein. The desired gene knockout can be induced by expressing RNP in the target cell or by delivering RNP itself to the target cell. In addition, by simultaneously delivering donor DNA, gene knock-in can also be induced. Therefore, by including the RNP composed of the guide RNA (gRNA) and Cas9 protein composed of crRNA and tracrRNA in the lipid nanoparticles according to the present invention, hepatocytes and spleen cells can be used as the gene vector for genome editing as target cells. Because RNP itself is directly loaded into the lipid nanoparticles and imported into the target cell, the off-target effect is smaller than the method of importing the gene encoding the DNA nuclease such as Cas9 protein into the target cell and expressing it.

[0077] In the present invention and this specification, the DNA nuclease carried by the lipid nanoparticles binds to DNA in a gRNA-dependent manner and recognizes and cuts the double-stranded DNA formed by pairing with part of the gRNA. Examples of the DNA nuclease include Cas9, Cpf1, and the like.

[0078] In the present invention and this specification, the Cas9 protein is a protein that binds to DNA in a gRNA-dependent manner and has at least one of RuvC nuclease activity and HNH nuclease activity. The Cas9 protein having both RuvC nuclease activity and HNH nuclease activity cuts the double strands of genomic DNA. The Cas9 protein having only either RuvC nuclease activity or HNH nuclease activity cuts only either of the double strands of genomic DNA.

[0079] The Cas9 protein used in the present invention can be a wild-type Cas9 protein derived from a bacterium with a CRISPR system, or it can be a variant protein that changes the wild-type protein. Examples of bacteria with a CRISPR system include: Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, Campylobacter jejuni, Geobacillus stearothermophilus, Streptococcus thermophilus, Treponema denticola, etc. In addition, as a variant protein that has changed the wild-type Cas9 protein, a variant that has been introduced to inactivate any variation in RuvC nuclease activity or HNH nuclease activity can be cited. The example of the variant, including the 10th aspartic acid of the wild-type Cas9 protein is replaced by alanine variant (Cas9 (D10A)). Because it has the function of a DNA nickase, Cas9 (D10A) is also called Cas9 nickase (Cas9n). In addition, it can also be a mutant protein, into which a mutation that does not affect the nuclease activity of the wild-type Cas9 protein is introduced.

[0080] The Cas9 protein used in the present invention can be obtained by adding various peptides to the wild-type Cas9 protein or its variant protein, or it can be a chimeric protein fused with other proteins. As examples of the peptide, label peptides such as HIs tags, Myc tags, Flag tags, nuclear localization signal peptides, etc. can be listed. As other proteins fused to wild-type or variant Cas9 proteins, for example, GST, fluorescent proteins, etc. can be listed.

[0081] In the present invention and this specification, Cpf1 protein is a protein that binds to DNA in a gRNA-dependent manner and has only RuvC nuclease activity. Cas9 protein having both RuvC nuclease activity and HNH nuclease activity cuts the double strands of genomic DNA to form smooth ends, while Cpf1 protein forms 5' overhangs.

[0082] The Cpf1 protein used in the present invention may be a wild-type Cpf1 protein derived from a bacterium having a CRISPR / Cpf1 system, or a variant protein having a wild-type protein. Examples of bacteria having a CRISPR / Cpf1 system include: Acidaminococcus sp., Lachnospilraceae bacterIum, etc. Furthermore, as a variant protein having a wild-type Cpf1 protein, a variant having a variant that increases nuclease activity and a variant protein having a variant that does not affect nuclease activity can be cited.

[0083] The Cpf1 protein used in the present invention may be a wild-type Cpf1 protein or a variant protein thereof to which various peptides are added, or a chimeric protein fused with other proteins. As the peptide or other protein, those similar to those listed for the Cas9 protein can be used.

[0084] In the present invention and this specification, gRNA is an RNA having a base sequence that can be paired with a target sequence on the genomic DNA cut by DNA nuclease (the base sequence of the object of genome editing). "Base sequence that can be paired with a target sequence" generally refers to a base sequence that is homologous (identical) or complementary to the target sequence to inhibit recognition of regions outside the target sequence. gRNA can be composed of a single RNA or two or more RNAs that form a complex.

[0085] When the DNA nuclease loaded on the lipid nanoparticle is Cas9 protein, gRNA can use crRNA and tracrRNA.CrRNA is derived from bacteria with CRISPR system, which is a single-stranded RNA, including a region composed of a base sequence complementary to part of tracrRNA (binding region with tracrRNA), and a region composed of a base sequence homologous or complementary to the target sequence on genomic DNA (genomic DNA binding region).TracrRNA is derived from bacteria with the same CRISPR system, which is a single-stranded RNA, having a region composed of a base sequence complementary to part of crRNA (binding region with crRNA), and hybridizing with crRNA in this region to form a hairpin structure.Cas9 protein recognizes the hairpin structure and forms RNP.CrRNA and tracrRNA can be single-stranded RNAs that are independent of each other, or they can be single-stranded RNAs that are connected by appropriate RNA joints.In addition, as bacteria with CRISPR system, the above-mentioned bacteria can be listed.Cas9 protein, crRNA and tracrRNA can all be derived from bacteria of the same type, or they can be derived from bacteria of different types. Furthermore, crRNA and tracrRNA may consist solely of natural RNA, or may partially or completely contain modified RNA or artificial nucleic acid, as long as the function of the CRISPR / Cas9 system is not impaired.

[0086] When the DNA nuclease loaded on the lipid nanoparticle is Cpf1 protein, the gRNA can be any RNA containing the target sequence like crRNA, and tracrRNA is not necessary. Thus, the gRNA can be shorter than when the Cas9 protein is used. In addition, the gRNA can be composed only of natural RNA, or it can partially or completely contain modified RNA or artificial nucleic acid, as long as the function of the CRISPR / Cpf1 system is not impaired.

[0087] The target sequence usually selects the base sequence of the region immediately following the PAM sequence. The PAM sequence is a sequence recognized by a DNA nuclease such as the Cas9 protein, and is determined according to the DNA nuclease such as the Cas9 protein used. For example, the PAM sequence recognized by the Cas9 protein includes 5'-NGG (N: A, G, C, T), and the PAM sequence recognized by the Cpf1 protein includes 5'-TTTV (V: A, G, C), 5'-TTTN (N: A, G, C, T). The base length of the target sequence in the gRNA such as crRNA is not particularly limited, for example, it can be about 15-30 bases in length, preferably 18-22 bases in length.

[0088] When the lipid nanoparticle according to the present invention is used as a gene carrier for genome editing using the CRISPR / Cas9 system, in order to further improve the genome editing efficiency, preferably, the lipid nanoparticle according to the present invention further comprises: DNA nuclease, gRNA, and a single-stranded oligonucleotide (ssON) comprising a region that can be paired with the partial gRNA. The region that can be paired with gRNA in ssON is called "gRNA pairing region", and the region paired with partial ssON in gRNA is called "ssON pairing region". RNP is formed by pairing the gRNA pairing region and the ssON pairing region, which is a complex of DNA nuclease, gRNA and ssON.

[0089] ssON can be an oligonucleotide consisting only of DNA, or an oligonucleotide consisting only of RNA, or a chimeric oligonucleotide containing both DNA and RNA. Furthermore, ssON can be composed only of natural RNA or DNA, or can partially or completely contain modified nucleic acids or artificial nucleic acids, as long as the function of the CRISPR system used is not impaired.

[0090] Unlike nucleic acids, Cas9 protein and Cpf1 protein have positive charges, so the loading efficiency of lipid nanoparticles containing pH-sensitive cationic lipids in the lipid components constituting the lipid membrane is low. In contrast, if the nucleic acid complexed with DNA nucleases such as Cas9 protein contains not only gRNAs such as crRNA and tracrRNA but also ssON, the positive charge of RNP is suppressed and the negative charge becomes stronger due to the large amount of nucleic acid contained in RNP, so that it can be effectively loaded on lipid nanoparticles containing pH-sensitive cationic lipids.

[0091] The base length of the ssON used in the present invention can be long enough to make the RNP negatively charged, which can be appropriately determined by considering the base length of gRNA such as crRNA and tracrRNA, the type of Cas9 protein, etc. The length of ssON can be, for example, about 50-500 bases in length.

[0092] When the DNA nuclease loaded on the lipid nanoparticle is Cas9 protein, in crRNA, a partial region except the binding region with tracrRNA and a region composed of complementary base sequences are included. ssON and crRNA are hybridized by the region complementary to the crRNA (binding region with crRNA). That is, the three-person complex formed by the mutual hybridization of crRNA and tracrRNA, crRNA and ssON forms a complex with the Cas9 protein. The region hybridized with ssON in crRNA (binding region with ssON) is not particularly limited, as long as crRNA can simultaneously hybridize with tracrRNA and ssON to form a three-person complex and the three-person complex can form a hairpin structure recognized by Cas9 protein. For example, the binding region with ssON in crRNA can include part or all of the genomic DNA binding region, or it can be the same as the genomic DNA binding region. And, in the case of the double incision method of the Cas9 nickase (Cas9n) in which RuvC nuclease activity or HNH nuclease activity is inactivated, two types of guide RNAs are used. Therefore, in the case where the DNA nuclease loaded on the lipid nanoparticle is Cas9n and the lipid nanoparticle is loaded with RNP for the double nicking method, two types of ssONs hybridized with their respective guide RNAs are used.

[0093] When the target gene fragment is knocked into the cut genomic DNA by the CRISPR system, ssON can also be used as the donor DNA for the knock-in. For example, in addition to the binding region with gRNA such as crRNA, ssON also has a 40-60bp homologous sequence region (homology arms) added to both ends of the knocked-in gene fragment for homologous recombination, and functions as a donor DNA.

[0094] The determination of the target sequence on the genomic DNA, the design of the gRNA base sequence such as crRNA and tracrRNA, the design of the ssON base sequence, etc. can be based on the base sequence information of the genomic DNA and the commonly used molecular biology tools by conventional methods. For example, gRNA design can be performed using design tools such as CRISPR design tools (Horizon Discovery), TrueDesign genome editor (Invitrogen), etc.

[0095] In addition, the lipid nanoparticles encapsulating the RNP as the complex of the DNA nuclease, gRNA and ssON can be lipid nanoparticles other than the lipid nanoparticles of the present invention. For example, it can also be used as a gene carrier suitable for genome editing by being encapsulated in the lipid nanoparticles disclosed in patent document 1 or patent document 2.

[0096] The gRNA pairing region in the ssON used in the present invention can be a base sequence (completely matched sequence) that is completely complementary to the ssON pairing region in gRNA, but can also be a base sequence with some non-complementary structural bases (mismatched bases). When the sequence that the gRNA pairing region is completely matched with the ssON pairing region, the stability of the RNP of the complex of the DNA nuclease, gRNA and ssON is excellent, although the lipid nanoparticles containing the RNP can be stably manufactured, when the lipid nanoparticles are incorporated into cells, RNP may be difficult to dissociate in cells. When the gRNA pairing region has an appropriate amount of mismatched bases relative to the ssON pairing region, RNP has a good balance between stability in the manufacturing process and being easy to dissociate in the cell, and can be used as a gene carrier with excellent manufacturing stability and genome editing efficiency.

[0097] As the gRNA pairing region in ssON, preferably 30-60% of the bases are complementary to the corresponding bases in the ssON pairing region, and more preferably 35-55% of the bases are complementary to the corresponding bases in the ssON pairing region. In addition, the Tm value between the gRNA pairing region in ssON and the ssON pairing region in gRNA is preferably 25-40°C, more preferably 25-35°C, and further preferably 25-30°C. In addition, for example, if it is less than 18 bases, the Tm value can be determined by the Wallace method, and if it is more than 18 bases, the Tm value can be determined by the nearest neighbor base pair method.

[0098] As a gene carrier for genome editing using the CRISPR / Cas9 system, instead of or in addition to the pH-sensitive cationic lipid of the present invention, a lipid nanoparticle containing the DNA nuclease, gRNA, and a single-stranded oligonucleotide (ssON) in a region capable of pairing with a portion of the gRNA is preferably used, wherein the constituent lipid is a pH-sensitive cationic lipid represented by the following general formula (II) (hereinafter, sometimes referred to as "pH-sensitive cationic lipid (II)") and / or a pH-sensitive cationic lipid represented by the following general formula (III) (hereinafter, sometimes referred to as "pH-sensitive cationic lipid (III)"). Even when the lipid nanoparticle containing these pH-sensitive cationic lipids is used as a gene carrier, as ssON, preferably 30-60% of the bases in the gRNA pairing region are complementary to the corresponding bases in the ssON pairing region, and more preferably 35-55% of the bases are complementary to the corresponding bases in the ssON pairing region. Furthermore, the Tm value between the gRNA pairing region in ssON and the ssON pairing region in gRNA is preferably 25-40°C, more preferably 25-35°C, and further preferably 25-30°C.

[0099] Chemical formula 7 (R 21 )(R 22 )C(OH)-(CH2)a-(O-CO)bX (II)

[0100] In the general formula (II), a represents an integer of 3-5, but is preferably 4. b represents 0 or 1. When b is 0, it means that there is no -O-CO- group and it is a single bond.

[0101] In the general formula (II), R 21 and R 22 Each independently represents a group represented by the following general formula (A'). In the general formula (A'), q represents an integer of 1-9; r represents 0 or 1; s represents an integer of 1-3; t represents 0 or 1; u represents an integer of 1-8; f represents 0 or 1; and v represents an integer of 4-12. However, when b and f are both 0, q is an integer of 3-5, r and t are 1, s is 1, and except when u+v is an integer of 6-10.

[0102] Chemical formula 8 CH3-(CH2)q-(CH=CH)r-(CH2)s-(CH=CH)t-(CH2)u-(CO-O)f-(CH2)v- (A')

[0103] As the pH-sensitive cationic lipid (II), R21 and R 22 Specifically, in the pH-sensitive cationic lipid (II), R 21 and R 22 Preferably, it is a group having 20 or more carbon atoms, that is, in the general formula (A'), q+2r+s+2t+u+f+v is preferably an integer greater than 19. In particular, as a pH-sensitive cationic lipid (II), q+2r+s+2t+u+f+v is preferably an integer of 19-33, more preferably an integer of 19-31, further preferably an integer of 21-31, and further preferably an integer of 21-27.

[0104] R as pH-sensitive cationic lipid (II) 21 and R 22 Preferably, the following groups are selected: in the general formula (A'), r is 1, t is 0, q is an integer of 5-11, preferably an integer of 6-10, s+u is an integer of 5-11, preferably an integer of 6-10, f is 1, v is an integer of 4-12, and q+s+u+v is an integer greater than 16; r is 0, t is 1, q+s is an integer of 5-11, preferably an integer of 6-10, u is an integer of 5-8, f is 1, v is an integer of 4-12, and q+s+u+v is an integer greater than 16. is an integer from 4 to 12, q+s+u+v is an integer greater than 16; r and t are 0, q+s+u is an integer from 13 to 23, preferably from 15 to 21, f is 1, v is an integer from 4 to 12, and q+s+u+v is an integer greater than 18; r and t are 0, q+s+u is an integer from 13 to 23, preferably from 15 to 21, f is 1, v is an integer from 6 to 10, and q+s+u+v is an integer greater than 18.

[0105] In pH-sensitive cationic lipids (II), R 21 and R 22 The groups may be the same group as the general formula (A') or different groups from each other, but are preferably the same group.

[0106] In the general formula (II), X is the same as X in the general formula (I).

[0107] The pH-sensitive cationic lipid (II) is preferably the following lipid: In the general formula (II), a is an integer of 3 to 5, b is 1, and X is a 5-7 membered non-aromatic heterocyclic group (but it is bonded to (O-CO)b- via a carbon atom in the heterocyclic group), preferably 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl (it is bonded to (O-CO)b- via a carbon atom in the ring, and one hydrogen atom may be replaced by a C1-4 alkyl group or a C 2-4 Alkenyl substituted. ), R 21 and R 22Each independently represents the following groups: in the general formula (A'), r is 1, t is 0, q is an integer of 5-11, preferably an integer of 6-10, s+u is an integer of 5-11, preferably an integer of 6-10, f is 1, v is an integer of 4-12, and q+s+u+v is an integer greater than 16; in the general formula (II), a is an integer of 3-5, b is 1, and X is a 5-7 membered non-aromatic heterocyclic group (but is bonded to (O-CO)b- through a carbon atom in the heterocyclic group.), preferably a 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl (bonded to (O-CO)b- through a carbon atom in the ring, and one hydrogen atom may be a C1-4 alkyl or C 2-4 Alkenyl substituted. ), R 21 and R 22 are each independently the following groups: in the general formula (A'), r is 0, t is 1, q+s is an integer of 5-11, preferably an integer of 6-10, u is an integer of 5-8, f is 1, v is an integer of 4-12, and q+s+u+v is an integer greater than 16; in the general formula (II), a is an integer of 3-5, b is 0, when X is the general formula (B), d is 0, R 3 and R 4 Each independently is C 1-4 Alkyl or C 2-4 Alkenyl (in R 3 and R 4 The C 1-4 Alkyl or C 2-4 In the alkenyl group, one or two hydrogen atoms may be replaced by a phenyl group), R 21 and R 22 Each independently represents the following groups: In the general formula (A'), r is 1, t is 0, q is an integer of 5-11, preferably an integer of 6-10, s+u is an integer of 5-11, preferably an integer of 6-10, f is 1, v is an integer of 4-12, and q+s+u+v is an integer greater than 16; in the general formula (II), a is an integer of 3-5, b is 0, when X is the general formula (B), d is 0, R 3 and R 4 Each independently is C 1-4 Alkyl or C 2-4 Alkenyl (in R 3 and R 4 The C 1-4 Alkyl or C 2-4 In the alkenyl group, one or two hydrogen atoms may be replaced by a phenyl group), R 21 and R 22Each independently represents the following group: In the general formula (A'), r is 0, t is 1, q+s is an integer of 5-11, preferably an integer of 6-10, u is an integer of 5-8, f is 1, v is an integer of 4-12, and q+s+u+v is an integer greater than 16. Among these lipids, as the pH-sensitive cationic lipid represented by the general formula (II), R 21 and R 22 The lipids are the same group, especially preferably R 21 and R 22 are lipids of the same group and a is 4.

[0108] Chemical formula 9 (R 31 )(R 32 )C(OH)-(CH2)a-(O-CO)bX (III)

[0109] In the general formula (III), a represents an integer of 3-5, but is preferably 4. b represents 0 or 1. When b is 0, it means that there is no -O-CO- group and it is a single bond.

[0110] In the general formula (III), R 31 and R 32 Each independently represents a group represented by the following general formula (A”). In the general formula (A”), R 33 and R 34 Each independently represents a linear or branched C5-15 alkyl group (alkyl group having 5 to 15 carbon atoms); g represents 0 or 1; and h represents an integer of 4 to 12.

[0111] Chemical formula 10 (R 33 )(R 34 )-CH-(CO-O)g-(CH2)h- (A”)

[0112] As the linear or branched C5-15 alkyl group, 11 The same bases as those listed in .

[0113] In the general formula (A"), R 33 and R 34 Each is independently preferably a linear or branched C5-12 alkyl group (alkyl group having 5 to 12 carbon atoms), more preferably a linear or branched C5-10 alkyl group (alkyl group having 5 to 10 carbon atoms), and further preferably a linear or branched C6-9 alkyl group (alkyl group having 6 to 9 carbon atoms). 31 and R 32They may be groups represented by the general formula (A"), may be the same groups as each other, or may be different groups.

[0114] In the general formula (III), X is the same as X in the general formula (I).

[0115] The pH-sensitive cationic lipid (III) is more preferably the following compound: In the general formula (III), R 1 and R 2 Each independently is a group (in the general formula (A"), R 33 and R 34 Each independently represents a linear C6-9 alkyl group, g represents 1, h represents an integer of 6-10), a represents an integer of 3-5, b represents 1, X represents 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl (bonded to (O-CO)b- via a carbon atom in the ring, and one hydrogen atom may be C 1-4 Alkyl or C 2-4 In the general formula (III), R 1 and R 2 Each independently is a group (in the general formula (A"), R 33 and R 34 Each independently represents a branched C6-9 alkyl group, g represents 1, h represents an integer of 6-10), a represents an integer of 3-5, b represents 1, X represents 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl (bonded to (O-CO)b- via a carbon atom in the ring, and one hydrogen atom may be C 1-4 Alkyl or C 2-4 In the general formula (III), R 1 and R 2 Each independently is a group (in the general formula (A"), R 33 and R 34 Each independently represents a linear C6-9 alkyl group, g represents 1, h represents an integer of 6-10), a represents an integer of 3-5, b represents 0, and when X represents the general formula (B), d represents 0, R 3 and R 4 Each independently is C 1-4 Alkyl; in the general formula (III), R 1 and R 2 Each independently is a group (in the general formula (A"), R 33 and R 34 Each independently represents a branched C6-9 alkyl group, g represents 1, h represents an integer of 6-10), a represents an integer of 3-5, b represents 0, and when X represents the general formula (B), d represents 0, R 3 and R 4 Each independently is C 1-4 alkyl.

[0116] As the pH-sensitive cationic lipid (III), the following compounds are particularly preferred: In the general formula (III), R 1 and R 2 are the same group, and in the general formula (A"), R 33 and R 34 Each independently represents a linear C6-9 alkyl group, g represents 1, h represents an integer from 6 to 10, a represents an integer from 3 to 5, b represents 1, X represents 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl (bonded to (O-CO)b- via a carbon atom in the ring, and one hydrogen atom may be C 1-4 Alkyl or C 2-4 In the general formula (III), R 1 and R 2 are the same group, and in the general formula (A"), R 33 and R 34 Each independently represents a branched C6-9 alkyl group, g represents 1, h represents an integer from 6 to 10, a represents an integer from 3 to 5, b represents 1, X represents 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl (bonded to (O-CO)b- via a carbon atom in the ring, and one hydrogen atom may be C 1-4 Alkyl or C 2-4 In the general formula (III), R 1 and R 2 are the same group, and in the general formula (A"), R 33 and R 34 Each is independently a linear C6-9 alkyl group, g is 1, h is an integer of 6-10, a is an integer of 3-5, b is 0, and when X is of the general formula (B), d is 0, R 3 and R 4 Each independently is C 1-4 Alkyl; in the general formula (III), R 1 and R 2 are the same group, and in the general formula (A"), R 33 and R 34 Each independently represents a branched C6-9 alkyl group, g represents 1, h represents an integer of 6-10, a represents an integer of 3-5, b represents 0, and when X represents the general formula (B), d represents 0, R 3 and R 4 Each independently is C 1-4 alkyl.

[0117] The pKa of pH-sensitive cationic lipid (II) and pH-sensitive cationic lipid (III) is not particularly limited, and can be, for example, selected from about 4.0-9.0, preferably from about 4.5-8.5, and more preferably from about 6-8. The type of each substituent is preferably selected to provide a pKa within this range.

[0118] pH-sensitive cationic lipids (II) and pH-sensitive cationic lipids (III) can be easily produced, for example, by the method specifically shown in the examples of this specification. With reference to this production method, by appropriately selecting raw material compounds, reagents and reaction conditions, a person skilled in the art can easily produce any lipid falling within the scope of general formula (II) or general formula (III). Example

[0119] The present invention is further described in detail below by examples, but the present invention is not limited to the following examples.

[0120] NMR In the following experiments, unless otherwise stated, the ECA500 or ECS400 apparatus of JEOL was used and tetramethylsilane was used as the internal standard (0 ppm) to obtain 1 H and 13C NMR spectra. d is the abbreviation for doublet, t is the abbreviation for triplet, and m is the abbreviation for multiplet. 1 Chemical shifts in the H NMR spectra are reported in ppm, expressed on the σ scale, downfield from tetramethylsilane.

[0121] Thin layer chromatography (TLC) In the following experiments, unless otherwise stated, the reactants of all reactions were monitored by TLC using pre-coated TLC plates (manufactured by Millipore). Visualization of TLC was performed by ultraviolet light (254 nm), phosphomolybdic acid staining or p-anisaldehyde staining. The reaction products were purified by an automated combiflash (registered trademark) Rf chromatography system (manufactured by Teledyne ISCO) or a Selekt system (manufactured by Biotage).

[0122] Lipids that make up lipid nanoparticles Cholesterol was produced by SIGMA Aldrich, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), polyethylene glycol 2000-modified 2-dimyristoyl-rac-glycerol, and polyethylene glycol 2000-modified dimyristoylglycerol (PEG-DMG) were produced by NOF Corporation. SM-102 (hereinafter, abbreviated as "SM") was produced by Cayman Chemical (catalog number: P / N 33474), ALC-0315 (hereinafter, abbreviated as "ALC") was produced by Ambeed (catalog number: P / N A1375212), and DLin-MC3-DMA (MC3) was produced by MedChemExpress (catalog number: P / N HY-112251).

[0123] mRNA Among the nucleic acids contained in the lipid nanoparticles, mRNA encoding firefly luciferase (Fluc) (CleanCap FLuc mRNA, 5 moU) manufactured by TriLink Biotechnologies was used.

[0124] Preparation of RNP complexes The recombinant spCas9 nuclease (TrueCut Cas9 protein v2) used products manufactured by Thermo Fisher Scientific, and the single guide RNA (sgRNA) and single-stranded oligodeoxyribonucleotide (ssODN) used products manufactured by Integrated DNA Technologies. As sgRNA, two sgRNAs targeting the TTR gene (sgTTR-G211, sgTTR-G269) were used. The base sequences of sgRNAs are shown in Table 1.

[0125] Table 1

[0126] As ssODNs paired with sgTTR-G211, 7 types described in Table 2 (ssODN-TTR_3-100%, ssODN-TTR_3-80%, ssODN-TTR_3-60%, ssODN-TTR_3-50%, ssODN-TTR_3-40%, ssODN-TTR_3-30%, ssODN-TTR_3-0%) having sequences matching 30%-100% with the ssON pairing region in sgRNA were used. As ssODNs paired with sgTTR-G269, four types (ssODN-TTR_4-60%, ssODN-TTR_4-50%, ssODN-TTR_4-40%, ssODN-TTR_4-30%) described in Table 2 having sequences that match 30% to 100% with the ssON pairing region in sgRNA were used.

[0127] Table 2

[0128] As ssODNs paired with sgTTR-G211, the six types described in Table 3 (ssODN-TTR_3-10°C-3', ssODN-TTR_3-10°C-5', ssODN-TTR_3-24°C-3', ssODN-TTR_3-24°C-5', ssODN-TTR_3-50°C-3', ssODN-TTR_3-50°C-5') having mismatch sequences with the ssON pairing region in sgRNA present on either the 5' side or the 3' side were used.

[0129] When the Tm values ​​of sgRNA and ssODN in the RNP complex were measured using the fluorescence quenching method, sgRNA modified with the fluorescent substance FAM at the 5' end and ssODN modified with the quenching substance IBFQ (Iowa Black (registered trademark) FQ) at the 3' end were used. As FAM-modified sgRNA, RNA with FAM linked to the 5' end of sgTTR-G211 (sgTTR-G211-5FAM) was used. As IBFQ-modified ssODN, an IBFQ-modified RNA (ssODN-80%-3IBFQ) having IBFQ bound to the 3' end of a sequence that matches 80% of the ssON pairing region in sgRNA (ssODN-80%), an IBFQ-modified RNA (ssODN-40%-3IBFQ) having IBFQ bound to the 3' end of a sequence that matches 40% of the ssON pairing region (ssODN-40%), or an IBFQ-modified RNA (ssODN-0%-3IBFQ) having IBFQ bound to the 3' end of an RNA having a sequence that matches 0% of the ssON pairing region (ssODN-0%) was used.

[0130] Table 3

[0131] Preparation of lipid nanoparticles In the following experiments, unless otherwise specified, lipid nanoparticles were produced by the alcohol dilution method using a flow channel. As the flow channel, a microfluidic device "iLiNP" (manufactured by Lilacpharma) with a built-in mixer was used. The flow rate of the device was controlled using a syringe pump (Harvard apparatus).

[0132] Specifically, the preparation of lipid nanoparticles containing only nucleic acids is as follows. First, an ethanol solution containing each lipid component at a predetermined molar ratio adjusted to a lipid concentration of 8 mM and an acetate buffer (25 mM, pH 4.0) adjusted to a nucleic acid concentration of 48.9 ng / mL are introduced into the microchannel at 0.125 mL / min and 0.375 mL / min (total flow rate 0.5 mL / min), respectively, and the lipid nanoparticle solution discharged from the channel is recovered. The lipid nanoparticle solution is placed in a dialysis membrane (MWCO 12000-14000), and after dialysis at 4° C. with PBS (-) (pH 7.4) as the external aqueous phase for 2 hours or more, the lipid nanoparticle solution is recovered from the dialysis membrane.

[0133] The preparation of lipid nanoparticles containing RNPs is as follows. First, an equal amount of sgRNA solution (10 μM) was added dropwise to the Cas protein solution (10 μM) while vigorously mixing to prepare an RNP solution (5 μM). The RNP solution was mixed with an equal amount of ssODN solution (5 μM) to obtain an RNP-ssODN complex (2.5 μM). Next, an ethanol solution containing each lipid component at a predetermined molar ratio adjusted to a lipid concentration of 8 mM and an RNP-ssODN solution diluted to 160 nM with 20 mM MES buffer (pH 6.0, 50 mM NaCl) were fed into the microchannel at 0.050 mL / min and 0.450 mL / min (total flow rate 0.5 mL / min), respectively, to recover the lipid nanoparticle solution discharged from the channel. The lipid nanoparticle solution was placed in a dialysis membrane (MWCO 12000-14000), and after dialysis for 2 hours or more at 4°C with PBS (-) (pH 7.4) as the external aqueous phase, the lipid nanoparticle solution was recovered from the dialysis membrane. The recovered lipid nanoparticle solution was concentrated by ultrafiltration using a centrifugal filter unit ("Amicon Ultra-15", MWCO 100 kDa, manufactured by Millipore). When preparing fluorescently labeled RNP-containing lipid nanoparticles, an appropriate amount of fluorescent molecules is added to an ethanol solution in which the lipid concentration is adjusted to 8 mM, for example, in the case of the near-infrared fluorescent cyanine dye DIR, it is prepared by containing 0.5 mol%.

[0134] Determination of the average particle size and zeta potential of lipid nanoparticles The average particle size (number average), ζ average particle size and polydispersity index (PdI) of the lipid nanoparticles in PBS(-) (pH 7.4) and the zeta potential in 10 mM HEPES buffer (pH 7.4) were measured using the analysis device "Zetasizer Nano ZS ZEN3600" (manufactured by Malvern) using the dynamic light scattering method.

[0135] Determination of the pKa of lipid nanoparticles The pKa of lipid nanoparticles is determined by using p-ToluenesulfonIc acId (TNS). First, TNS (final concentration: 0.75 μM) and lipid nanoparticles (final concentration: 30 μM) are mixed in a buffer (200 mL) adjusted to each pH (pH 3.5-9.5). The buffer uses 20 mM citric acid buffer, 20 mM sodium phosphate buffer or 20 mM Tris-HCl buffer containing 130 mM NaCl. The fluorescence intensity of the prepared mixed solution is measured using a spectrofluorophotometer (VarioskanLux, manufactured by Thermo Fisher Scientific), set to λex = 321 nm and λem = 447 nm. In the measured values, the highest and lowest values ​​are taken as 100% and 0% charge rates, respectively, and the pH showing 50% charge rate is calculated as the expressed pKa. The content ratio (%) of pH-sensitive cationic lipids with cationic charge is calculated using the Henderson-Hasselbalch equation.

[0136] Encapsulation efficiency of lipid nanoparticles The encapsulation efficiency of the lipid nanoparticles was determined by measuring the amount of encapsulated nucleic acid (the amount of ssODN in the case of RNP-ssODN) using Ribogreen (product manufactured by Life Technologies) which is an RNA intercalator.

[0137] Metabolic evaluation of lipid nanoparticles (determination of lipid content in the liver) Liquid chromatography-mass spectrometry (LC / MS) for lipid quantification used liver samples stored at -80°C prior to analysis. Liver tissues were recovered from mice after intravenous injection of lipid nanoparticles (0.5 mg RNA / kg body weight). The tissue was homogenized with 200 μL of water, and the resulting tissue homogenate (50 μL) was extracted with 400 μL of acetonitrile / isopropanol (50:50 (v / v)). After centrifugation of the extract (4000 rpm, 10 minutes), the supernatant was filtered, and the filtrate was diluted 50 times with water / acetonitrile / isopropanol (1:8:8 (v / v)), transferred to a vial for LC / MS analysis using Accucore, and LC / MS was performed under the following conditions. Detection was performed under the optimal conditions for detecting parent lipids and predicted metabolites. Each lipid molecule was monitored using the mass of [M+H]+→916 (CL4H6), 864 (CL4F6), 982 (CL4F10-b-7), and 752 (CL4F10-e-2).

[0138] (LC / MS analysis conditions) LC / MS apparatus: LTQ-Orbitrap XL (manufactured by Thermo Scientific) Chromatographic column: RP-MS LC column (2.6 μm, 2.1 mm × 50 mm, manufactured by Thermo Scientific) Temperature: 40℃ Mobile phase: A gradient of solvent A (acetonitrile / water (60:40 (v / v)) containing 10 mM ammonium formate and 0.1% formic acid) and solvent B (isopropanol / acetonitrile (90:10 (v / v)) containing 10 mM ammonium formate and 0.1% formic acid) was used. Gradient conditions (solvent B concentration): 0 min (50%) → 2 min (90%) → 4 min (90%) → 4.5 min (50%) → 8 min (50%) Mode: Positive ion mode Injection volume: 1 μL Flow rate: 150μL / min

[0139] Determination of Fluc activity First, BALB / c mice (female, 4 weeks old) were intravenously injected with lipid nanoparticles (loaded with FlucmRNA) encapsulated with FlucmRNA at a dose of 0.01mg mRNA / kg body weight. Six hours after intravenous injection, mice were euthanized, each tissue was recovered, frozen with liquid nitrogen, and stored at -80°C. Bead-type cell disruption device (MicroSmash MS-100R, manufactured by TOMY SeIko) was used to homogenize tissue with passive lysis buffer (manufactured by Promega). Tissue fragments were removed by centrifugation and supernatant was recovered. According to the manufacturer's protocol, the Fluc activity in the supernatant was measured using a luciferase assay system (manufactured by E1500, Promega). Luminescence was measured using a luminometer (manufactured by Luminescencer-PSN, ATTO). Protein concentration was determined using a commercially available assay kit (BCA protein test kit, manufactured by PIerce). Fluc activity was expressed as relative light units (RLU) per milligram of protein.

[0140] Determination of TTR-KO activity BALB / c mice (female, 4 weeks old) were intravenously injected with lipid nanoparticles encapsulating Cas9 / sgTTR-RNP (Cas9 / sgTTR-RNP loaded lipid nanoparticles) at a specified dose. One week after intravenous injection, the mice were euthanized, blood was collected, and serum was separated by centrifugation. The TTR protein concentration in the serum was determined using a commercially available ELISA kit (Prealbumin ELISA kit (mouse), manufactured by Aviva Systems Biology) according to the manufacturer's protocol. The relative value (%) of the TTR protein concentration in the serum of mice given each lipid nanoparticle, with the serum TTR protein concentration of untreated mice set as 100%, was set as the knockout activity (TTR-KO activity) of the lipid nanoparticle.

[0141] In vitro DNA cleavage activity assay Genomic DNA was recovered and purified from BALB / c mouse liver tissue using a commercially available nucleic acid extraction kit (NucleoSpin Tissue, manufactured by MACHEREY-NAGEL) according to the manufacturer's protocol. Using this genomic DNA as a template and the primer set described in Table 4, dsDNA from the TTR locus was amplified by PCR (using KAPA HiFiHotStart DNA polymerase (manufactured by KAPA Biosystems)), and purified using a commercially available nucleic acid purification kit (ISOSPIN PCR product, manufactured by Nippon Gene).

[0142] Table 4

[0143] ssODN (5 equivalents of lipid nanoparticles) was added to Cas9 / sgTTR-RNP to form a TTR-RNP-ssODN complex. The TTR-RNP-ssODN complex and purified PCR product (0.2 equivalents of RNP) were mixed with reaction buffer (50mMTris-HCl, 100mM NaCl, 10mM MgCl2, 1mM DTT, pH7.9) and incubated at 37°C for 30 minutes. Next, the reaction solution was heated at 65°C for 5 minutes to inactivate Cas9-RNP, and the dsDNA was run in a 3% agarose gel at 100V for 40 minutes. The run gel was stained (GelRed, manufactured by Biotium) and visualized using a fluorescent / visible light gel photography system (Printgraph CMOS I, manufactured by ATTO). The bands were quantified using image analysis software (ImageJ).

[0144] DNA cleavage was calculated using the following formula: where Int_full and Int_cleaved represent the intensities from full-length (uncleaved) and cleaved DNA, respectively.

[0145] [DNA cleavage] = [Int cleaved ] / [Int full ]

[0146] Determination of Tm values ​​of sgRNA and ssODN by fluorescence quenching method When the Tm value of sgRNA and ssODN in the RNP complex is measured using the fluorescence quenching method, FAM-modified sgRNA whose 5' end is modified with the fluorescent substance FAM and 20 bases of IBFQ-modified ssODN whose 3' end is modified with the quenching substance IBFQ are used. Specifically, IBFQ-modified ssODN (5 equivalents of RNP) was added to Cas9 RNP containing FAM-modified sgRNA (final RNP concentration: 2.5 μM) to form an RNP-ssODN complex. The resulting solution (10 μL) was incubated at 10°C, and then the temperature was raised by 1°C at 10 second intervals. The fluorescence intensity was measured at various temperatures of 11-70°C using a thermal cycler ("Dice (registered trademark) Real Time System III", manufactured by TaKaRaBIo). The temperature at which the fluorescence intensity reached the maximum was set as the measurement Tm value (°C).

[0147] T7E1 trial BALB / c mice (female, 4 weeks old) were intravenously injected with 2 mg RNP / kg body weight of lipid nanoparticles encapsulating TTR-RNP-ssODN complexes (lipid nanoparticles loaded with TTR-RNP-ssODN) for 2 consecutive days. One week after the last injection, the mice were euthanized, and the liver tissues were recovered, quickly frozen in liquid nitrogen, and stored at -80°C. Genomic DNA was recovered and purified from the recovered liver tissue using a commercially available nucleic acid extraction kit (NucleoSpin Tissue, manufactured by MACHEREY-NAGEL) according to the manufacturer's protocol. The T7E1 test was performed using a commercially available genome editing detection kit (Alt-R (registered trademark), manufactured by Integrated DNA Technologies). The dsDNA treated with T7E1 was electrophoresed on an agarose gel in the same manner as described in the <Determination of in vitro DNA cleavage activity>, the gel after electrophoresis was stained, and the bands were quantified.

[0148] Evaluation of body distribution For fluorescent labeling of lipid nanoparticles, the RNP-ssODN solution was mixed with a 3-fold molar amount of fluorescent molecules (DyLight 650NHS ester) in DMSO and incubated for 30 minutes at ambient temperature in a dark room. Unreacted fluorescent molecules were removed by ultrafiltration (Amicon Ultra-0.5, MWCO 3kDa, manufactured by Millipore).

[0149] BALB / c mice (female, 4 weeks old) were intravenously injected with 2 mg RNP / kg body weight of lipid nanoparticles encapsulated with the obtained fluorescently labeled RNP-ssODN (fluorescently labeled RNP-ssODN loaded lipid nanoparticles). One hour after intravenous injection, the mice were euthanized and various tissues including blood were collected. The collected blood was immediately diluted with 1% sodium dodecyl sulfate (SDS) solution. After the recovered tissue was weighed immediately, it was homogenized in 1% sodium dodecyl sulfate (SDS) solution. The resulting homogenate was transferred to a black 96-well plate and fluorescence was measured at λex=652nm and λem=672nm using a microplate reader (VarioskanLux, manufactured by Thermo Fisher Scientific). A calibration curve was created for the fluorescently labeled RNP-ssODN loaded lipid nanoparticles and blood or tissue homogenates prepared in the same manner from untreated mice. Distribution in vivo is expressed as a percentage of the injected amount of each tissue or blood (% injected amount).

[0150] Toxicity evaluation BALB / c mice (female, 4 weeks old) were intravenously injected with lipid nanoparticles loaded with TTR-RNP-ssODN at 2 mg RNP / kg body weight for 2 consecutive days. One week after the last injection, the mice were anesthetized, weighed and blood samples were collected. Blood was collected from the inferior vena cava and serum was processed. Hematological parameters in serum were determined by Oriental Yeast Industries Co., Ltd.

[0151] Synthesis Example 1: Synthesis of CL4F6 [7-(4-(diisopropylamino)butyl)-13-((2-hexylnonanoyl)oxy)-7-hydroxytridecyl 2-hexyldecanoate]

[0152] Chemical formula 11

[0153] 7-(4-(diisopropylamino)butyl)tridecane-1,7,13-triol (1.0 mmol) synthesized according to the method described in Patent Document 1 was dissolved in 5 mL of dichloromethane, followed by the addition of 2-hexyldecanoic acid (2.20 mmol), DMAP (N,N-dimethyl-4-aminopyridine) (0.20 mmol) and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mmol), and the reaction was allowed to proceed overnight at room temperature. After the solvent was distilled off using a rotary evaporator, the mixture was suspended in ethyl acetate and the insoluble matter was removed by filtration. The filtrate was washed with a 0.5 N aqueous sodium hydroxide solution and saturated salt water. Anhydrous sodium sulfate was added to the organic layer for dehydration. After filtering, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by silica gel chromatography [eluting solvent; dichloromethane: methanol (continuous gradient)] to obtain CL4F6.

[0154] Synthesis Example 2: Synthesis of CL4F6-b-3 [7-(4-(dipropylamino)butyl)-7-hydroxy-13-((3-propylhexanoyl)oxy)tridecyl 3-ethylheptanoic acid]

[0155] Chemical formula 12

[0156] (1) Synthesis of ethyl 3-propylhex-2-enoate To triethyl phosphonoacetate (2.1 mL, 10.5 mmol) dissolved in THF (tetrahydrofuran, 20 mL) was added NaH (60% in mineral oil, 0.40 g, 10.0 mmol), and the resulting reaction mixture was stirred on ice for 30 minutes. 4-Heptanone (0.57 g, 5.0 mmol) was added to the reaction mixture and stirred overnight at 65 ° C. The reaction mixture was cooled to room temperature, diluted with EtOAc, washed with water and brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. Thus, 3-propylhex-2-enoic acid ethyl ester (1.76 g, yield 191%) was obtained as a yellow oil. The crude product can be used for the next step reaction without further purification.

[0157] (2) Synthesis of ethyl 3-propylhexanoate 3-propyl hex-2-enoic acid ethyl ester (1.76g) is dissolved in MeOH (10mL) and DCM (dichloromethane, 10mL) mixed solvent after under Ar atmosphere, adds Pd / C (176mg, 10wt%). Gained mixture is hydrogenated overnight at ambient temperature under balloon pressure. Reaction mixture is filtered through the small pad of diatomite, after washing with DCM, evaporates filtrate. Gained residue is loaded on normal phase chromatographic column (Sfaer Silica HC D, Biotage company manufactures), is purified by flash chromatography (Selekt, Biotage company manufactures) with the gradient mobile phase of hexane and EtOAc. Thus, obtain colorless oily 3-propyl hexanoic acid ethyl ester (0.59g, productive rate 63%).

[0158] (3) Synthesis of 3-propylhexanoic acid 3-propyl hexanoic acid ethyl ester (0.59g) was dissolved in a mixed solvent of 2-propanol (10.7mL) and water (5.3mL), KOH (1.77g, 31.5mmol) was added, and the reaction mixture was stirred overnight at 70°C. The reaction mixture was cooled to room temperature, and after adding hexane, it was washed with 1N HCl aqueous solution, water and brine, and dried over anhydrous Na2SO4. The reaction solution after the drying was filtered, and the filtrate was evaporated. Thus, colorless oily 3-propyl hexanoic acid (0.46g, yield 92%) was obtained. The crude product can be used for the next step reaction without further purification.

[0159] (4) Synthesis of CL4F6-b-3 3-Propyl hexanoic acid (348 mg, 2.2 mmol) was dissolved in anhydrous DCM (10 mL) and 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (388 mg, 1.0 mmol), and DMAP (12.2 mg, 0.10 mmol) and EDCI-HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (479 mg, 2.5 mmol) were added to the resulting mixture, and stirred overnight at ambient temperature. Then, the reaction mixture was evaporated, the residue was suspended with EtOAc, washed with 0.5 N NaOH aqueous solution and brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. The residue was loaded onto a reverse phase chromatography column (HPC18Aq, manufactured by Teledyne ISCO) and purified by flash chromatography (CombIFlash Rf, manufactured by Teledyne ISCO) using a gradient mobile phase of acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA aqueous solution and 0.1% TFA. Thus, CL4F6-b-3 (340 mg, yield 52%) was obtained as a colorless oil.

[0160] Synthesis Example 3: Synthesis of CL4F7-b-4 [7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diyl bis(3-butylheptanoate)]

[0161] Chemical formula 13

[0162] (1) Synthesis of ethyl 3-butylhept-2-enoate The reaction was carried out in the same manner as in Synthesis Example 2(1) except that 5-nonanone (0.71 g, 5.0 mmol) was used instead of 4-heptanone to obtain ethyl 3-butylhept-2-enoate (1.65 g, yield 155%) as a yellow oil. The crude product was used in the next reaction without further purification.

[0163] (2) Synthesis of ethyl 3-butylheptanoate The same reaction as in Synthesis Example 2 (2) was carried out except that 3-butylhept-2-enoic acid ethyl ester (1.65 g) was used instead of 3-propylhex-2-enoic acid ethyl ester and Pd / C 165 mg (10 wt %) was used to obtain colorless oily 3-butylhept-2-enoic acid ethyl ester (0.50 g, yield 47%).

[0164] (3) Synthesis of 3-butylheptanoic acid After 3-butylheptanoic acid ethyl ester (0.54g) was dissolved in a mixed solvent of 2-propanol (7.8mL) and water (3.9mL), KOH (1.29g, 23mmol) was added and the reaction mixture was stirred overnight at 70°C. The reaction mixture was cooled to room temperature, and after adding hexane, it was washed with 1N HCl aqueous solution, water and brine, and dried over anhydrous Na2SO4. The reaction solution after drying was filtered and the filtrate was evaporated. Thus, a colorless oily 3-butylheptanoic acid (0.41g, yield 95%) was obtained. The crude product can be used for the next step reaction without further purification.

[0165] (4) Synthesis of CL4F7-b-4 3-Butylheptanoic acid (261mg, 1.4mmol) was dissolved in anhydrous DCM (10mL) and 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (247mg, 0.64mmol), and then, in the resulting mixture, DMAP (7.8mg, 0.064mmol) and EDCI-HCl (307mg, 1.6mmol) were added and stirred overnight at ambient temperature. Then, the reaction mixture was evaporated, the residue was suspended in EtOAc, washed with 0.5NNaOH aqueous solution and brine, and dried over anhydrous Na2SO4. The reaction solution after the drying was filtered and the filtrate was evaporated. The residue was loaded onto a reverse phase column (HP C18Aq, manufactured by Teledyne ISCO) and purified by flash chromatography (CombIFlash Rf, manufactured by Teledyne ISCO) using a gradient mobile phase of acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA aqueous solution and 0.1% TFA. Thus, CL4F7-b-4 (150 mg, yield 32%) was obtained as a colorless oil.

[0166] Synthesis Example 4: Synthesis of CL4F8-b-5 [7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-pentyloctanoate))]

[0167] Chemical formula 14

[0168] (1) Synthesis of ethyl 3-pentyloct-2-enoate The reaction was carried out in the same manner as in Synthesis Example 2(1) except that 6-undecane (0.85 g, 5.0 mmol) was used instead of 4-heptanone to obtain ethyl 3-pentyloct-2-enoate (3.94 g, yield 164%) as a yellow oil. The crude product was used in the next reaction without further purification.

[0169] (2) Synthesis of ethyl 3-pentyloctanoate The same reaction as in Synthesis Example 2 (2) was carried out except that 3-pentyloctanoic acid ethyl ester (1.65 g) was used instead of 3-propylhex-2-enoic acid ethyl ester and 394 mg (10 wt %) of Pd / C was used to obtain colorless oily 3-pentyloctanoic acid ethyl ester (1.11 g, yield 46%).

[0170] (3) Synthesis of 3-pentyloctanoic acid After ethyl 3-pentyl octanoate (1.11 g) was dissolved in a mixed solvent of 2-propanol (15.6 mL) and water (7.8 mL), KOH (2.58 g, 46 mmol) was added and the reaction mixture was stirred overnight at 70 ° C. The reaction mixture was cooled to room temperature, and after adding hexane, it was washed with 1N HCl aqueous solution, water and brine, and dried over anhydrous Na2SO4. The reaction solution after the drying was filtered and the filtrate was evaporated. Thus, a colorless oily 3-pentyl octanoic acid (0.94 g, yield 97%) was obtained. The crude product can be used for the next step reaction without further purification.

[0171] (4) Synthesis of CL4F8-b-5 3-Amyl octanoic acid (461 mg, 2.15 mmol) was dissolved in anhydrous DCM (10 mL) and 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (379 mg, 0.98 mmol). DMAP (11.9 mg, 0.10 mmol) and EDCI-HCl (469 mg, 2.45 mmol) were added to the resulting mixture and stirred overnight at ambient temperature. Then, the reaction mixture was evaporated, the residue was suspended with EtOAc, washed with 0.5 N NaOH aqueous solution and brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. The residue was loaded onto a reverse phase column (HP C18Aq, manufactured by Teledyne ISCO) and purified by flash chromatography (CombIFlash Rf, manufactured by Teledyne ISCO) using a gradient mobile phase of acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA aqueous solution and 0.1% TFA. Thus, CL4F8-b-5 (320 mg, yield 42%) was obtained as a colorless oil.

[0172] Synthesis Example 5: Synthesis of CL4F9-b-6 [7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-hexylnonanoate))]

[0173] Chemical formula 15

[0174] (1) Synthesis of ethyl 3-hexylnon-2-enoate The reaction was carried out in the same manner as in Synthesis Example 2(1) except that 9-tridecanone (0.99 g, 5.0 mmol) was used instead of 4-heptanone to obtain ethyl 3-hexylnon-2-enoate (1.63 g, yield 121%) as a colorless oil. The crude product was used in the next reaction without further purification.

[0175] (2) Synthesis of ethyl 3-hexylnonanoate The same reaction as in Synthesis Example 2 (2) was carried out except that 3-hexylnon-2-enoic acid ethyl ester (1.63 g) was used instead of 3-propylhex-2-enoic acid ethyl ester and 163 mg (10 wt %) of Pd / C was used to obtain colorless oily 3-hexylnon-2-enoic acid ethyl ester (0.54 g, yield 40%).

[0176] (3) Synthesis of 3-hexylnonanoic acid 3-Hexyl nonanoic acid ethyl ester (0.54g) was dissolved in a mixed solvent of 2-propanol (6.8mL) and water (3.4mL), KOH (1.12g, 20mmol) was added, and the reaction mixture was stirred overnight at 70°C. The reaction mixture was cooled to room temperature, and after adding hexane, it was washed with 1N HCl aqueous solution, water and brine, and dried over anhydrous Na2SO4. The reaction solution after the drying was filtered, and the filtrate was evaporated. Thus, a colorless oily 3-hexyl nonanoic acid (0.44g, yield 92%) was obtained. The crude product can be used for the next step reaction without further purification.

[0177] (4) Synthesis of CL4F9-b-6 3-Hexyl nonanoic acid (339mg, 1.4mmol) was dissolved in anhydrous DCM (10mL) and 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (247mg, 0.64mmol), and then, in the resulting mixture, DMAP (7.8mg, 0.064mmol) and EDCI-HCl (307mg, 1.6mmol) were added and stirred overnight at ambient temperature. Then, the reaction mixture was evaporated, the residue was suspended in EtOAc, washed with 0.5NNaOH aqueous solution and brine, and dried over anhydrous Na2SO4. The reaction solution was filtered and the filtrate was evaporated. The residue was loaded onto a reverse phase column (HP C18Aq, manufactured by Teledyne ISCO) and purified by flash chromatography (CombIFlash Rf, manufactured by Teledyne ISCO) using a gradient mobile phase of acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA aqueous solution and 0.1% TFA. Thus, CL4F9-b-6 (250 mg, yield 46%) was obtained as a colorless oil.

[0178] Synthesis Example 6: Synthesis of CL4F10-b-7 [7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-heptyldecanoate))]

[0179] Chemical formula 16

[0180] (1) Synthesis of ethyl 3-heptyldec-2-enoate NaH (60%, 0.22 g, 5.5 mmol in mineral oil) was added to triethyl phosphonoacetate (1.20 mL, 6.0 mmol) dissolved in THF (20 mL), and the resulting reaction mixture was stirred on ice for 3 hours. After adding 8-pentadecanone (1.13 g, 5.0 mmol) to the reaction mixture, refluxed overnight. The reaction mixture was cooled to room temperature, diluted with EtOAc, washed with water and brine, and dried over anhydrous Na2SO4. The reaction solution after the drying was filtered and the filtrate was evaporated. Thus, a mixture of yellow oily 8-pentadecanone and 3-heptyldec-2-enoic acid ethyl ester (1.63 g, yield 110%) was obtained. The crude product can be used for the next step reaction without further purification.

[0181] (2) Synthesis of ethyl 3-heptyldecanoate The reaction was carried out in the same manner as in Synthesis Example 2 (2), except that 3-heptyldec-2-enoic acid ethyl ester (1.63 g) was used instead of 3-propylhex-2-enoic acid ethyl ester and 163 mg (10 wt %) of Pd / C was used, to obtain a colorless oily mixture of 8-pentadecanone and 3-heptyldecanoic acid ethyl ester (1.16 g, yield 45%).

[0182] (3) Synthesis of 3-heptyldecanoic acid A mixture of 8-pentadecanone and ethyl 3-heptyldecanoate (1.16 g) was dissolved in a mixed solvent of 2-propanol (15.3 mL) and water (7.7 mL), and KOH (1.54 g, 27.4 mmol) was added, and the reaction mixture was stirred overnight at 70 ° C. The reaction mixture was cooled to room temperature, and after adding hexane, it was washed with 1N HCl aqueous solution, water and brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. Thus, a mixture of 8-pentadecanone and ethyl 3-heptyldecanoate (1.05 g, yield 97%) was obtained as a black oil. The crude product can be used for the next step without further purification.

[0183] (4) Synthesis of CL4F10-b-7 A mixture of 8-pentadecanone and 3-heptyldecanoic acid (1.05 g, 2.20 mmol of 3-heptyldecanoic acid) was dissolved in anhydrous DCM (10 mL) and 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (388 mg, 1.0 mmol). DMAP (12.2 mg, 0.1 mmol) and EDCI-HCl (49 mg, 2.5 mmol) were added to the resulting mixture and stirred overnight at ambient temperature. Next, the reaction mixture was evaporated, the residue was suspended with EtOAc, washed with 0.5 N NaOH aqueous solution and brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. The residue was loaded onto a reverse phase column (HP C18Aq, manufactured by Teledyne ISCO) and purified by flash chromatography (CombIFlash Rf, manufactured by Teledyne ISCO) using a gradient mobile phase of acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA aqueous solution and 0.1% TFA. Thus, CL4F10-b-7 (360 mg, 40% yield) was obtained as a colorless oil.

[0184] Synthesis Example 7: Synthesis of CL4F11-b-8 [7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-octyl undecanoate))]

[0185] Chemical formula 17

[0186] (1) Synthesis of ethyl 3-octylundec-2-enoate The reaction was carried out in the same manner as in Synthesis Example 2 (1), except that 9-heptadecanone (1.27 g, 5.0 mmol) was used instead of 4-heptanone, to obtain reddish brown oily 3-octylundec-2-enoic acid ethyl ester (2,12 g, yield 131%). The crude product was used in the next reaction without further purification.

[0187] (2) Synthesis of ethyl 3-octyl undecanoate The same reaction as in Synthesis Example 2 (2) was carried out except that 3-octylundec-2-enoic acid ethyl ester (2.12 g) was used instead of 3-propylhex-2-enoic acid ethyl ester and Pd / C212 mg (10 wt %) was used to obtain colorless oily 3-octylundecanoic acid ethyl ester (1.05 g, yield 64%).

[0188] (3) Synthesis of 3-octylundecanoic acid After ethyl 3-octyl undecanoate (1.05 g) was dissolved in a mixed solvent of 2-propanol (10.8 mL) and water (5.4 mL), KOH (1.79 g, 32 mmol) was added and the reaction mixture was stirred overnight at 70 ° C. The reaction mixture was cooled to room temperature, and after adding hexane, it was washed with 1N HCl aqueous solution, water and brine, and dried over anhydrous Na2SO4. The reaction solution after drying was filtered and the filtrate was evaporated. Thus, 3-octyl undecanoic acid (0.84 g, yield 88%) was obtained as a yellow oil. The crude product can be used for the next step reaction without further purification.

[0189] (4) Synthesis of CL4F11-b-8 3-Octyl undecanoic acid (418 mg, 1.4 mmol) was dissolved in anhydrous DCM (10 mL) and 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (247 mg, 0.64 mmol). DMAP (7.8 mg, 0.064 mmol) and EDCI-HCl (307 mg, 1.6 mmol) were added to the resulting mixture and stirred overnight at ambient temperature. Then, the reaction mixture was evaporated, the residue was suspended with EtOAc, washed with 0.5 N NaOH aqueous solution and brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. The residue was loaded onto a reverse phase column (HP C18Aq, manufactured by Teledyne ISCO) and purified by flash chromatography (CombIFlash Rf, manufactured by Teledyne ISCO) using a gradient mobile phase of acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA aqueous solution and 0.1% TFA. Thus, CL4F11-b-8 (384 mg, yield 63%) was obtained as a yellow oil.

[0190] Synthesis Example 8: Synthesis of CL4F9-b [7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(2-cycloheptyl acetate))]

[0191] Chemical formula 18

[0192] (1) Synthesis of ethyl 2-cycloheptylene acetate The reaction was carried out in the same manner as in Synthesis Example 2(1) except that cycloheptanone (0.56 g, 5.0 mmol) was used instead of 4-heptanone to obtain ethyl 2-cycloheptylideneacetate (1.72 g, yield 189%) as a colorless oil. The crude product was used in the next reaction without further purification.

[0193] (2) Synthesis of ethyl 2-cycloheptyl acetate The same reaction as in Synthesis Example 2 (2) was carried out except that ethyl 2-cycloheptylidene acetate (1.72 g) was used instead of ethyl 3-propylhex-2-enoate and 172 mg (10 wt %) of Pd / C was used to obtain ethyl 2-cycloheptylidene acetate (0.80 g, yield 87%) as a colorless oil.

[0194] (3) Synthesis of 2-cycloheptylacetic acid After ethyl 2-cycloheptyl acetate (0.80 g) was dissolved in a mixed solvent of 2-propanol (14.8 mL) and water (7.4 mL), KOH (2.45 g, 43.7 mmol) was added and the reaction mixture was stirred overnight at 70 ° C. The reaction mixture was cooled to room temperature, and after adding hexane, it was washed with 1N HCl aqueous solution, water and brine, and dried over anhydrous Na2SO4. The reaction solution after drying was filtered and the filtrate was evaporated. Thus, colorless oily 2-cycloheptyl acetic acid (0.60 g, yield 77%) was obtained. The crude product can be used for the next step reaction without further purification.

[0195] (4) Synthesis of CL4F9-b 2-cycloheptyl acetic acid (600mg, 3.8mmol) was dissolved in anhydrous DCM (10mL) and 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (670mg, 1.73mmol), and then, in the resulting mixture, DMAP (21mg, 0.10mmol) and EDCI-HCl (830mg, 2.5mmol) were added and stirred overnight at ambient temperature. Then, the reaction mixture was evaporated, the residue was suspended in EtOAc, washed with 0.5NNaOH aqueous solution and saline, and dried over anhydrous Na2SO4. The reaction solution was filtered and the filtrate was evaporated. The residue was loaded onto a reverse phase column (HP C18Aq, manufactured by Teledyne ISCO) and purified by flash chromatography (CombIFlash Rf, manufactured by Teledyne ISCO) using a gradient mobile phase of acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA aqueous solution and 0.1% TFA. Thus, CL4F9-b (430 mg, yield 27%) was obtained as a colorless oil.

[0196] Synthesis Example 9: Synthesis of CL4F14-b [7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(2-cyclododecyl acetate))]

[0197] Chemical formula 19

[0198] (1) Synthesis of ethyl 2-cyclododecyl acetate The reaction was carried out in the same manner as in Synthesis Example 2(1) except that cyclododecanone (0.91 g, 5.0 mmol) was used instead of 4-heptanone to obtain ethyl 2-cyclododecyl acetate (2.0 g, yield 162%) as a yellow oil. The crude product was used in the next reaction without further purification.

[0199] (2) Synthesis of ethyl 2-cyclododecyl acetate The same reaction as in Synthesis Example 2 (2) was carried out except that ethyl 2-cyclododecyl acetate (2.0 g) was used instead of ethyl 3-propylhex-2-enoate and 200 mg (10 wt %) of Pd / C was used to obtain ethyl 2-cyclododecyl acetate (1.06 g, yield 84%) as a colorless oil.

[0200] (3) Synthesis of 2-cyclododecyl acetic acid After ethyl 2-cyclododecyl acetate (1.06 g) was dissolved in a mixed solvent of 2-propanol (14.1 mL) and water (7.1 mL), KOH (2.35 g, 41.8 mmol) was added, and the reaction mixture was stirred at 70 ° C overnight. The reaction mixture was cooled to room temperature, hexane was added, washed with 1N HCl aqueous solution, water and brine, and dried over anhydrous Na2SO4. The reaction solution after drying was filtered and the filtrate was evaporated. Thus, a white solid 2-cyclododecyl acetic acid (0.92 g, yield 82%) was obtained. The crude product can be used for the next step without further purification.

[0201] (4) Synthesis of CL4F14-b 2-Cyclododecylacetic acid (920mg, 4.1mmol) was dissolved in anhydrous DCM (10mL) and 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (720mg, 1.86mmol), and then DMAP (23mg, 0.18mmol) and EDCI-HCl (890mg, 4.66mmol) were added to the resulting mixture and stirred overnight at ambient temperature. Then, the reaction mixture was evaporated, the residue was suspended with EtOAc, washed with 0.5N NaOH aqueous solution and brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. The residue was loaded onto a reverse phase column (HP C18Aq, manufactured by Teledyne ISCO) and purified by flash chromatography (CombIFlash Rf, manufactured by Teledyne ISCO) using a gradient mobile phase of acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA aqueous solution and 0.1% TFA. Thus, CL4F14-b (770 mg, yield 52%) was obtained as a colorless oil.

[0202] Synthesis Example 10: Synthesis of CL4F17-b [7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(2-cyclopentadecanyl acetate))]

[0203] Chemical formula 20

[0204] (1) Synthesis of ethyl 2-cyclopentadecylideneacetate The reaction was carried out in the same manner as in Synthesis Example 2(1) except that cyclopentadecanone (1.12 g, 5.0 mmol) was used instead of 4-heptanone to obtain ethyl 2-cyclopentadecylideneacetate (2.3 g, yield 156%) as a yellow oil. The crude product was used in the next reaction without further purification.

[0205] (2) Synthesis of ethyl 2-cyclopentadecylacetate The same reaction as in Synthesis Example 2 (2) was carried out except that ethyl 2-cyclopentadecylideneacetate (2.3 g) was used instead of ethyl 3-propylhex-2-enoate and 30 mg (10 wt %) of Pd / C2 was used to obtain ethyl 2-cyclopentadecylate (0.84 g, yield 57%) as a colorless oil.

[0206] (3) Synthesis of 2-cyclopentadecanacetic acid 2-cyclopentadecanyl ethyl acetate (0.84g) was dissolved in a mixed solvent of 2-propanol (7.5mL) and water (4.7mL), KOH (1.57g, 28mmol) was added, and the reaction mixture was stirred overnight at 70°C. The reaction mixture was cooled to room temperature, and after adding hexane, it was washed with 1N HCl aqueous solution, water and brine, and dried over anhydrous Na2SO4. The reaction solution after filtering and drying was evaporated. Thus, white solid 2-cyclopentadecanyl acetic acid (0.69g, yield 91%) was obtained. The crude product can be used for the next step reaction without further purification.

[0207] (4) Synthesis of CL4F17-b 2-Cyclopentadecylacetic acid (690mg, 2.56mmol) was dissolved in anhydrous DCM (10mL) and 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (450mg, 1.16mmol), and then, DMAP (14.2mg, 0.12mmol) and EDCI-HCl (560mg, 2.9mmol) were added to the resulting mixture and stirred overnight at ambient temperature. Then, the reaction mixture was evaporated, the residue was suspended with EtOAc, washed with 0.5N NaOH aqueous solution and brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. The residue was loaded onto a reverse phase column (HP C18Aq, manufactured by Teledyne ISCO) and purified by flash chromatography (CombIFlash Rf, manufactured by Teledyne ISCO) using a gradient mobile phase of acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA aqueous solution and 0.1% TFA. Thus, CL4F17-b (280 mg, yield 27%) was obtained as a colorless oil.

[0208] Synthesis Example 11: Synthesis of CL4F10-e-2 [7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(6-ethyldecanoate))]

[0209] Chemical formula 21

[0210] (1) Synthesis of 6-ethyldec-4-enoic acid ethyl ester Under an Ar atmosphere cooled to 0°C, 1M potassium tert-butoxide (THF solution) (6.0mL) was added dropwise to triphenylphosphine bromide (2.74g, 6.0mmol) suspended in anhydrous THF (6mL), and the resulting reaction mixture was stirred at room temperature for 30 minutes. Then, 2-ethylhexanal (0.64g, 5.0mmol) was added to the reaction mixture and stirred overnight at room temperature. After the reaction mixture was quenched with a saturated NH4Cl aqueous solution, it was diluted with EtOAc, further washed with brine, and dried over anhydrous Na2SO4. The reaction solution after filtration and drying was evaporated. The residue was dissolved in hexane, and after passing through silica gel, the solvent was evaporated. The residue was loaded onto a normal phase chromatographic column (Sfaer Silica HC D, manufactured by Biotage), and purified by flash chromatography (Selekt, manufactured by Biotage) with a gradient mobile phase of hexane and EtOAc. Thus, colorless oily 6-ethyldec-4-enoic acid ethyl ester (0.38g, 34%) was obtained.

[0211] (2) Synthesis of 6-ethyldecanoate The reaction was carried out in the same manner as in Synthesis Example 2 (2), except that 6-ethyldec-4-enoic acid ethyl ester (0.38 g, 1.69 mmol) was used instead of 3-propylhex-2-enoic acid ethyl ester and Pd / C 38 mg (10 wt%) was used to obtain colorless oily 6-ethyldecanoic acid ethyl ester (0.38 g, yield 99%). The crude product was used in the next step without further purification.

[0212] (3) Synthesis of 6-ethyldecanoic acid After 6-ethyldecanoate (0.38 g, 1.67 mmol) was dissolved in a mixed solvent of 2-propanol (5.6 mL) and water (2.8 mL), KOH (0.94 g, 16.7 mmol) was added and the reaction mixture was stirred overnight at 70 ° C. The reaction mixture was cooled to room temperature, and after adding hexane, it was washed with 1N HCl aqueous solution, water and brine, and dried over anhydrous Na2SO4. The reaction solution after the drying was filtered and the filtrate was evaporated. Thus, a colorless oily 6-ethyldecanoic acid (0.31 g, yield 90%) was obtained. The crude product can be used for the next step reaction without further purification.

[0213] (4) Synthesis of CL4F10-e-2 6-ethyldecanoic acid (305mg, 1.5mmol) was dissolved in anhydrous DCM (10mL) and 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (264mg, 0.68mmol), and then, in the resulting mixture, DMAP (8.3mg, 0.064mmol) and EDCI-HCl (326mg, 1.7mmol) were added and stirred overnight at ambient temperature. Then, the reaction mixture was evaporated, the residue was suspended in EtOAc, washed with 0.5NNaOH aqueous solution and brine, and dried over anhydrous Na2SO4. The reaction solution was filtered and the filtrate was evaporated. The residue was loaded onto a reverse phase column (HP C18Aq, manufactured by Teledyne ISCO) and purified by flash chromatography (CombIFlash Rf, manufactured by Teledyne ISCO) using a gradient mobile phase of acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA aqueous solution and 0.1% TFA. The residue was placed in a normal phase column (Sfaer Silica HC D, Biotage) and purified by flash chromatography (Slekt, manufactured by Biotage) using a gradient mobile phase of DCM and MeOH. Thus, CL4F10-e-2 (270 mg, yield 52%) was obtained as a colorless oil.

[0214] Synthesis Example 12: Synthesis of CL4F11-e-3 [7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(6-propylundecanoate))]

[0215] Chemical formula 22

[0216] (1) Synthesis of 2-propylheptanal 2-Propylheptanol-1-ol (1.58 g, 10 mmol) was dissolved in anhydrous DCM (30 mL), TEMPO (2,2,6,6-tetramethylpiperidinyl 1-oxyl) (15.5 mg, 0.10 mmol) and Bu4NHSO4 (0.17 g, 0.50 mmol) were added, and the resulting mixture was cooled on ice. Then, NaOCl5H2O (1.98 g, 12.0 mmol) was added to the mixture, and the reaction mixture was stirred on ice for 3 hours. The reaction mixture was quenched with saturated Na2S2O3 aqueous solution, diluted with EtOAc, further washed with brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. Thus, 2-propylheptanal (1.46 g, 93%) was obtained as a colorless oil. The crude product can be used for the next step reaction without further purification.

[0217] (2) Synthesis of 6-butyl dodec-4-enoic acid ethyl ester Under an Ar atmosphere cooled to -78 ° C, (4-ethoxy-4-oxobutyl) triphenylphosphonium bromide (5.12g, 11.2mmol) suspended in anhydrous THF (8mL) was added dropwise 1M potassium tert-butoxide (THF solution) (11.2mL), and the resulting reaction mixture was stirred at -78 ° C for 30 minutes. Then, 2-propyl heptanal (1.46g) was added to the reaction mixture, and the temperature was gradually raised to room temperature overnight. After the reaction mixture was quenched with a saturated NH4Cl aqueous solution, it was diluted with Et2O, further washed with brine, and dried over anhydrous Na2SO4. The reaction solution after drying was filtered and the filtrate was evaporated. The residue was dissolved in hexane, and after passing through silica gel, the solvent was evaporated. The residue was loaded onto a normal phase chromatographic column (Sfaer Silica HC D, manufactured by Biotage) and purified by flash chromatography (Selekt, manufactured by Biotage) with a gradient mobile phase of hexane and EtOAc. Thus, ethyl 6-butyldodec-4-enoate (0.98 g, 41%) was obtained as a yellow oil.

[0218] (3) Synthesis of 6-butyldodecanoic acid ethyl ester Except that 6-butyl dodec-4-enoic acid ethyl ester (0.98 g, 3.85 mmol) was used instead of 3-propyl hex-2-enoic acid ethyl ester and Pd / C 98 mg (10 wt%) was used, the reaction was carried out in the same manner as in Synthesis Example 2 (2) to obtain 6-butyl dodecanoic acid ethyl ester (0.91 g, yield 92%) as a turbid white oil. The crude product was used for the next step reaction without further purification.

[0219] (4) Synthesis of 6-butyldodecanoic acid 6-Butyl dodecanoic acid ethyl ester (0.91g, 3.5mmol) was dissolved in a mixed solvent of 2-propanol (11.8mL) and water (5.9mL), KOH (1.96g, 35mmol) was added, and the reaction mixture was stirred overnight at 70°C. The reaction mixture was cooled to room temperature, and after adding hexane, it was washed with 1N HCl aqueous solution, water and brine, and dried over anhydrous Na2SO4. The reaction solution after the drying was filtered, and the filtrate was evaporated. Thus, yellow oily 6-butyl dodecanoic acid (0.78g, yield 97%) was obtained. The crude product can be used for the next step reaction without further purification.

[0220] (5) Synthesis of CL4F11-e-3 6-Butyl dodecanoic acid (692 mg, 2.7 mmol) was dissolved in anhydrous DCM (10 mL) and 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (460 mg, 1.2 mmol), and DMAP (14.7 mg, 0.12 mmol) and EDCI-HCl (575 mg, 3.0 mmol) were added to the resulting mixture and stirred overnight at ambient temperature. Then, the reaction mixture was evaporated, the residue was suspended with EtOAc, washed with 0.5 N NaOH aqueous solution and brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. The residue was loaded onto a reverse phase column (HP C18Aq, manufactured by Teledyne ISCO) and purified by flash chromatography (CombIFlash Rf, manufactured by Teledyne ISCO) using a gradient mobile phase of acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA aqueous solution and 0.1% TFA. The resulting residue was loaded onto a normal phase column (Sfaer Silica HC D, manufactured by Biotage) and purified by flash chromatography (Selekt, manufactured by Biotage) using a gradient mobile phase of DCM and MeOH. Thus, CL4F11-e-3 (445 mg, 46% yield) was obtained as a colorless oil.

[0221] Synthesis Example 13: Synthesis of CL4F12-e-4[7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(6-butyldodecanoate))]

[0222] Chemical formula 23

[0223] (1) Synthesis of 2-butyloctanal 2-Butyl-1-n-octanol (1.86 g, 10 mmol) was dissolved in anhydrous DCM (30 mL), TEMPO (15.5 mg, 0.10 mmol) and Bu4NHSO4 (0.17 g, 0.50 mmol) were added, and the resulting mixture was cooled on ice. Then, NaOCl 5H2O (1.98 g, 12.0 mmol) was added to the mixture, and the reaction mixture was stirred on ice for 3 hours. After the reaction mixture was quenched with a saturated Na2S2O3 aqueous solution, it was diluted with EtOAc, further washed with water and brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. The residue was loaded onto a normal phase chromatographic column (Sfaer Silica HC D, manufactured by Biotage) and purified by flash chromatography (Selekt, manufactured by Biotage) with a gradient mobile phase of hexane and EtOAc. Thus, a colorless oily 2-butyloctanal (1.86 g, 101%) was obtained.

[0224] (2) Synthesis of 6-hexyldodec-4-enoic acid ethyl ester Under an Ar atmosphere cooled to 0°C, 1M potassium tert-butoxide (THF solution) (12.0mL) was added dropwise to (4-ethoxy-4-oxobutyl) triphenylphosphonium bromide (5.49g, 12.0mmol) suspended in anhydrous THF (12mL), and the resulting reaction mixture was stirred at room temperature for 30 minutes. Then, 2-butyloctanal (1.86g, 10.0mmol) was added to the reaction mixture, and the reaction mixture was stirred at room temperature overnight. After the reaction mixture was quenched with a saturated NH4Cl aqueous solution, it was diluted with Et2O, further washed with brine, and dried over anhydrous Na2SO4. The reaction solution after filtration and drying was evaporated. The residue was dissolved in hexane, and after passing through silica gel, the solvent was evaporated. The residue was loaded onto a normal phase chromatographic column (Sfaer Silica HC D, manufactured by Biotage) and purified by flash chromatography (Selekt, manufactured by Biotage) with a gradient mobile phase of hexane and EtOAc. Thus, ethyl 6-butyldodec-4-enoate (1.75 g) was obtained as a colorless oil.

[0225] (3) Synthesis of 6-butyldodecanoic acid ethyl ester The reaction was carried out in the same manner as in Synthesis Example 2 (2), except that 6-butyl dodec-4-enoic acid ethyl ester (1.75 g) was used instead of 3-propyl hex-2-enoic acid ethyl ester and Pd / C 175 mg (10 wt%) was used to obtain 6-butyl dodecanoic acid ethyl ester (1.75 g) as a turbid white oil. The crude product was used in the next reaction without further purification.

[0226] (4) Synthesis of 6-butyldodecanoic acid After 6-butyl dodecanoic acid ethyl ester (1.75g) was dissolved in a mixed solvent of 2-propanol (4.0mL) and water (2.0mL), KOH (0.67g, 12mmol) was added and the reaction mixture was stirred overnight at 70°C. The reaction mixture was cooled to room temperature, and after adding hexane, it was washed with 1N HCl aqueous solution, water and brine, and dried over anhydrous Na2SO4. The reaction solution after drying was filtered and the filtrate was evaporated. Thus, yellow oily 6-butyl dodecanoic acid (1.59g) was obtained. The crude product can be used for the next step reaction without further purification.

[0227] (5) Synthesis of CL4F12-e-4 6-Butyl dodecanoic acid (1.59 g) was dissolved in anhydrous DCM (10 mL) and 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (210 mg, 0.55 mmol). DMAP (6.7 mg, 0.055 mmol) and EDCI-HCl (260 mg, 1.38 mmol) were added to the resulting mixture and stirred overnight at ambient temperature. Then, the reaction mixture was evaporated, the residue was suspended with EtOAc, washed with 0.5 N NaOH aqueous solution and brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. The residue was loaded onto a reverse phase column (HP C18Aq, manufactured by Teledyne ISCO) and purified by flash chromatography (CombIFlash Rf, manufactured by Teledyne ISCO) using a gradient mobile phase of acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA aqueous solution and 0.1% TFA. The resulting residue was loaded onto a normal phase column (SfaerSilica HC D, manufactured by Biotage) and purified by flash chromatography (Selekt, manufactured by Biotage) using a gradient mobile phase of DCM and MeOH. Thus, CL4F12-e-4 (85 mg, 18% yield) was obtained as a colorless oil.

[0228] Synthesis Example 14: Synthesis of CL4F12-e-6[7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(6-hexyldodecanoate)]

[0229] Chemical formula 24

[0230] (1) Synthesis of 2-hexyloctanal The reaction was carried out in the same manner as in Synthesis Example 13(1) except that 2-hexyl-1-n-octanol (2.14 g, 10 mmol) was used instead of 2-butyl-1-n-octanol to obtain 2-hexyloctanal (1.81 g, 85%) as a colorless oil.

[0231] (2) Synthesis of 6-hexyldodec-4-enoic acid ethyl ester Under an Ar atmosphere cooled to -78 ° C, (4-ethoxy-4-oxobutyl) triphenylphosphonium bromide (4.67g, 10.2mmol) suspended in anhydrous THF (8mL) was added dropwise 1M potassium tert-butoxide (THF solution) (10.2mL), and the resulting reaction mixture was stirred at -78 ° C for 30 minutes. Then, 2-hexyloctanal (1.81g) was added to the reaction mixture, and the reaction mixture was gradually warmed to room temperature overnight. After the reaction mixture was quenched with a saturated NH4Cl aqueous solution, it was diluted with Et2O, further washed with brine, and dried over anhydrous Na2SO4. The reaction solution after drying was filtered and the filtrate was evaporated. The residue was dissolved in hexane, and after passing through silica gel, the solvent was evaporated. The residue was loaded onto a normal phase chromatographic column (Sfaer Silica HC D, manufactured by Biotage) and purified by flash chromatography (Selekt, manufactured by Biotage) with a gradient mobile phase of hexane and EtOAc. Thus, ethyl 6-hexyldodec-4-enoate (1.93 g, yield 73%) was obtained as a colorless oil.

[0232] (3) Synthesis of 6-hexyldodecanoic acid ethyl ester The reaction was carried out in the same manner as in Synthesis Example 2 (2), except that 6-hexyldodec-4-enoic acid ethyl ester (1.93 g, 6.2 mmol) was used instead of 3-propylhex-2-enoic acid ethyl ester and Pd / C193 mg (10 wt%) was used to obtain 6-hexyldodecanoic acid ethyl ester (1.75 g) as a turbid white oil. The crude product was used in the next step without further purification.

[0233] (4) Synthesis of 6-hexyldodecanoic acid 6-Hexyldodecanoic acid ethyl ester (1.95g, 6.2mmol) was dissolved in a mixed solvent of 2-propanol (21mL) and water (10.5mL), KOH (3.48g, 62mmol) was added, and the reaction mixture was stirred overnight at 70°C. The reaction mixture was cooled to room temperature, and after adding hexane, it was washed with 1N HCl aqueous solution, water and brine, and dried over anhydrous Na2SO4. The reaction solution after the drying was filtered, and the filtrate was evaporated. Thus, yellow oily 6-hexyldodecanoic acid (1.73g, yield 98%) was obtained. The crude product can be used for the next step reaction without further purification.

[0234] (5) Synthesis of CL4F12-e-6 6-Hexyldodecanoic acid (1.2 g, 4.2 mmol) was dissolved in anhydrous DCM (10 mL) and 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (757 mg, 1.95 mmol), and DMAP (24.4 mg, 0.2 mmol) and EDCI-HCl (930 mg, 4.9 mmol) were added to the resulting mixture, and the mixture was stirred overnight at ambient temperature. Next, the reaction mixture was evaporated, the residue was suspended with EtOAc, washed with 0.5 N NaOH aqueous solution and brine, and dried over anhydrous Na2SO4. The residue was loaded onto a reverse phase column (HP C18Aq, manufactured by Teledyne ISCO) and purified by flash chromatography (CombIFlash Rf, manufactured by Teledyne ISCO) using a gradient mobile phase of acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA aqueous solution and 0.1% TFA. The obtained residue was loaded onto a normal phase chromatography column (Sfaer Silica HC D, manufactured by Biotage), and purified by flash chromatography (Selekt, manufactured by Biotage) using a gradient mobile phase of DCM and MeOH to obtain CL4F12-e-6 (320 mg, yield 18%) as a yellow oil.

[0235] Synthesis Example 15] Synthesis of CL4F11-ζ-2[7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(7-ethylundecanoate)]

[0236] Chemical formula 25

[0237] (1) Synthesis of 7-ethylundec-5-enoic acid Under an Ar atmosphere cooled to -78°C, 1M potassium tert-butoxide (THF solution) (12mL) was added dropwise to 4-(carboxybutyl)triphenylphosphine bromide (2.66g, 6.0mmol) suspended in anhydrous THF (6mL), and the resulting reaction mixture was stirred at -78°C for 30 minutes. Then, 2-ethylhexanal (0.64g, 5.0mmol) was added to the reaction mixture, and the reaction mixture was gradually warmed to room temperature overnight. After the reaction mixture was quenched with a saturated NH4Cl aqueous solution, it was diluted with Et2O, further washed with brine, and dried over anhydrous Na2SO4. The reaction solution after drying was filtered and the filtrate was evaporated. Thus, a colorless oily ethyl undecyl-5-enoic acid (0.69g, yield 65%) was obtained. The crude product can be used for the next step reaction without further purification.

[0238] (2) Synthesis of 7-ethylundecanoic acid Under Ar atmosphere, ethyl undecyl-5-enoic acid (0.69 g, 3.2 mmol) was dissolved in a mixed solvent of MeOH (10 mL) and DCM (10 mL), and Pd / C (69 mg, 10 wt%) was added. The resulting mixture was hydrogenated overnight at ambient temperature under balloon pressure. The reaction mixture was filtered through a small pad of diatomaceous earth, washed with DCM, and the filtrate was evaporated. Thus, 7-ethyl undecanoic acid (0.69 g, 100% yield) was obtained as a turbid white oil. The crude product was used for the next step without further purification.

[0239] (3) Synthesis of CL4F11-ζ-2 7-Ethyl undecanoic acid (0.69 g, 3.2 mmol) was dissolved in anhydrous DCM (10 mL), 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (562 mg, 1.45 mmol) and EDCI-HCl (690 mg, 3.6 mmol), and the reaction mixture was stirred overnight at ambient temperature. Then, the reaction mixture was evaporated, the residue was suspended with EtOAc, washed with 0.5 N NaOH aqueous solution and brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. The residue was loaded into a reverse phase chromatographic column (Sfaer C18, manufactured by Biotage) and purified by flash chromatography (Slekt, manufactured by Biotage) using a gradient mobile phase of a mixed solvent of 0.1% TFA aqueous solution and acetonitrile / 2-propanol (1:1 (volume ratio)) containing 0.1% TFA. The residue was placed on a normal phase chromatography column (Sfaer Silica HC D, manufactured by Biotage), and purified by flash chromatography (Slekt, manufactured by Biotage) using a gradient mobile phase of DCM and MeOH to obtain CL4F 11-ζ-2 (472 mg, yield 42%) as a colorless oil.

[0240] Synthesis Example 16: Synthesis of CL4F12-η-2[7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(8-ethyldodecanoate)]

[0241] Chemical formula 26

[0242] (1) Synthesis of 8-ethyldodec-6-enoic acid The reaction was carried out in the same manner as in Synthesis Example 15 (1), except that 5-(carboxybutyl)triphenylphosphonium bromide (2.74 g, 6.0 mmol) was used instead of 4-(carboxybutyl)triphenylphosphonium bromide (2.66 g, 6.0 mmol), to obtain colorless oily 8-ethyldodec-6-enoic acid (0.85 g, yield 75%). The crude product was used in the next step without further purification.

[0243] (2) Synthesis of 8-ethyldodecanoic acid The reaction was carried out in the same manner as in Synthesis Example 15 (2), except that ethyl dodec-6-enoic acid (0.85 g, 3.7 mmol) was used instead of ethyl undec-5-enoic acid (0.69 g, 3.2 mmol) and Pd / C (85 mg, 10 wt%) was used instead of Pd / C (69 mg, 10 wt%) to obtain turbid white oily 8-ethyl dodecanoic acid (0.86 g, yield 102%). The crude product was used in the next step without further purification.

[0244] (3) Synthesis of CL4F12-η-2 The reaction was carried out in the same manner as in Synthesis Example 15 (3), except that 8-ethyldodecanoic acid (0.55 g, 2.4 mmol) was used instead of 7-ethylundecanoic acid (0.69 g, 3.2 mmol), and anhydrous DCM (10 mL) and a mixture of 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (430 mg, 1.1 mmol) and DMAP (13 mg, 0.11 mmol) and EDCI-HCl (527 mg, 2.75 mmol) were used instead of anhydrous DCM (10 mL) and a mixture of 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (562 mg, 1.45 mmol) and EDCI-HCl (690 mg, 3.6 mmol) to obtain yellow oily CL4F12-η-2 (202 mg, yield 23%).

[0245] Synthesis Example 17: Synthesis of CL4F11-η-3(3)[7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(8,10,10-trimethylundecanoate)]

[0246] Chemical formula 27

[0247] (1) Synthesis of 3,5,5-trimethylhexanal 3,5,5-trimethylhexan-1-ol (1.44 g, 10 mmol) was dissolved in anhydrous DCM (30 mL), TEMPO (15.5 mg, 0.10 mmol) and Bu4NHSO4 (0.17 g, 0.50 mmol) were added, and the resulting mixture was cooled on ice. Then, NaOCl 5H2O (1.98 g, 12.0 mmol) was added to the mixture, and the reaction mixture was stirred on ice for 3 hours. After the reaction mixture was quenched with a saturated aqueous solution of Na2S2O3, it was diluted with EtOAc, further washed with brine, and dried over anhydrous Na2SO4. The dried reaction solution was filtered and the filtrate was evaporated. Thus, 3,5,5-trimethylhexanal (0.91 g, 64% yield) was obtained as a yellow oil. The crude product can be used for the next step reaction without further purification.

[0248] (2) Synthesis of 8,10,10-trimethylundec-5-enoic acid Under an Ar atmosphere cooled to -78°C, 1M potassium tert-butoxide (THF solution) (12mL) was added dropwise to 5-(carboxybutyl)triphenylphosphonium bromide (2.74g, 6.0mmol) suspended in anhydrous THF (6mL), and the resulting reaction mixture was stirred at -78°C for 30 minutes. Then, 3,5,5-trimethylhexanal (0.91g) was added to the reaction mixture, and the reaction mixture was gradually warmed to room temperature overnight. After the reaction mixture was quenched with a saturated NH4Cl aqueous solution, it was diluted with Et2O, further washed with brine, and dried over anhydrous Na2SO4. The reaction solution after drying was filtered and the filtrate was evaporated. The residue was loaded into a reverse phase chromatographic column (Sfaer C18, manufactured by Biotage) and purified by flash chromatography (Slekt, manufactured by Biotage) using a gradient mobile phase of 0.1% TFA aqueous solution and acetone containing 0.1% TFA. Thus, 8,10,10-trimethylundec-5-enoic acid (0.42 g, yield 37%) was obtained as a colorless oil.

[0249] (3) Synthesis of 8,10,10-trimethylundecanoic acid The reaction was carried out in the same manner as in Synthesis Example 15 (2), except that 8,10,10-trimethylundecanoic acid (0.42 g, 1.9 mmol) was used instead of ethylundecanoic acid (0.69 g, 3.2 mmol) and Pd / C (42 mg, 10 wt%) was used instead of Pd / C (69 mg, 10 wt%) to obtain turbid white oily 8,10,10-trimethylundecanoic acid (0.42 g, yield 99%). The crude product was used in the next step without further purification.

[0250] (4) Synthesis of CL4F11-η-3(3) The reaction was carried out in the same manner as in Synthesis Example 15(3), except that 8,10,10-trimethylundecanoic acid (0.42 g, 1.8 mmol) was used instead of 7-ethylundecanoic acid (0.69 g, 3.2 mmol), and anhydrous DCM (10 mL) and a mixture of 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (318 mg, 0.8 mmol) and DMAP (10 mg, 0.1 mmol) and EDCI-HCl (383 mg, 2.0 mmol) were used instead of anhydrous DCM (10 mL) and a mixture of 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (562 mg, 1.45 mmol) and EDCI-HCl (690 mg, 3.6 mmol) to obtain colorless oily CL4F11-η-3(3) (251 mg, yield 38%).

[0251] Synthesis Example 18: Synthesis of CL4F10-ζ-3(3)[7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(7,9,9-trimethyldecanoate)]

[0252] Chemical formula 28

[0253] (1) Synthesis of 3,5,5-trimethylhexanal The reaction was carried out in the same manner as in Synthesis Example 17(1) to obtain 3,5,5-trimethylhexanal (1.18 g, yield 83%) as a yellow oil. The crude product was used in the next reaction without further purification.

[0254] (2) Synthesis of 7,9,9-trimethyldec-4-enoic acid Under an Ar atmosphere cooled to -78°C, 1M potassium tert-butoxide (THF solution) (20mL) was added dropwise to 3-(carboxypropyl)triphenylphosphonium (4.3g, 10.0mmol) suspended in anhydrous THF (6mL), and the resulting reaction mixture was stirred at -78°C for 30 minutes. Then, 3,5,5-trimethylhexanal (1.18g) was added to the reaction mixture, and the reaction mixture was gradually warmed to room temperature overnight. After the reaction mixture was quenched with a saturated NH4Cl aqueous solution, it was diluted with Et2O, further washed with brine, and dried over anhydrous Na2SO4. The reaction solution after drying was filtered and the filtrate was evaporated. The residue was loaded into a reverse phase chromatographic column (Sfaer C18, manufactured by Biotage) and purified by flash chromatography (Slekt, manufactured by Biotage) using a gradient mobile phase of 0.1% TFA aqueous solution and acetone containing 0.1% TFA. Thus, 7,9,9-trimethyldec-4-enoic acid (0.51 g, yield 29%) was obtained as a yellow oil.

[0255] (3) Synthesis of 7,9,9-trimethyldecanoic acid The reaction was carried out in the same manner as in Synthesis Example 15 (2), except that 7,9,9-trimethyldec-4-enoic acid (0.51 g, 2.4 mmol) was used instead of ethyl undec-5-enoic acid (0.69 g, 3.2 mmol) and Pd / C (51 mg, 10 wt%) was used instead of Pd / C (69 mg, 10 wt%) to obtain 7,9,9-trimethyldecanoic acid (0.51 g, yield 99%) as a turbid white oil. The crude product was used in the next step without further purification.

[0256] (4) Synthesis of CL4F10-ζ-3(3) The reaction was carried out in the same manner as in Synthesis Example 15 (3), except that 7,9,9-trimethyldecanoic acid (0.51 g, 2.4 mmol) was used instead of 7-ethylundecanoic acid (0.69 g, 3.2 mmol), and anhydrous DCM (10 mL) and a mixture of 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (426 mg, 1.1 mmol) and DMAP (13 mg, 0.1 mmol) and EDCI-HCl (518 mg, 2.7 mmol) were used instead of anhydrous DCM (10 mL) and a mixture of 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (562 mg, 1.45 mmol) and EDCI-HCl (690 mg, 3.6 mmol) to obtain yellow oily CL4F10-ζ-3(3) (75 mg, 9% yield).

[0257] Synthesis Example 19: Synthesis of CL4F12-θ-3(3)[7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(9,11,11-trimethyldodecanoate)]

[0258] Chemical formula 29

[0259] (1) Synthesis of 3,5,5-trimethylhexanal The reaction was carried out in the same manner as in Synthesis Example 17(1) to obtain 3,5,5-trimethylhexanal (1.37 g, yield 94%) as a yellow oil. The crude product was used in the next reaction without further purification.

[0260] (2) Synthesis of 9,11,11-trimethyldodec-6-enoic acid Under an Ar atmosphere cooled to -78°C, 1M potassium tert-butoxide (THF solution) (23mL) was added dropwise to 5-(carboxybutyl)triphenylphosphonium bromide (5.2g, 11.3mmol) suspended in anhydrous THF (6mL), and the resulting reaction mixture was stirred at -78°C for 30 minutes. Then, 3,5,5-trimethylhexanal (1.37g) was added to the reaction mixture, and the reaction mixture was gradually warmed to room temperature overnight. After the reaction mixture was quenched with a saturated NH4Cl aqueous solution, it was diluted with Et2O, further washed with brine, and dried over anhydrous Na2SO4. The reaction solution after drying was filtered and the filtrate was evaporated. The residue was loaded into a reverse phase chromatographic column (Sfaer C18, manufactured by Biotage) and purified by flash chromatography (Slekt, manufactured by Biotage) using a gradient mobile phase of 0.1% TFA aqueous solution and acetone containing 0.1% TFA. Thus, 9,11,11-trimethyldodec-6-enoic acid (0.47 g, yield 21%) was obtained as a colorless oil.

[0261] (3) Synthesis of 9,11,11-trimethyldodecanoic acid The reaction was carried out in the same manner as in Synthesis Example 15 (2), except that 9,11,11-trimethyldodec-6-enoic acid (0.47 g, 2.0 mmol) was used instead of ethylundec-5-enoic acid (0.69 g, 3.2 mmol) and Pd / C (47 mg, 10 wt%) was used instead of Pd / C (69 mg, 10 wt%) to obtain turbid white oily 9,11,11-trimethyldodecanoic acid (0.47 g, yield 96%). The crude product was used in the next step without further purification.

[0262] (4) Synthesis of CL4F12-θ-3(3) The reaction was carried out in the same manner as in Synthesis Example 15 (3), except that 9,11,11-trimethyldodecanoic acid (0.47 g, 1.9 mmol) was used instead of 7-ethylundecanoic acid (0.69 g, 3.2 mmol), and anhydrous DCM (10 mL) and a mixture of 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (333 mg, 0.9 mmol) and DMAP (11 mg, 0.1 mmol) and EDCI-HCl (414 mg, 2.2 mmol) were used instead of anhydrous DCM (10 mL) and a mixture of 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (562 mg, 1.45 mmol) and EDCI-HCl (690 mg, 3.6 mmol) to obtain yellow oily CL4F12-θ-3(3) (233 mg, yield 32%).

[0263] Synthesis Example 20: Synthesis of CL4F12-ζ-2(2)[7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(7,11-dimethyldodecanoate)]

[0264] Chemical formula 30

[0265] (1) Synthesis of 3,7-dimethyloctanal The reaction was carried out in the same manner as in Synthesis Example 17 (1), except that 3,7-dimethyloctan-1-ol (1.58 g, 10.0 mmol) was used instead of 3,5,5-trimethylhexan-1-ol (1.44 g, 10 mmol), to obtain 3,7-dimethyloctanal (1.59 g, yield 102%) as a colorless oil. The crude product was used in the next reaction without further purification.

[0266] (2) Synthesis of 7,11-dimethyldodec-4-enoic acid The reaction was carried out in the same manner as in Synthesis Example 17 (2), except that 3-(carboxypropyl)triphenylphosphonium bromide (5.2 g, 12.0 mmol) was used instead of 5-(carboxybutyl)triphenylphosphonium bromide (5.2 g, 11.3 mmol) and 3,7-dimethyloctanal (1.59 g) was used instead of 3,5,5-trimethylhexanal (1.37 g) to obtain colorless oily 7,11-dimethyldodec-4-enoic acid (0.90 g, yield 40%).

[0267] (3) Synthesis of 7,11-dimethyldodecanoic acid The reaction was carried out in the same manner as in Synthesis Example 15 (2), except that 7,11-dimethyldodec-4-enoic acid (0.90 g, 4.0 mmol) was used instead of ethylundec-5-enoic acid (0.69 g, 3.2 mmol) and Pd / C (90 mg, 10 wt%) was used instead of Pd / C (69 mg, 10 wt%) to obtain 7,11-dimethyldodecanoic acid (0.90 g, yield 99%) as a turbid white oil. The crude product was used in the next step without further purification.

[0268] (4) Synthesis of CL4F12-ζ-2(2) The reaction was carried out in the same manner as in Synthesis Example 15 (3), except that 7,11-dimethyldodecanoic acid (0.90 g, 4.0 mmol) was used instead of 7-ethylundecanoic acid (0.69 g, 3.2 mmol), and anhydrous DCM (10 mL) and a mixture of 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (698 mg, 1.8 mmol) and DMAP (22 mg, 0.2 mmol) and EDCI-HCl (861 mg, 4.5 mmol) were used instead of anhydrous DCM (10 mL) and a mixture of 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (562 mg, 1.45 mmol) and EDCI-HCl (690 mg, 3.6 mmol) to obtain yellow oily CL4F12-ζ-2(2) (306 mg, yield 21%).

[0269] Synthesis Example 21: Synthesis of CL4F13-η-2(2)[7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(8,12-dimethyltridecanoate)]

[0270] Chemical formula 31

[0271] (1) Synthesis of 3,7-dimethyloctanal The reaction was carried out in the same manner as in Synthesis Example 19(1) to obtain 3,7-dimethyloctanal (1.63 g, yield 104%) as a colorless oil. The crude product was used in the next reaction without further purification.

[0272] (2) Synthesis of 8,12-dimethyltridecanoic acid The reaction was carried out in the same manner as in Synthesis Example 17 (2), except that 4-(carboxybutyl)triphenylphosphonium bromide (5.3 g, 12.0 mmol) was used instead of 5-(carboxybutyl)triphenylphosphonium bromide (5.2 g, 11.3 mmol) and 3,7-dimethyloctanal (1.63 g) was used instead of 3,5,5-trimethylhexanal (1.37 g) to obtain colorless oily 8,12-dimethyltridecanoic acid (0.77 g, yield 32%).

[0273] (3) Synthesis of 8,12-dimethyltridecanoic acid The reaction was carried out in the same manner as in Synthesis Example 15 (2), except that 7,11-dimethyldodec-4-enoic acid (0.90 g, 4.0 mmol) was used instead of ethylundec-5-enoic acid (0.69 g, 3.2 mmol) and Pd / C (90 mg, 10 wt%) was used instead of Pd / C (69 mg, 10 wt%) to obtain turbid white oily 8,12-dimethyltridecanoic acid (0.76 g, yield 98%). The crude product was used in the next step without further purification.

[0274] (4) Synthesis of CL4F13-η-2(2) The reaction was carried out in the same manner as in Synthesis Example 15(3), except that 8,12-dimethyltridecanoic acid (0.76 g, 3.1 mmol) was used instead of 7-ethylundecanoic acid (0.69 g, 3.2 mmol), and anhydrous DCM (10 mL) and a mixture of 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (553 mg, 1.4 mmol) and DMAP (17 mg, 0.1 mmol) and EDCI-HCl (684 mg, 3.1 mmol) were used instead of anhydrous DCM (10 mL) and a mixture of 7-(4-dipropylamino)butyl)tridecane-1,7,13-triol (562 mg, 1.45 mmol) and EDCI-HCl (690 mg, 3.6 mmol) to obtain yellow oily CL4F13-η-2(2) (558 mg, yield 47%).

[0275] Example 1 Lipid nanoparticles were prepared using the lipids synthesized in Synthesis Example 1-14 as constituent lipids. In addition, 7-(4-(diisopropylamino)butyl)-7-hydroxytridecane-1,13-diyl dioleate (CL4H6) was also used as a constituent lipid. CL4H6 was synthesized according to the method described in Patent Document 1. As neutral lipids, DSPC, cholesterol (chol), and PEG-DMG were used.

[0276] Specifically, pH-sensitive cationic lipids, DSPC, cholesterol and PEG-DMG in a molar ratio of 50 / 10 / 40 / 1.5 (mol%) were used to prepare lipid nanoparticles loaded with Fluc mRNA (lipid nanoparticles loaded with Fluc mRNA) by an alcohol dilution method. The type of pH-sensitive cationic lipids used in the preparation, the encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI, ζ potential (mV) and pKa of the prepared lipid nanoparticles were measured.

[0277] The results of lipid nanoparticles containing a pH-sensitive cationic lipid (a pH-sensitive cationic lipid branched at the β position) in which c in the general formula (A) is 1 among the pH-sensitive cationic lipids of the present invention are shown in Table 5.

[0278] Table 5

[0279] Regardless of which pH-sensitive cationic lipid is used, lipid nanoparticles encapsulating nucleic acids can be prepared. It was observed that as the number of carbon atoms of the pH-sensitive cationic lipid increases, the encapsulation rate (%) increases, the particle size decreases, and the pKa tends to decrease. In addition, when comparing chain-type pH-sensitive cationic lipids (CL4F6-b-3, CL4F7-b-4, CL4F8-b-5, CL4F9-b-6, CL4F10-b-7, CL4F11-b-8) and cyclic pH-sensitive cationic lipids (CL4F9-b, CL4F14-b, CL4F17-b), it was observed that the cyclic type tends to have a higher pKa than the chain type.

[0280] Next, the prepared lipid nanoparticles loaded with Fluc mRNA were administered to Balb / c mice, and the Fluc activity (RLU / mg protein) was measured. Figure 1 As shown. Regardless of which lipid nanoparticles were administered to mice, Fluc activity was confirmed in the liver and spleen. From this result, it can be confirmed that all pH-sensitive cationic lipids used this time are suitable as constituent lipids of lipid nanoparticles that can be used as gene carriers. In particular, CL4F8-B-5, CL4F9-B-6, and CL4F10-B-7 showed gene transfer capabilities that were equal to or higher than CL4F6.

[0281] In addition, the results of lipid nanoparticles containing a pH-sensitive cationic lipid (a pH-sensitive cationic lipid branched at the ε position) in which c in the general formula (A) is 4 among the pH-sensitive cationic lipids of the present invention are shown in Table 6.

[0282] Table 6

[0283] Regardless of which pH-sensitive cationic lipid is used, lipid nanoparticles encapsulating nucleic acids can be prepared. Even for pH-sensitive cationic lipids branched at the ε position, it was observed that the encapsulation rate (%) increased, the particle size decreased, and the pKa decreased as the number of carbon atoms increased.

[0284] Next, the prepared lipid nanoparticles loaded with Fluc mRNA were administered to Balb / c mice, and the Fluc activity (RLU / mg protein) was measured. Figure 2 As shown. Regardless of which lipid nanoparticles were administered to mice, Fluc activity was confirmed in the liver and spleen. From this result, it can be confirmed that all pH-sensitive cationic lipids used this time are suitable as constituent lipids of lipid nanoparticles that can be used as gene carriers. In particular, lipid nanoparticles containing all pH-sensitive cationic lipids also showed higher gene introduction activity in the spleen compared to lipid nanoparticles containing CL4F6. These results indicate that among the pH-sensitive cationic lipids of the present invention, lipid nanoparticles composed of pH-sensitive cationic lipids branched at the ε position can be used as gene carriers targeting the spleen.

[0285] Example 2 For lipid nanoparticles containing CL4F11-e-3 as the pH-sensitive cationic lipid, the effects of the type and content ratio of neutral lipids were studied.

[0286] Specifically, CL4F11-e-3, DSPC or DOPE, cholesterol and PEG-DMG with a molar ratio of 50 / 10 / 40 / 0.75 (mol%) were used to prepare lipid nanoparticles loaded with Fluc mRNA by alcohol dilution method. The pH-sensitive cationic lipid species used in the preparation, the encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI and ζ potential (mV) of the prepared lipid nanoparticles were measured. The results are shown in Table 7.

[0287] Table 7

[0288] The results showed that lipid nanoparticles containing nucleic acids could be prepared regardless of the content ratio of neutral lipids in either lipid nanoparticles containing DSPC or lipid nanoparticles containing DOPE. Lipid nanoparticles containing DOPE showed a more uniform and higher encapsulation rate.

[0289] Next, the prepared lipid nanoparticles loaded with Fluc mRNA were administered to Balb / c mice, and the Fluc activity (RLU / mg protein) was measured. Figure 3 As shown. Regardless of which lipid nanoparticle was administered to mice, high Fluc activity was confirmed in the liver and spleen, confirming that it can be used as a gene delivery vehicle targeting the liver or spleen. In particular, lipid nanoparticles containing DOPE showed stable activity over a wide range of DOPE content.

[0290] Example 3 Among the lipid nanoparticles loaded with Fluc mRNA prepared in Example 1, metabolism in the liver of lipid nanoparticles prepared by containing CL4H6, CL4F6, or CL4F10-e-2 was examined.

[0291] Specifically, BALB / c mice (female, 4 weeks old) were intravenously injected with lipid nanoparticles encapsulating Fluc mRNA (lipid nanoparticles loaded with Fluc mRNA) at a dose of 0.5 mg mRNA / kg body weight. 0.5 hours, 4 hours or 24 hours after intravenous injection, the mice were euthanized, and the amount of lipid derived from each lipid nanoparticle in the liver was measured. The measurement results are shown in FIG. Figure 4 As shown. The results show that, as for the content in the liver, CL4H6 was metabolized and almost disappeared from the liver within 24 hours after administration, while CL4F6 was almost not metabolized within 24 hours after administration and the content in the liver did not change. On the other hand, the metabolism of CL4F10-e-2 in the body was similar to that of CL4H6, and most of it was degraded within 24 hours after administration. These results show that among the pH-sensitive cationic lipids of the present invention, the pH-sensitive cationic lipids branched at the ε position have good biodegradability.

[0292] Example 4 Lipid nanoparticles encapsulating TTR-RNP-ssODN complexes (lipid nanoparticles loaded with TTR-RNP-ssODN) were prepared using CL4H6, CL4F6, CL4F8-b-5, CL4F9-b-6, CL4F10-b-7, CL4F11-e-3 or CL4F12-e-6 as pH-sensitive cationic lipids. As ssODN in lipid nanoparticles loaded with TTR-RNP-ssODN, ssODN-TTR_3-40% was used.

[0293] Specifically, pH-sensitive cationic lipids, DSPC, cholesterol and PEG-DMG in a molar ratio of 50 / 10 / 40 / 3.5 (mol%) were used to prepare lipid nanoparticles loaded with TTR-RNP-ssODN by an alcohol dilution method. The type of pH-sensitive cationic lipids used in the preparation, the encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI and ζ potential (mV) of the prepared lipid nanoparticles were measured. The measurement results are shown in Table 8.

[0294] Table 8

[0295] Next, each lipid nanoparticle loaded with TTR-RNP-ssODN was administered to mice, and the TTR-KO activity was measured. The results of the measurement of TTR activity (TTR protein content) (μg / mL) in the mouse serum 7 days after administration of each lipid nanoparticle are as follows: Figure 5 As shown. In the figure, "NT" indicates the results of mice not given lipid nanoparticles. The results are as follows: compared with TTR-RNP-ssODN-loaded lipid nanoparticles containing CL4F6 in which the hydrocarbon chain of the side chain is branched at the α position, TTR-RNP-ssODN-loaded lipid nanoparticles containing CL4F8-b-5, CL4F9-b-6, CL4F10-b-7, CL4F11-e-3 or CL4F12-e-6 have lower serum TTR activity and induce higher gene knockout. In particular, when the number of carbon atoms of the pH-sensitive cationic lipid is small, the knockout activity is higher, and in particular, more than 90% knockout was achieved with CL4F11-e-3.

[0296] Similarly, CL4F11-e-3 or CL4F12-e-4 was used as a pH-sensitive cationic lipid to prepare lipid nanoparticles loaded with TTR-RNP-ssODN, and the encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI and ζ potential (mV) of the prepared lipid nanoparticles were measured. The results are shown in Table 9.

[0297] Table 9

[0298] Each lipid nanoparticle loaded with TTR-RNP-ssODN was administered to mice, and the TTR activity (μg / mL) in the mouse serum was measured 7 days after administration. Figure 6 As shown. Although the lipid nanoparticles containing CL4F12-e-4 also showed excellent knockout activity, it is clear that the lipid nanoparticles containing CL4F11-e-3 showed the highest knockout activity. These results show that in the delivery of lipid nanoparticles, from the perspective of delivery efficiency, as a pH-sensitive cationic lipid, in the general formula (A), R 11 and R 12 The number of carbon atoms in the group with the smaller number of carbon atoms is Nc S Preferably, c is 3-6, more preferably 3-5, and preferably c is 4 or more. And it is also shown that: in the general formula (A), R 11 , R 12 The sum of and c is preferably about 9-16, and optimally about 11-14.

[0299] Example 5 The effect of the matching ratio between the ssODN and sgRNA used in the study on the knockout activity of lipid nanoparticles loaded with TTR-RNP-ssODN. Specifically, TTR-RNP-ssODN complexes were prepared using sgTTR-G211 or sgTTR-G269 as gRNA and the 11 species described in Table 2 as ssODN. Lipid nanoparticles were prepared by an alcohol dilution method using CL4H6, DSPC, cholesterol, and PEG-DMG in a molar ratio of 50 / 10 / 40 / 3.5 (mol%).

[0300] Next, each lipid nanoparticle loaded with TTR-RNP-ssODN was administered to mice, and the TTR-KO activity was measured to determine the TTR-KO activity. The obtained TTR-KO activity, as well as the GC content (%) and Tm value of the ssODN used are shown in Table 10 (n = 2). In addition, the GC content (%) of ssODN is the GC content (%) of the bases in the ssODN that match the sgRNA.

[0301] Table 10

[0302] As shown in Table 10, among the sgRNAs matching sgTTR-G211, the ssODN-TTR_3-40% with a Tm value of 26°C had the highest TTR-KO activity, and among the sgRNAs matching sgTTR-G269, the ssODN-TTR_4-40% with a Tm value of 24°C had the highest TTR-KO activity. These results indicate that TTR-KO activity is affected by the Tm value of ssODN and can be improved by using ssODN with a Tm value in the range of 20-40°C.

[0303] Example 6 Lipid nanoparticles loaded with TTR-RNP-ssODN were prepared using sgTTR-G211 and ssODN-TTR_3-0%, ssODN-TTR_3-40%, or ssODN-TTR_3-80%. As a control, lipid nanoparticles encapsulating TTR-RNP (TTR-RNP-loaded lipid nanoparticles) were prepared without using ssODN. The lipid nanoparticles were prepared with CL4H6, DSPC, cholesterol, and PEG-DMG in a composition of a molar ratio of 50 / 10 / 40 / 3.5 (mol%).

[0304] The encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI and ζ potential (mV) of the produced lipid nanoparticles were measured. The measurement results are shown in Table 11.

[0305] Table 11

[0306] For each TTR-RNP produced, the in vitro DNA cleavage activity (%) was measured (n=3). Statistical analysis was performed using non-repeated analysis of variance followed by SNK test. The measurement results (%) (mean ± SD) are shown in Figure 7 As shown. In the figure, "**" indicates p < 0.01. In the figure, "ssODN (-)" indicates the result of TTR-RNP produced without using ssODN, and "0%" indicates the result of TTR-RNP-ssODN containing ssODN-TTR_3-0%. "40%" and "80%" indicate the results of TTR-RNP-ssODN containing ssODN-TTR_3-40% or ssODN-TTR_3-80%, respectively.

[0307] like Figure 7 As shown in the figures, TTR-RNP-ssODN containing ssODN has lower DNA cleavage activity in vitro compared with TTR-RNP without ssODN (ssODN(-)). TTR-RNP-ssODN containing ssODN-TTR_3-80% has very low DNA cleavage activity, but TTR-RNP-ssODN containing ssODN-TTR_3-40% has DNA cleavage activity comparable to that of TTR-RNP-ssODN containing ssODN-TTR_3-0%. These results indicate that DNA cleavage activity can be increased by controlling the Tm value of ssODN.

[0308] Each of the manufactured lipid nanoparticles (lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-0%, lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-40%, and lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-80%) were administered to mice, and the in vivo distribution was evaluated. As a control, TTR-RNP without ssODN was administered to mice in the same manner without being encapsulated in lipid nanoparticles (free RNP), and the in vivo distribution was evaluated. The evaluation results are shown in Table 12. When lipid nanoparticles encapsulating TTR-RNP-ssODN containing ssODN were administered, the amount transferred to the liver and spleen was higher than when TTR-RNP without ssODN (free RNP) was directly administered (in the table, "ssODN (-) (free)"). It has been determined that the presence or absence of ssODN incorporated into lipid nanoparticles or in RNPs affects metastasis to the liver or spleen.

[0309] Table 12

[0310] T7E1 test was performed on lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-40%. Specifically, 2 mg RNP / kg body weight was intravenously injected for 2 consecutive days. One week after the last injection, the mice were euthanized and blood and liver were collected.

[0311] Genomic DNA was recovered from the recovered liver and subjected to T7E1 assay (n=3). As a control, the liver recovered from mice that had not been given lipid nanoparticles was similarly subjected to T7E1 assay. The results showed that administration of lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-40% achieved 27% KO activity at the DNA level.

[0312] Serum was prepared from the recovered blood, and the TTR protein concentration in the serum was measured to determine the activity of TTR-KO. As a control, the TTR protein concentration in the serum of mice not given lipid nanoparticles was also measured. The results showed that the TTR protein concentration in the serum of mice given lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-40% was reduced by 81% compared with untreated mice, confirming the high activity of TTR-KO.

[0313] In addition, the effect of the difference in mismatch sites in the ssON pairing region in sgRNA on gene knockout activity was studied. Specifically, sgTTR-G211 and ssODN-TTR_3-10℃-3', ssODN-TTR_3-10℃-5', ssODN-TTR_3-24℃-3', ssODN-TTR_3-24℃-5', ssODN-TTR_3-50℃-3' or ssODN-TTR_3-50℃-5' were used to prepare lipid nanoparticles loaded with TTR-RNP-ssODN in the same manner as above (CL4H6 / DSPC / cholesterol / PEG-DMG=50 / 10 / 40 / 3.5 (mol%)). For the manufactured lipid nanoparticles, the encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI and ζ potential (mV) were determined. The results are shown in Table 13.

[0314] Table 13

[0315] Each lipid nanoparticle loaded with TTR-RNP-ssODN was administered to mice, and the TTR protein content (μg / mL) in the mouse serum was measured 7 days after administration. Figure 8 As shown. In the figure, "NT" represents the results of mice not given lipid nanoparticles. And, "10℃-3'", "10℃-5'", "24℃-3'", "24℃-5'", "50℃-3'" and "50℃-5'" represent the results of mice given lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-10℃-3', ssODN-TTR_3-10℃-5', ssODN-TTR_3-24℃-3', ssODN-TTR_3-24℃-5', ssODN-TTR_3-50℃-3' or ssODN-TTR_3-50℃-5', respectively. The serum TTR of lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-10℃-3' and lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-10℃-5' were comparable. Similarly, the serum TTR of lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-24℃-3' and lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-24℃-5', lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-50℃-3' and lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-50℃-5' were also comparable. These results indicate that differences in mismatch sites have little effect on gene knockout activity and that the Tm value is very important.

[0316] Example 7 Lipid nanoparticles were prepared using CL4F6 synthesized in Synthesis Example 1, CL4F9-b-6 synthesized in Synthesis Example 5, CL4F14-b synthesized in Synthesis Example 9, CL4F12-e-4 synthesized in Synthesis Example 13, CL4F12-e-6 synthesized in Synthesis Example 14, or MC3 as constituent lipids. As neutral lipids, DSPC, cholesterol (chol), and PEG-DMG were used.

[0317] Specifically, pH-sensitive cationic lipids, DSPC, cholesterol and PEG-DMG in a molar ratio of 50 / 10 / 40 / 1.5 (mol%) were used to prepare lipid nanoparticles loaded with Fluc mRNA (lipid nanoparticles loaded with Fluc mRNA) by an alcohol dilution method. However, the composition of lipid nanoparticles containing MC3 as a pH-sensitive cationic lipid is MC3, DSPC, cholesterol and PEG-DMG in a molar ratio of 50 / 10 / 38.5 / 1.5 (mol%).

[0318] Next, lipid nanoparticles containing CL4F6, CL4F9-b-6, CL4F14-b, or CL4F12-e-4 were administered to Balb / c mice, and the Fluc activity (RLU / mg protein) was measured (n=2). Fig. 9 As shown. Regardless of which lipid nanoparticles were administered to mice, Fluc activity was confirmed in the liver, spleen, lung, brain, heart, kidney, and muscle. In particular, the Fluc activity in the liver and spleen was very high. These results indicate that all pH-sensitive cationic lipids used this time are suitable as constituent lipids of lipid nanoparticles that can be used as gene carriers for various tissues, and are particularly useful as constituent lipids for gene carriers for the liver and spleen.

[0319] In addition, for mice given lipid nanoparticles loaded with Fluc mRNA containing CL4F12-e-4, blood was collected 24 hours after administration to examine each component. As a control, mice given PBS instead of lipid nanoparticles loaded with Fluc mRNA were also subjected to similar blood tests. The results are shown in Table 14 (n = 3, unpaired t test). In the table, TP represents total protein; AST represents aspartate aminotransferase; ALB represents albumin; LDH represents lactose dehydrogenase; BUN represents blood urea nitrogen; γ-GT represents γ-glutamyl transpeptidase; CRE represents creatinine; T-CHO represents total cholesterol; IP represents inorganic phosphate; TG represents triglycerides; HDL-C represents high-density lipoprotein cholesterol; T-BIL represents total bilirubin; GLU represents glucose. In addition, ** represents P < 0.01; * represents P < 0.05. The results showed that no particular toxicity was observed in mice administered with lipid nanoparticles containing CL4F12-e-4 and loaded with Fluc mRNA.

[0320] Table 14

[0321] In addition, among the prepared lipid nanoparticles loaded with Fluc mRNA, for the lipid nanoparticles containing CL4F12-e-6 or MC3, the encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI, ζ potential (mV) and pKa of the prepared lipid nanoparticles were measured. The results are shown in Table 15.

[0322] Table 15

[0323] Lipid nanoparticles loaded with Fluc mRNA containing CL4F12-e-6 or MC3 were administered to Balb / c mice, and the Fluc activity (RLU / mg protein) was measured (n=2). Fig.10 As shown. In the liver, spleen, lung and kidney, the lipid nanoparticles containing CL4F12-e-6 were confirmed to have higher Fluc activity than the lipid nanoparticles containing MC3. These results show that the lipid nanoparticles such as CL4F12-e-6 according to the present invention, which use pH-sensitive cationic lipids as constituent lipids, are good gene carriers, and have higher gene expression activity than the lipid nanoparticles containing MC3 currently used in clinical practice.

[0324] The lipid nanoparticles loaded with Fluc mRNA containing CL4F12-e-6 were further similarly administered to mice subjected to freeze-thaw treatment and untreated mice to examine the gene expression activity in various tissues.

[0325] Specifically, lipid nanoparticles loaded with Fluc mRNA were prepared as follows. First, a 1 mg / mL mRNA solution was diluted to 73.3 ng / μL with a citric acid buffer (25 mM, pH 4.0) and used as an mRNA solution. Dissolved in ethanol, the total lipid concentration was 8 mM, and the molar ratio of each lipid was CL4F12-e-6 / DSPC / cholesterol / PEG-DMG = 50 / 10 / 40 / 1.5 (mol%), and a lipid solution (N / P = 6) was prepared. A 10 mL container syringe (manufactured by HAMILTON) was connected to a microfluidic channel with a built-in baffle mixer, and flow was performed at a total flow rate of 2.0 mL / min and a flow rate ratio of mRNA solution: lipid solution = 3:1 (1.5 mL / min: 0.5 mL / min). The solution flowing out of the flow channel was recovered, and an appropriate amount was transferred to a dialysis membrane ("Spectra / Por4 diagnostic membrane", MWCO: 12000-15000), dialyzed twice at 4°C for 2 hours in Tris buffer containing sucrose (20 mM Tris-HCl, 9% sucrose by mass, pH 7.4), and the lipid nanoparticles loaded with Fluc mRNA were recovered.

[0326] After a portion of the obtained lipid nanoparticles loaded with Fluc mRNA was frozen at -80°C, it was dissolved at room temperature and referred to as a "frozen sample". In addition, the sample that was not subjected to freeze-thaw treatment was referred to as a "non-frozen sample". For the frozen sample and the non-frozen sample, the encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI, ζ potential (mV) and pKa of the prepared lipid nanoparticles were measured. The results are shown in Table 16. In the "Frozen" column, "-" is the result of the non-frozen sample, and "+" is the result of the frozen sample.

[0327] Table 16

[0328] Furthermore, the frozen samples and the non-frozen samples were administered to Balb / c mice, and the Fluc activity (RLU / mg protein) was measured (n=3, unpaired t-test). Fig.11 As shown. Fig.11 As shown, no significant difference was observed in gene expression activity in each tissue between the frozen samples and the non-frozen samples, confirming that lipid nanoparticles containing CL4F12-e-6 were hardly affected by freeze-thaw treatment.

[0329] Example 8 Using CL4F11-e-3 synthesized in Synthesis Example 12, CL4F11-ζ-2 synthesized in Synthesis Example 15, CL4F10-ζ-3(3) synthesized in Synthesis Example 18, CL4F12-η-2 synthesized in Synthesis Example 16, CL4F11-η-3(3) synthesized in Synthesis Example 17, CL4F12-ζ-2(2) synthesized in Synthesis Example 20, CL4F12-θ-3(3) synthesized in Synthesis Example 19, and CL4F13-η-2(2) synthesized in Synthesis Example 21 as constituent lipids, lipid nanoparticles encapsulating the TTR-RNP-ssODN complex (lipid nanoparticles loaded with TTR-RNP-ssODN) were prepared in the same manner as in Example 4. As the ssODN in the lipid nanoparticles loaded with TTR-RNP-ssODN, ssODN-TTR_3-40% was used. As neutral lipids, DSPC, cholesterol (chol), and PEG-DMG were used.

[0330] Specifically, lipid nanoparticles loaded with TTR-RNP-ssODN were prepared by alcohol dilution method using pH-sensitive cationic lipids, DSPC, cholesterol and PEG-DMG in a molar ratio of 50 / 10 / 40 / 3.5 (mol%). The encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI, ζ potential (mV) and pKa of each prepared lipid nanoparticle were measured. The results are shown in Table 17. In the table, "side chain carbon number" refers to the total number of carbon atoms constituting the side chain of the pH-sensitive cationic lipid contained (the number of carbon atoms from the end of the main chain to the branch position is subtracted from the total number of carbon atoms constituting the molecule).

[0331] Table 17

[0332] Next, each lipid nanoparticle loaded with TTR-RNP-ssODN was administered to mice, and the TTR-KO activity was measured. The results of the measurement of TTR protein content (μg / mL) in the mouse serum 7 days after administration of each lipid nanoparticle are as follows: Fig.12 As shown. In the figure, "NT" represents the results of mice that were not given lipid nanoparticles. The same applies to "NT" in the following figures. The results: lipids with fewer scaffold carbon atoms have lower serum TTR activity and show higher gene expression knockout activity. Lipid nanoparticles loaded with TTR-RNP-ssODN containing CL4F11-ζ-2 (in the figure, "CL4F11-ζ-2") have the lowest TTR protein content in serum and show the highest knockout activity. Therefore, lipid nanoparticles loaded with TTR-RNP-ssODN containing CL4F11-ζ-2 were used in subsequent experiments.

[0333] For lipid nanoparticles loaded with TTR-RNP-ssODN containing CL4F11-ζ-2, the PEG-DMG content ratio was 1.5-3.5 mol%, and lipid nanoparticles loaded with TTR-RNP-ssODN were prepared in the same manner except that the RNP weight ratio ([RNP weight] / [total lipid weight]) relative to the total weight of lipids constituting the lipid nanoparticles was 2, 4 or 5.6 mass%. The encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI, ζ potential (mV) and pKa of each prepared lipid nanoparticle loaded with TTR-RNP-ssODN were measured. The results are shown in Table 18. In the table, "RNP / lipid" means [RNP weight] / [total lipid weight]; "PEG" means the content ratio (mol%) relative to the total amount of PEG-DMG lipids.

[0334] Table 18

[0335] As shown in Table 18, when the PEG-DMG content ratio was reduced, a tendency of a reduction in the encapsulation efficiency was observed. On the other hand, although a tendency was observed that the smaller the weight ratio of [RNP weight] / [total lipid weight], the smaller the particle size of the lipid nanoparticles, no significant effect was observed.

[0336] Each lipid nanoparticle loaded with TTR-RNP-ssODN was administered to mice (0.2 mg RNP / kg body weight), and the TTR protein content (μg / mL) in the mouse serum was measured 7 days after administration. Fig.13 As shown. Fig.13 As shown, the serum TTR activity of mice administered with any TTR-RNP-ssODN-loaded lipid nanoparticles was at the same level, confirming that the PEG-DMG content ratio and the weight ratio of [RNP weight] / [total lipid weight] did not significantly affect the gene knockout activity.

[0337] For TTR-RNP-ssODN-loaded lipid nanoparticles containing CL4F11-ζ-2, lipid nanoparticles loaded with TTR-RNP-ssODN were prepared in the same manner except that the content ratio of CL4F11-ζ-2 was 40, 50 or 60 mol% and the content ratio of DSPC was 5 or 10 mol%. That is, when the content ratio of CL4F11-ζ-2 was x% (x=40, 50 or 60) and the content ratio of DSPC was y% (y=5 or 10), the lipid composition ratio of the prepared TR-RNP-ssODN-loaded lipid nanoparticles was CL4F11-ζ-2 / DSPC / cholesterol / PEG-DMG=x / y / 100-xy / 3.5 (mol%). The encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI, ζ potential (mV) and pKa of each prepared lipid nanoparticle loaded with TTR-RNP-ssODN were measured. The results are shown in Table 19. In the table, "CL (mol%)" represents the content ratio (mol%) of CL4F11-ζ-2; "PC (mol%)" represents the content ratio (mol%) of DSPC. As shown in Table 19, even if the content ratio of CL4F11-ζ-2 to DSPC is changed, the physical properties such as encapsulation efficiency (%) are not significantly affected.

[0338] Table 19

[0339] Each lipid nanoparticle loaded with TTR-RNP-ssODN was administered to mice (0.2 mg RNP / kg body weight), and the TTR protein content (μg / mL) in the mouse serum was measured 7 days after administration. Fig.14 In the figure, "CL (mol%)" and "PC (mol%)" are the same as those in Table 19. Fig.14 As shown, the serum TTR activity of mice administered with any lipid nanoparticles loaded with TTR-RNP-ssODN was at the same level, confirming that the content ratio of pH-sensitive cationic lipids and neutral lipids in lipid nanoparticles had little effect on gene knockout activity.

[0340] TTR-RNP-ssODN-loaded lipid nanoparticles containing CL4F11-ζ-2 (CL4F11-ζ-2 / DSPC / cholesterol / PEG-DMG=50 / 10 / 40 / 3.5 (mol%)) were administered to mice (0.2, 0.75 or 2.0 mg RNP / kg body weight), and the TTR protein content (μg / mL) in the mouse serum was measured 7 days after administration. The results of the measurement of TTR protein content in serum are shown in Fig.15 The results of the knockout efficiency (%) calculated from the TTR protein content in the serum are shown in (A). Fig.15 As shown in part (B) of Fig.15 As shown in part (A) of FIG, when CL4F11-ζ-2-containing TTR-RNP-ssODN-loaded lipid nanoparticles were administered at 2.0 mg RNP / kg body weight, 98% inhibition of TTR gene expression was achieved even with a single administration. Furthermore, it was confirmed that the knockout activity increased in a dose-dependent manner ( Fig.15 (B)).

[0341] Lipid nanoparticles loaded with TTR-RNP-ssODN containing CL4F11-ζ-2 (CL4F11-ζ-2 / DSPC / cholesterol / PEG-DMG=50 / 10 / 40 / 3.5 (mol%)) were administered to mice (2.0 mg RNP / kg body weight), and the TTR protein content (μg / mL) in the mouse serum was measured 1, 4 or 12 weeks after administration. As a control, Cas9 / sgGFP-RNP prepared using sgRNA (sgGFP) targeting the GFP gene instead of sgTTR and lipid nanoparticles loaded with GFP-RNP-ssODN loaded on lipid nanoparticles containing CL4F11-ζ-2 were administered to mice in the same manner, and the TTR protein content (μg / mL) in the serum was measured. The measurement results are shown in the following table. Fig.16 In the figure, "TTR" is the result of administering lipid nanoparticles loaded with TTR-RNP-ssODN to mice, and "GFP" is the result of administering lipid nanoparticles loaded with GFP-RNP-ssODN to mice.

[0342] The gene knockdown activity of TTR-RNP-ssODN-loaded lipid nanoparticles was compared between the case where CL4F11-ζ-2 was included as the constituent lipid of the lipid nanoparticle and the case where SM, ALC, or MC3 was included as the constituent lipid of the gene carrier. ALC and SM are referred to as constituent lipids of the gene carrier.

[0343] The encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI, ζ potential (mV) and pKa of each prepared TTR-RNP-ssODN-loaded lipid nanoparticle were measured. The results are shown in Table 20. As shown in Table 20, there was no significant difference in physical properties such as encapsulation efficiency (%) for any of the TTR-RNP-ssODN-loaded lipid nanoparticles.

[0344] Table 20

[0345] Each lipid nanoparticle loaded with TTR-RNP-ssODN was administered to mice (2.0 mg RNP / kg body weight), and the TTR protein content (μg / mL) in the mouse serum was measured 7 days after administration (n=3). Fig.17 As shown. Fig.17 As shown, TTR-RNP-ssODN-loaded lipid nanoparticles containing CL4F11-ζ-2 showed significantly superior gene knockdown activity than other TTR-RNP-ssODN-loaded lipid nanoparticles.

[0346] The gene expression activity of the Fluc mRNA-loaded lipid nanoparticles was compared between the case where CL4F11-ζ-2 was contained as the constituent lipid of the lipid nanoparticles and the case where SM, ALC, or MC3 was contained as the constituent lipid of the gene carrier.

[0347] The encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI, ζ potential (mV) and pKa of each prepared lipid nanoparticle loaded with Fluc mRNA were measured. The results are shown in Table 21. As shown in Table 21, there was no significant difference in the physical properties of any lipid nanoparticle loaded with Fluc mRNA, such as encapsulation efficiency (%).

[0348] Table 21

[0349] Each lipid nanoparticle loaded with Fluc mRNA was administered to Balb / c mice (0.01 mg mRNA / kg body weight), and the Fluc activity (RLU / mg protein) of the mice was measured 6 hours after administration (n=3, unpaired t-test). Fig.18As shown. Fig.18 As shown, the lipid nanoparticles loaded with Fluc mRNA containing CL4F11-ζ-2 showed comparable or higher gene expression activity than other lipid nanoparticles loaded with Fluc mRNA. In particular, the gene expression activity in the spleen was better than any lipid nanoparticles loaded with Fluc mRNA containing SM, ALC or MC3, and the lipid nanoparticles containing CL4F11-ζ-2 were suitable as gene carriers targeting the spleen.

[0350] In addition, for lipid nanoparticles loaded with TTR-RNP-ssODN containing CL4F11-ζ-2, CL4F11-e-3 or CL4F12-e-4 as pH-sensitive cationic lipids, the effects of freeze-thaw treatment on the physical properties of the lipid nanoparticles themselves and the effects on gene knockout activity when administered to mice were studied.

[0351] Specifically, lipid nanoparticles loaded with TTR-RNP-ssODN were prepared as follows. First, while stirring a 10 μM gRNA solution with a vortex mixer, a 10 μM Cas9 solution was gradually added to prepare a 5 μM RNP solution. An equal amount of 5 μM RNP solution was mixed with a 5 μM ssODN solution to obtain a 2.5 μM RNP-ssODN solution. The 2.5 μM RNP-ssODN solution was diluted to 160 nM with a MES buffer (pH 6.0) containing 50 mM NaCl and used as an RNP solution. A lipid solution was prepared by dissolving each lipid in an ethanol solution to have a desired molar ratio composition and a total lipid concentration of 8 mM. A 10 mL container syringe (manufactured by HAMILTON) was connected to a microfluidic channel with a built-in baffle mixer and flowed at a total flow rate of 2.0 mL / min and a flow rate ratio of RNP solution: lipid solution = 9:1 (1.8 mL / min: 0.2 mL / min). The solution flowing out of the flow channel was recovered, and an appropriate amount was transferred to a dialysis membrane ("Spectra / Por4 diagnostic membrane", MWCO: 12000-15000), dialyzed twice at 4°C for 2 hours in Tris buffer containing sucrose (20 mM Tris-HCl, 9% sucrose by mass, pH 7.4), and the lipid nanoparticles loaded with TTR-RNP-ssODN were recovered.

[0352] After a portion of the obtained lipid nanoparticles loaded with TTR-RNP-ssODN was frozen at -80°C, it was dissolved at room temperature and referred to as a "frozen sample". In addition, the sample that was not subjected to freeze-thaw treatment was referred to as a "non-frozen sample". For the frozen samples and the non-frozen samples, the encapsulation efficiency (%), ζ average particle size (nm), number average particle size (nm), PdI, ζ potential (mV) and pKa of the prepared lipid nanoparticles were measured. The results are shown in Table 22. In the table, in the "Frozen" column, "-" is the result of the non-frozen sample, and "+" is the result of the frozen sample. For any of the lipid nanoparticles loaded with TTR-RNP-ssODN, there was no significant difference in the physical properties between the frozen sample and the non-frozen sample.

[0353] Table 22

[0354] Furthermore, the frozen samples and the non-frozen samples were administered to Balb / c mice, and the TTR protein content (μg / mL) in the mouse serum was measured 7 days after administration (n=3). Fig.19 As shown in the figure. In the figure, "CL" indicates the pH-sensitive cationic lipid contained in the administered lipid nanoparticles loaded with TTR-RNP-ssODN. And, in the "Frozen" column in the figure, "None" indicates a non-frozen sample, and "Yes" indicates a frozen sample. Fig.19 As shown, no significant difference was observed in the gene knockdown activity between the frozen samples and the non-frozen samples, confirming that lipid nanoparticles containing CL4F11-ζ-2 and the like were hardly affected by freeze-thaw treatment.

[0355] The tissue distribution of lipid nanoparticles containing CL4F11-ζ-2 when administered to mice was studied in the case where TTR-RNP-ssODN was loaded and the case where sgTTR was directly loaded as siRNA without forming RNP.

[0356] As the TTR-RNP-ssODN-loaded lipid nanoparticles containing CL4F11-ζ-2, DIR-labeled (DIR-labeled TTR-RNP-ssODN-loaded lipid nanoparticles) were used.

[0357] Lipid nanoparticles loaded with sITTR loaded with sgTTR as siRNA were prepared as follows. First, a 1 mg / mL siRNA solution was diluted to 48.9 ng / μL with a 25 mM acetate buffer (pH 4.0) and used as a siRNA solution. A lipid solution (N / P=9) was prepared by dissolving each lipid in an ethanol solution to have a desired composition (mol%) and a total lipid concentration of 8 mM. In order to fluorescently label the lipid nanoparticles, a DIR solution was added to the lipid solution at 0.5 mol%. A 10 mL container syringe (manufactured by HAMILTON) was connected to a microfluidic channel with a built-in baffle mixer, and flow was performed at a total flow rate of 0.5 mL / min and a flow rate ratio of siRNA solution: lipid solution = 3:1 (0.375 mL / min: 0.125 mL / min). The solution flowing out of the flow channel was recovered, and an appropriate amount was transferred to a dialysis membrane ("Spectra / Por4 diagnostic membrane", MWCO: 12000-15000), dialyzed twice in PBS (-) at 4°C for 2 hours, and the DIR-labeled lipid nanoparticles loaded with sITTR were recovered.

[0358] BALB / c mice (female, 4 weeks old) were administered DIR-labeled lipid nanoparticles loaded with TTR-RNP-ssODN or DIR-labeled lipid nanoparticles loaded with sITTR diluted to have the same DIR fluorescence intensity as 0.75 mg RNP / kg body weight via the tail vein. One hour after administration, blood and organs were recovered, and the DIR fluorescence intensity was measured using the imaging system IVIS (in vivo imaging system, manufactured by PerkinElmer). The measurement results are shown in Figure 2. Fig. 20 In the figure, "RNP-LNP" is the result of mice administered with DIR-labeled lipid nanoparticles loaded with TTR-RNP-ssODN, and "siRNA-LNP" is the result of mice administered with DIR-labeled lipid nanoparticles loaded with sITTR.

[0359] like Fig. 20 As shown, when administered in vivo, DIR-labeled lipid nanoparticles loaded with TTR-RNP-ssODN and DIR-labeled lipid nanoparticles loaded with sITTR had approximately the same tissue distribution, but in detail, the results showed that DIR-labeled lipid nanoparticles loaded with TTR-RNP-ssODN had higher spleen selectivity than DIR-labeled lipid nanoparticles loaded with sITTR.

[0360] Example 9 In the lipid nanoparticles loaded with TTR-RNP-ssODN, the effect of the Tm value of the ssODN used was studied.

[0361] First, sgTTR-G211-5FAM was used as sgRNA, and ssODN-80%-3IBFQ, ssODN-40%-3IBFQ, or ssODN-0%-3IBFQ was used as ssODN, and the Tm values ​​of sgRNA and ssODN were measured by the fluorescence quenching method (n=3). Fig.21 Part (A) shows the plot of fluorescence intensity at various temperatures from 11 to 70°C. Fig.21 Part (B) shows the change in fluorescence intensity at each temperature. In the figure, "80%", "40%" and "0%" are the results of measurement using ssODN-80%-3IBFQ, ssODN-40%-3IBFQ and ssODN-0%-3IBFQ, respectively.

[0362] Since ssODN-0%-3IBFQ is always separated from sgTTR-G211-5FAM, in the reaction solution containing ssODN-0%-3IBFQ, Fig.21 As shown in part (A), the fluorescence intensity is high at any temperature and hardly changes with temperature. In contrast, in the reaction solution containing ssODN-80%-3IBFQ and the reaction solution containing ssODN-40%-3IBFQ, the fluorescence intensity increases as the temperature rises and approaches the fluorescence intensity of the reaction solution containing ssODN-0%-3IBFQ. In other words, it was confirmed that the dissociation of the RNP-ssODN complex occurs in a temperature-dependent manner.

[0363] Since the Tm value is the temperature at which the fluorescence intensity changes the most, the Tm value for ssODN-80%-3IBFQ is 62°C; the Tm value for ssODN-40%-3IBFQ is 32°C ( Fig.21 (B)). At around 40°C, almost all ssODN-40%-3IBFQ dissociated from RNP, but almost all ssODN-80%-3IBFQ formed a complex with RNP. These results indicate that ssODN-40%-3IBFQ dissociated rapidly from RNP in cells (37°C).

[0364] Next, the DNA cleavage activity of the RNP-ssODN complex was measured in vitro when ssODN with a matching rate of 0, 40, or 80% in the ssON pairing region in the sgRNA was used. sgTTR-G211 was used as the sgRNA. ssODN-TTR_3-80%, ssODN-TTR_3-40%, or ssODN-TTR_3-0% was used. The Tm values ​​of sgTTR-G211 and ssODN-TTR_3-80% were 50°C, and the Tm values ​​of sgTTR-G211 and ssODN-TTR_3-40% were 26°C.

[0365] In vitro DNA cleavage activity was measured at a reaction temperature of 37°C or 15°C (n=3). As a control, the DNA cleavage activity of RNP without ssODN in the reaction system was also measured in the same manner. The DNA cleavage activity was determined as a relative activity value, with the activity without ssODN (ssODN(-)) being 100%. The results of the measurement at 37°C are shown in Table 1. Fig. 22 As shown in part (A), the results of the measurement at 15°C are as follows Fig. 22 In the figure, "80%", "40%" and "0%" are the results of measurement using ssODN-TTR_3-80%, ssODN-TTR_3-40% and ssODN-TTR_3-0%, respectively, and "ssODN(-)" is the result of measurement without ssODN.

[0366] At 37°C, the relative DNA cleavage activity of RNP containing ssODN-TTR_3-40% was comparable to that of RNP containing ssODN-TTR 3-0%. The relative DNA cleavage activity of RNP containing ssODN-TTR 3-80% was only about 20%, which was significantly lower. On the other hand, at 15°C, the relative DNA cleavage activity of RNP containing ssODN-TTR_3-40% was higher than that of RNP containing ssODN-TTR_3-80%, but significantly lower than that of RNP containing ssODN-TTR_3-0%. The DNA cleavage activity when using ssODN-TTR_3-40% at 37°C and 15°C is significantly different in that the Tm value at 37°C (26°C) is higher than the Tm value of ssODN-TTR_3-40%. Therefore, most of ssODN-TTR_3-40% is dissociated from RNP, in order to exert the original DNA cleavage activity of RNP. At 15°C, which is lower than the Tm value, most of ssODN-TTR_3-40% forms a complex with RNP, which is presumably due to the reduction in DNA cleavage activity.

[0367] These results indicate that by designing ssODN so that the Tm values ​​of sgRNA and ssODN are lower than the animal body temperature (about 37°C), the gene knockout activity in animals can be further enhanced.

[0368] In addition, the effect of temperature on intracellular gene knockout activity during the preparation of lipid nanoparticles loaded with TTR-RNP-ssODN was studied. Specifically, first, lipid nanoparticles loaded with TTR-RNP-ssODN having a lipid composition of CL4H6 / DSPC / cholesterol / PEG-DMG = 50 / 10 / 40 / 3.5 (mol%) were obtained in the same manner as Example 8, except that sgTTR-G211 was used as sgRNA, ssODN-TTR_3-30% or ssODN-TTR_3-40% was used as ssODN, and the temperature when mixing the RNP solution and the lipid solution in the microchannel with a built-in baffle mixer was 4°C.

[0369] The obtained lipid nanoparticles loaded with TTR-RNP-ssODN were administered to mice (2.0 mg RNP / kg body weight), and the TTR protein content (μg / mL) in the mouse serum was measured 7 days after administration. Fig.23 As shown in the figure, "30%" and "40%" are the results of administering lipid nanoparticles loaded with TTR-RNP-ssODN containing ssODN-TTR_3-30% and ssODN-TTR_3-40% to mice, respectively. Fig.23 As shown, lipid nanoparticles loaded with TTR-RNP-ssODN using ssODN-TTR_3-30% and lipid nanoparticles loaded with TTR-RNP-ssODN using ssODN-TTR_3-40% both have low serum TTR activity and fully exert gene knockout activity. In particular, when prepared in an environment without temperature control, the relative gene knockout activity is not very high (Table 10), while the gene knockout activity is high when prepared at 4°C, because the manufacturing temperature is below the Tm value, it is speculated that this is ssODN and sgRNA are effectively loaded on lipid nanoparticles in the state of forming a complex. These results show that: by designing ssODN so that the Tm value of sgRNA and ssODN is lower than the animal body temperature (about 37°C), and further by setting the manufacturing temperature when loaded on lipid nanoparticles to be sufficiently lower than the Tm value, RNP-ssODN-loaded lipid nanoparticles with sufficiently high gene knockout activity can be effectively obtained.

Claims

1. A lipid nanoparticle, characterized in that: It comprises a pH-sensitive cationic lipid represented by the following formula (I): Chemical formula 1 (R 1 )(R 2 )C(OH)-(CH2)a-(O-CO)bX (I) In formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R 1 and R 2 Each independently represents a group represented by the following general formula (A), Chemical formula 2 (R 11 )(R 12 )HC-(CH2)c-(CO-O)-(CH2)e- (A) In formula (A), R 11 and R 12 Each independently represents a linear or branched C 1-15 alkyl; c represents an integer from 1 to 7; e represents an integer from 4 to 12; but R 11 and R 12 can be connected to each other to form a ring; X represents a group represented by the following general formula (B) or a 5-7 membered non-aromatic heterocyclic group, but the group is combined with (O-CO)b- through a carbon atom, and one or two hydrogen atoms of the ring can be replaced by C 1-4 Alkyl or C 2-4 Alkenyl substitution, Chemical formula 3 -(CH2)d-N(R 3 )(R 4 ) (B) In formula (B), d represents an integer of 0-3; R 3 and R 4 Each independently represents C 1-4 Alkyl or C 2-4 alkenyl, wherein the C 1-4 Alkyl or C 2-4 In alkenyl, one or two hydrogen atoms can be replaced by phenyl, but R 3 and R 4 can combine with each other to form a 5-7 membered non-aromatic heterocyclic ring, wherein one or two hydrogen atoms of the ring can be replaced by C 1-4 Alkyl or C 2-4 Alkenyl substitution.

2. The lipid nanoparticle according to claim 1, characterized in that The c is an integer of 4-7.

3. The lipid nanoparticle according to claim 1 or 2, characterized in that The R 11 and the R 12 The sum of the carbon atoms of the group with more carbon atoms and c is 5-17.

4. The lipid nanoparticle according to claim 1 or 2, characterized in that It also includes sterol and polyalkylene glycol modified lipids.

5. The lipid nanoparticle according to claim 1 or 2, characterized in that It contains nucleic acid.

6. The lipid nanoparticle according to claim 5, characterized in that The nucleic acid is siRNA, mRNA or plasmid DNA.

7. The lipid nanoparticle according to claim 1 or 2, characterized in that It comprises a DNA nuclease, a guide RNA, and a single-stranded oligonucleotide containing a region that can pair with a portion of the guide RNA.

8. The lipid nanoparticle according to claim 7, characterized in that In the region where the single-stranded oligonucleotide can pair with a portion of the guide RNA, 30-60% of the bases in the region are complementary to the bases in the guide RNA.

9. The lipid nanoparticle according to claim 7, characterized in that The DNA nuclease is Cas9 protein, The guide RNA consists of crRNA and tracrRNA.

10. A lipid nanoparticle, characterized in that: The method comprises a lipid component, a DNA nuclease, a guide RNA, and a single-stranded oligonucleotide containing a region that can pair with a portion of the guide RNA. In the region where the single-stranded oligonucleotide can pair with a portion of the guide RNA, 30-60% of the bases in the region are complementary to the bases in the guide RNA.

11. A pharmaceutical composition, characterized in that The lipid nanoparticles according to claim 1, 2 or 10 are used as an active ingredient.

12. The pharmaceutical composition according to claim 11, characterized in that It is used in gene therapy.

13. A method for expressing an exogenous gene, characterized in that: A lipid nanoparticle as described in claim 1, 2 or 10, which encapsulates a target exogenous gene expressed in hepatocytes or spleen cells, is administered to a test animal, but the test animal does not include humans, and the exogenous gene is expressed in the liver or spleen of the test animal.

14. A genome editing method, characterized in that: The method comprises introducing the lipid nanoparticles according to claim 10 into cells.

15. A pH-sensitive cationic lipid, characterized in that It is represented by the following formula (I): Chemical formula 4 (R 1 )(R 2 )C(OH)-(CH2)a-(O-CO)bX (I) In formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R 1 and R 2 Each independently represents a group represented by the following general formula (A); Chemical formula 5 (R 11 )(R 12 )HC-(CH2)c-(CO-O)-(CH2)e- (A) In formula (A), R 11 and R 12 Each independently represents a linear or branched C 1-15 alkyl; c represents an integer of 1-7; e represents an integer of 4-12; but R 11 and R 12 can be connected to each other to form a ring; X represents a group represented by the following general formula (B) or a 5-7 membered non-aromatic heterocyclic group, but the group is combined with (O-CO)b- through a carbon atom, and one or two hydrogen atoms of the ring can be replaced by C 1-4 Alkyl or C 2-4 Alkenyl substitution; Chemical formula 6 -(CH2)d-N(R 3 )(R 4 ) (B) In formula (B), d represents an integer of 0-3; R 3 and R 4 Each independently represents C 1-4 Alkyl or C 2-4 alkenyl, wherein the C 1-4 Alkyl or C 2-4 In alkenyl, one or two hydrogen atoms are replaced by phenyl, but R 3 and R 4 can combine with each other to form a 5-7 membered non-aromatic heterocyclic ring, wherein one or two hydrogen atoms of the ring can be replaced by C 1-4 Alkyl or C 2-4 Alkenyl substitution.

16. The pH-sensitive cationic lipid according to claim 15, characterized in that It is: 7-(4-(dipropylamino)butyl)-7-hydroxy-13-((3-propylhexanoyl)oxy)tridecyl 3-ethylheptanoic acid, 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-butylheptanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-pentyloctanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-hexylnonanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-heptyldecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(3-octyl undecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(2-cycloheptyl acetate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(2-cyclododecyl acetate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(2-cyclopentadecanyl acetate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(6-ethyldecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(6-propyl undecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(6-butyldodecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(6-hexyldodecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(7-ethylundecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(8-ethyldodecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(8,10,10-trimethylundecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(7,9,9-trimethyldecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(9,11,11-trimethyldodecanoate), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(7,11-dimethyldodecanoate) or 7-(4-(Dipropylamino)butyl)-7-hydroxytridecane-1,13-diyl bis(8,12-dimethyltridecanoate).

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