Lipid compositions and methods of delivering therapeutic agents

Abstract

The purpose of the present invention is to provide a lipid composition capable of delivering a nucleic acid such as RNA to a hematopoietic stem / progenitor cell or a mesenchymal stem cell, and a method for delivering a therapeutic agent to a cell using the lipid composition. The present invention provides a lipid composition comprising (A) a therapeutic agent and (B) a lipid nanoparticle conjugated to a targeting molecule wherein the lipid nanoparticle comprises an ionizable lipid and the targeting molecule specifically binds to a marker of hematopoietic stem / progenitor cells or mesenchymal stem cells.

Description

Technical Field

[0001] The present invention relates to a lipid composition comprising a therapeutic agent and lipid nanoparticles (LNPs), and a method of delivering the therapeutic agent to a cell using the lipid composition. Background Art

[0002] Hematopoietic stem / progenitor cells (HSPCs) and mesenchymal stem cells (MSCs) are key targets for gene therapy. Current gene therapy protocols involve harvesting HSPCs or MSCs from donors / patients, culturing them in vitro, transducing them with retroviral vectors, and reimplanting them into patients whose bone marrow has been conditioned. In addition to technical complexity and manufacturing costs, disadvantages of this approach include the need to culture them in the presence of multiple cytokines that may affect the multipotency and engraftment of HSPCs. In addition, the need for myeloablative surgery such as total body irradiation (TBI) or lethal chemotherapy, including busulfan / cyclophosphamide (BU / CY), in patients with non-malignant diseases also poses additional risks.

[0003] In addition, in the case of allogeneic transplantation, donor HSC or MSC must avoid immune rejection by the recipient. The recognition of HLA incompatibility by the immune system is the main obstacle to allogeneic hematopoietic stem cell transplantation. Therefore, sibling donors with the same HLA genotype are the gold standard for transplantation purposes, but only 30% of patients have such donors. For the remaining 70% of patients, alternative sources of stem cells are matched unrelated adult unpaid donors, half-matched donors, or umbilical cord blood units.

[0004] On the other hand, it is known that CD117 is expressed in hematopoietic stem cells. Non-patent document 1 describes the effectiveness of anti-CD117 antibody modified nanoparticles and the combination of hematopoietic stem / progenitor cell recruitment and anti-antibody modified nanoparticles. Patent document 1 describes charged lipids / polymers and anti-CD117 antibodies. Patent document 2 describes liposomes coated with anti-CD117 antibodies. However, non-patent document 1 and patent documents 1 and 2 do not describe the combination of ionizable lipids and anti-CD117 antibodies.

[0005] Reference List

[0006] Patent Literature

[0007] Patent Document 1: WO2019 / 213308

[0008] Patent Document 2: WO2015 / 153805

[0009] Non-patent literature

[0010] Non-patent document 1: Paula Cannon et al., HUMAN GENE THERAPY, Vol. 32, Nos. 1 and 2 (pp. 31-43) DOI: 10.1089 / hum.2020.263 Summary of the invention

[0011] The purpose to be solved by the present invention

[0012] The present invention addresses this need by providing lipid compositions capable of delivering nucleic acids (such as RNA) to hematopoietic stem / progenitor cells and methods of using the lipid compositions to deliver therapeutic agents to cells.

[0013] Methods to solve this problem

[0014] As a result of extensive research to solve the above-mentioned purpose, the inventors have found that a therapeutic agent can be effectively delivered to hematopoietic stem / progenitor cells by administering lipid nanoparticles, the lipid nanoparticles being bound to a targeting molecule that specifically binds to a marker of hematopoietic stem / progenitor cells, and the therapeutic agent being encapsulated in the lipid nanoparticles. The present invention is accomplished based on the above-mentioned findings.

[0015] According to the present invention, the following inventions are provided.

[0016] <1> A lipid composition comprising (A) a therapeutic agent and (B) a lipid nanoparticle conjugated to a targeting molecule,

[0017] wherein the lipid nanoparticles comprise ionizable lipids, and

[0018] The targeting molecule specifically binds to a marker of hematopoietic stem / progenitor cells or mesenchymal stem cells.

[0019] <2> according to <1> The lipid composition, wherein the lipid nanoparticles comprise PEG-lipid conjugated to the targeting molecule.

[0020] <3> according to <1> The lipid composition, wherein the ionizable lipid has at least one ionizable amino group and at least one biodegradable group, and wherein the biodegradable group is represented by -O(CO)O-, -O(CO)-, -(CO)O- or SS.

[0021] <4> according to <1> The lipid composition, wherein the ionizable lipid is a compound represented by formula (4):

[0022] [Chemical formula 1]

[0023]

[0024] Where X represents NR 1 -or-O-,

[0025] R 1 represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a 21 -L 1 -R 22 - represents a group, R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 Represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or

[0026] [Chemical formula 2]

[0027]

[0028] R 22 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms,

[0029] R 2 and R 3 Each independently represents a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a 31 -L 2 -R 32 - represents a group, R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 Represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or

[0030] [Chemical formula 3]

[0031]

[0032] R 32 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms,

[0033] R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms,

[0034] R 4 With R 5 , R 10 With R 5 , R 5 With R 12 , R 4 With R6 , R 5 With R 6 , R 6 With R 7 , R 6 With R 10 , R 12 With R 7 , and R 7 With R 8 Any one or more groups in may be linked together to form a 4- to 7-membered ring which may contain an O atom,

[0035] The substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms represents a hydroxyl group, a carboxyl group, a NR 45 R 46 An amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 , or -OR 44 A group represented by 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0036] The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group represent alkyl groups having 1 to 18 carbon atoms, hydroxyl groups, carboxyl groups, groups consisting of -NR 45 R 46 An amino group represented by -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 , or -OR 44 represents a group, and R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0037] a, b, c and d each independently represent an integer of 0 to 3, wherein a+b is 1 or more, and c+d is 1 or more.

[0038] <5> according to <1> The lipid composition, wherein the ionizable lipid is a compound represented by formula (1):

[0039] [Chemical formula 4]

[0040]

[0041] In the formula,

[0042] R 1 and R 2 Each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, wherein R 1 , R 2 and R 3 The hydrocarbon group represented by may be one or more selected from -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 AND-OR 56 Substituents are substituted,

[0043] R 4 represents a hydrocarbon group having 1 to 8 carbon atoms,

[0044] R 5 and R 6 Each independently represents a hydrocarbon group having 1 to 8 carbon atoms or -R 8 -L 1 -R 9 , but R 5 and R 6 Except for the case where all of them are hydrocarbon groups having 1 to 8 carbon atoms,

[0045] R 7 Indicates -R 10 -L 2 -R 11 -L 3 -R 12 ,

[0046] R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms,

[0047] R 53 , R 54 , R 55 and R 56 each independently represents a hydrocarbon group having 1 to 24 carbon atoms,

[0048] By R 53 , R 54 , R 55 and R 56The hydrocarbon group represented by may be replaced by an aryl group having 6 to 20 carbon atoms or -SR 58 replace,

[0049] The above aromatic groups having 6 to 20 carbon atoms may be replaced by -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 、-OR 56 , or -(hydrocarbon group having 1 to 12 carbon atoms)-R 57 replace,

[0050] R 58 represents a hydrocarbon group having 1 to 12 carbon atoms, and

[0051] R 57 Indicates -OH, COOH, -NR 61 R 62 、-OC(O)OR 63 、-C(O)OR 64 、-OC(O)-R 65 , or -OR 66 ,

[0052] R 61 and R 62 each independently represents a hydrocarbon group having 1 to 8 carbon atoms,

[0053] R 63 , R 64 , R 65 and R 66 each independently represents a hydrocarbon group having 1 to 24 carbon atoms,

[0054] By R 63 , R 64 , R 65 and R 66 The hydrocarbon group represented by may be replaced by an aryl group having 6 to 20 carbon atoms or -SR 68 replace,

[0055] The above aromatic groups having 6 to 20 carbon atoms may be replaced by -OH, COOH, -NR 61 R 62 、-OC(O)OR 63 、-C(O)OR 64 、-OC(O)-R 65 、-OR 66 , or -(hydrocarbon group having 1 to 12 carbon atoms)-R 67 replace,

[0056] R 68 represents a hydrocarbon group having 1 to 12 carbon atoms, and

[0057] L 1 , L 2 and L 3 each independently represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-,

[0058] R 8 represents a hydrocarbon group having 1 to 12 carbon atoms,

[0059] R 9 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0060] R 10 represents a hydrocarbon group having 1 to 8 carbon atoms,

[0061] R 11 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0062] R 12 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0063] By R 9 and R 12 The hydrocarbon group represented by 53 、-C(O)OR 54 、-OC(O)-R 55 , or -SR 58 Substitution, where R 53 , R 54 , R 55 and R 58 is defined above, and

[0064] By R 11 The hydrocarbon group represented by -OC(O)OR 53 、-C(O)OR 54 , or -OC(O)-R 55 Substitution, where R 53 , R 54 and R 55 The definition of is as above.

[0065] <6> according to <1> The lipid composition, wherein the ionizable lipid is a compound represented by the following formula (5):

[0066] [Chemical formula 5]

[0067]

[0068] Where R51 and R 52 Each independently represents a hydrocarbon group having 1 to 21 carbon atoms, which may have a substituent A,

[0069] The substituent A represents a hydroxyl group, or -G 20 -CH(R 55 )(R 56 )、-N(R 58 )(R 59 ) or -G 20 -R 60 The group represented by

[0070] G 20 represents -O(CO)-, or -(CO)O-,

[0071] R 55 and R 56 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms,

[0072] R 58 and R 59 Each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms, which may have a substituent B,

[0073] The substituent B is -N(R 61 )(R 62 ),

[0074] R 61 and R 62 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms,

[0075] R 60 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0076] L 10 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0077] G 30 Indication-S-(CO)-NR 64 ,

[0078] R 64 Indicated by -L 30 -G 20 -CH(R 55 )(R 56 ) represents a group,

[0079] a represents 0 or 1,

[0080] L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms,

[0081] G 10 Represents -O(CO)-, -(CO)O-, -O(CO)O-, or -N(C(O)R 63 )-,

[0082] R 63 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0083] L 20 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0084] b represents 0 or 1,

[0085] R 53 , R 54 and R 57 Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms, which may have a substituent C,

[0086] The substituent C represents -(CO)OR 65 or -O(CO)-R 65 The group represented by

[0087] R 65 represents a hydrocarbon group having 1 to 18 carbon atoms or -L 40 -CH(R 66 )(R 67 ) represents a group,

[0088] L 40 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0089] R 66 and R 67 represents a hydrocarbon group or an alkoxy group having 1 to 10 carbon atoms.

[0090] <7> according to <1> The lipid composition, wherein the ionizable lipid is at least one selected from the compounds represented by the following formula:

[0091] [Chemical formula 6]

[0092] MC3

[0093]

[0094] L-319

[0095]

[0096] ALC-0315

[0097]

[0098] SM-102

[0099]

[0100] Lipid 5

[0101]

[0102] Lipid 29

[0103]

[0104] ATX-100

[0105]

[0106] Lipid A9

[0107]

[0108] Lp01

[0109]

[0110] TCL053

[0111]

[0112] Compound 1

[0113]

[0114] Compound 2

[0115]

[0116] Compound 3

[0117]

[0118] Compound 4

[0119]

[0120] cKK-E12

[0121]

[0122] C12-200

[0123]

[0124] 306Oi10

[0125]

[0126] 93-O17S

[0127]

[0128] YSK05

[0129]

[0130] CL4H6

[0131]

[0132] ssPalmOPhe

[0133]

[0134] Compound 5

[0135]

[0136] Compound 6

[0137]

[0138] Compound 7

[0139]

[0140] Compound 8

[0141]

[0142] Compound 9

[0143]

[0144] <8> according to <1> The lipid composition, wherein the lipid nanoparticles contain sterols.

[0145] <9> according to <1> The lipid composition, wherein the lipid nanoparticles comprise phospholipids.

[0146] <10> according to <1> The lipid composition, wherein the therapeutic agent comprises a polynucleotide.

[0147] <11> according to <9> The lipid composition, wherein the polynucleotide is DNA or RNA.

[0148] <12> according to <9> The lipid composition, wherein the polynucleotide is mRNA, sgRNA or siRNA.

[0149] <13> according to <1> The lipid composition, wherein the targeting molecule is at least one selected from nucleic acids, peptides, antibodies and small molecules.

[0150] <14> according to <12> The lipid composition, wherein the targeting molecule is an antibody.

[0151] <15> according to <1> In the lipid composition, the marker of hematopoietic stem / progenitor cells or mesenchymal stem cells is CD34, CD105, CD117, or CD184 (CXCR4).

[0152] <16> according to <1> In the lipid composition, the marker of hematopoietic stem / progenitor cells or mesenchymal stem cells is CD117.

[0153] <17> according to <1> In the lipid composition, the marker of mesenchymal stem cells is CD105.

[0154] <18> A method for delivering a therapeutic agent to cells expressing a marker of hematopoietic stem / progenitor cells or mesenchymal stem cells, comprising administering to an individual a composition according to <1> The lipid composition.

[0155] <19> according to <18> The method further comprises administering to the individual a therapeutically effective amount of an inflammation reducing agent prior to administering to the individual the lipid composition according to claim 1.

[0156] <20> according to <19> The method, wherein the inflammation reducing agent is selected from (a) corticosteroids, (b) antihistamines, (c) acetaminophen, (d) NSAIDS, (e) kinase inhibitors having CD117 kinase activity inhibitory activity, or (f) other immunosuppressants.

[0157] <21> A method for reducing adverse reactions associated with the administration of anti-CD117 antibody-modified LNPs, the method comprising administering to an individual a therapeutically effective amount of an inflammation reducing agent prior to administering the CD117 antibody-modified LNPs.

[0158] <22> according to <21> The method, wherein the inflammation reducing agent is selected from (a) corticosteroids, (b) antihistamines, (c) acetaminophen, (d) NSAIDS, (e) kinase inhibitors having CD117 kinase activity inhibitory activity, or (f) other immunosuppressants.

[0159] Also provided herein are methods of delivering a therapeutic agent to hematopoietic stem / progenitor cells (HSPCs) or mesenchymal stem cells in culture.

[0160] Effects of the Invention

[0161] According to the lipid composition and method of the present invention, a therapeutic agent can be effectively delivered to hematopoietic stem / progenitor cells or mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0162] Figure 1 Shown is an in vitro evaluation of CD45 gene silencing on EML cells using LNPs conjugated to aCD117.

[0163] Figure 2 Knockdown of CD45 via the interaction of the aCD117-receptor with ionizable lipids is shown.

[0164] Figure 3 Shown are CD45 expression levels in bone marrow LSK cells quantified by flow cytometry.

[0165] Figure 4 In vivo Cre mRNA delivery in bone marrow HSPCs using the Ai14 mouse model is shown.

[0166] Figure 5 Shown are LNP uptake in HSPCs or LT-HSCs at different alkyl chain lengths (left) and the level of functional gene silencing in HSPCs (right).

[0167] Figure 6 Shown are uptake in HSPCs measured as % of LSK cells that are DiR+ (left) and functional knockdown of CD45 in HSPCs (right).

[0168] Figure 7 Shown are LNP uptake in various cell populations of the bone marrow as measured by % DiR positivity (top) and the dose response of functional siCD45 knockdown using Ab-LNP formulations (bottom).

[0169] Figure 8 Shown are (top) LNP uptake in various cell populations in the bone marrow as measured by % DiR positivity, and (bottom) the dose response of functional siCD45 knockdown using Ab-LNP formulations.

[0170] Fig. 9 Shown (left) at 1 mg kg -1 Screening of surrogate markers for in vivo HSPC delivery at doses of siCD45 (n=3 mice for anti-CD49d condition, n=2 mice for all other conditions, one-way ANOVA and Dunnett's multiple comparison test). Data are presented as mean ± SD (*P<0.05, **P<0.01, ***P<0.001), and (right) at 1 mg / kg -1Treatment with ligand-modified LNPs at doses of siCD45 did not result in any depletion or elimination of HSCs. Statistical values ​​were determined by one-way ANOVA with Dunnett's multiple comparisons with the PBS control group (n=3 mice for the anti-CD49 group and n=2 mice for the other groups). Significance was defined as P<0.05.

[0171] Fig.10 In vitro RNA delivery to human primary HSPCs using a non-antagonistic antibody (clone LMJ729) is shown.

[0172] Fig.11 Shown is luciferase expression in human primary bone marrow CD34+ cells, quantified using the SteadyGlo Luciferase Assay System after treatment with LNPs or PBS at a dose of 100 ng / 5,000 cells for 24 hours.

[0173] Fig.12 Shown are Itgb1 mRNA levels in primary murine bone marrow mesenchymal stem cells, quantified using RT-qPCR 24 hours after LNP treatment at 100 nM siRNA. Remaining Itgb1 mRNA levels were normalized to the housekeeping gene B2m.

[0174] Fig.13 Shown is luciferase expression in human primary bone marrow CD34+ cells, quantified using the SteadyGlo Luciferase Assay System after treatment with LNPs or PBS at a dose of 100 ng / 5,000 cells for 24 hours.

[0175] Fig.14 Anti-CD117 LNPs encapsulating Cre mRNA show higher levels of editing in vivo. (a) Schematic diagram of the LoxP-flanked stop box of Ai14 transgenic mice that prevents TdTomato transcription. Upon delivery of Cre recombinase via Cre mRNA, the stop box is excised and the cells express TdTomato. (b) Timeline of the experimental workflow for bone marrow and blood analysis. Figures a and b were created with BioRender.com. (c) Representative flow cytometric dot plots of TdTomato expression in LT-HSCs at different doses of Cre mRNA. (d) Quantification of dose-response in both HSPCs and LT-HSCs (for 1 mg kg -1Ab-LNP group n = 4 mice, for other groups n = 3 mice). Statistics were performed using two-way ANOVA and Tukey's multiple comparison test. (e) Time course monitoring of TdTomato expression in mature immune cells (n = 3 mice). (f) TdTomato expression in T cell subsets (n = 3 mice). (g) TdTomato expression in erythrocytes (TER-119+) (n = 3 mice). Data are expressed as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001).

[0176] Fig.15 The effect of PEG-lipid alkyl length on mRNA delivery to HSPCs in the bone marrow is shown. DETAILED DESCRIPTION

[0177] Hereinafter, the present invention will be described in detail.

[0178] In the present specification, "to" represents a range including numerical values ​​described as a minimum value and a maximum value before and after it, respectively.

[0179] The present invention is a lipid composition comprising (A) a therapeutic agent and (B) a lipid nanoparticle bound to a targeting molecule.

[0180] wherein the therapeutic agent is encapsulated in lipid nanoparticles,

[0181] The lipid nanoparticle comprises an ionizable lipid, and

[0182] The targeting molecule is a molecule that specifically binds to a marker of hematopoietic stem / progenitor cells or mesenchymal stem cells.

[0183] In one embodiment, the lipid composition of the present invention is used in combination with an inflammation reducing agent. The inflammation reducing agent is selected from (a) corticosteroids, (b) antihistamines, (c) acetaminophen, (d) NSAIDS, (e) kinase inhibitors with CD117 kinase activity inhibitory activity, or (f) other immunosuppressants.

[0184] The present invention further relates to a method for delivering a therapeutic agent to a cell expressing a marker of a hematopoietic stem / progenitor cell or a mesenchymal stem cell, the method comprising administering the lipid composition of the present invention to an individual. Preferably, the method for delivering a therapeutic agent to a cell according to the present invention may further include administering an inflammatory reducing agent of an effective amount to an individual before administering the lipid composition of the present invention to the individual.

[0185] In the present invention, lipid nanoparticles bound to targeting molecules are used.

[0186] The present invention also relates to a method for reducing adverse reactions associated with the administration of anti-CD117 antibody modified LNP, the method comprising administering to an individual a therapeutically effective amount of an inflammation reducing agent prior to administering the CD117 antibody modified LNP. Preferably, the inflammation reducing agent is selected from (a) corticosteroids, (b) antihistamines, (c) acetaminophen, (d) NSAIDS, (e) kinase inhibitors having CD117 kinase activity inhibitory activity, or (f) other immunosuppressants.

[0187] <Targeting molecules>

[0188] The targeting molecule is a molecule that specifically binds to a hematopoietic stem / progenitor cell marker or a mesenchymal stem cell. By using a molecule that specifically binds to a hematopoietic stem / progenitor cell marker or a mesenchymal stem cell marker as a targeting molecule, the therapeutic agent encapsulated in the lipid nanoparticle can be effectively delivered to the hematopoietic stem / progenitor cell or the mesenchymal stem cell.

[0189] The type of targeting molecule is not particularly limited, as long as it is a molecule specifically combined with a marker of hematopoietic stem / progenitor cell or mesenchymal stem cell, and can include a molecule combined with a cell surface, a molecule combined with an extracellular matrix, etc. The molecule combined with the cell surface can include, for example, a molecule combined with a membrane protein (such as a receptor or channel exposed to the cell surface). As a targeting molecule, it is preferred to use a molecule combined with a marker of hematopoietic stem / progenitor cell or mesenchymal stem cell. As a targeting molecule, it is preferred to use a non-antagonistic molecule. As a targeting molecule, for example, at least one selected from a carbohydrate, a nucleic acid, a peptide, a protein, an antibody, an antibody fragment, an antigen binding domain, an immunoglobulin or an immunoglobulin fragment and a small molecule can be used. The targeting molecule is preferably an antibody.

[0190] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen or epitope. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, and can be immunoreactive portions of complete immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single-chain antibodies and humanized antibodies. The term "antibody fragment" refers to a part of a complete antibody, and refers to the antigen-determining variable region of a complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, VHH and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed by antibody fragments.

[0191] Markers for hematopoietic stem / progenitor cells may include CD13, CD27, CD33, CD34, CD45, CD49d (VLA-4, integrin α4), CD49e (VLA-5, integrin α5), CD49f (VLA-6, integrin α6), CD51 (integrin αV), CD59, CD84 (CD150 family), CD93, CD110 (thrombopoietin (TPO) receptor), CD114 (CSF3 receptor, G-CSF receptor), CD115 (CSF1 receptor), CD116 (GM-CSF receptor), CD117 (c-Kit / SCF receptor), CD121a (IL-1R), CD 123 (IL-3R), CD 124 (IL-4R), CD 125 (IL-5Rα), CD 126 (IL-6R), CD128 (IL-8Rα), CD129 (IL-9R), CD133 (Prominin 1), CD135 (Flt3 receptor), CD166 (ALCAM), CD184 (CXCR4), CD210 (IL-10R), CD218 (IL-18R1), CD244, CD360 (IL-21R), integrin β7, Prominin 2, erythropoietin R, endothelial cell selective adhesion molecule, Tie1, Tie2, MPL, gpl30, leukemia inhibitory factor receptor, oncostatin M receptor, Embigin, etc. Among the above, CD117 is preferred. As an exemplary targeting molecule, anti-CD117 antibodies can be used.

[0192] Hematopoietic stem / progenitor cells express c-Kit (CD117, a dimeric transmembrane receptor tyrosine kinase). Signal transduction involving CD117 is essential for many hematopoietic stem / progenitor cell functions, including homing, proliferation, adhesion, maintenance, and survival. CD117 is also expressed in other cell types such as cancer cells.

[0193] Markers for mesenchymal stem cells may include CD105.

[0194] Examples of antibody clones for each marker are shown below.

[0195] <Mouse CD117>

[0196] Antagonism: ACK2 (BioXCell)

[0197] Non-antagonistic: 2B8 (BioXCell)

[0198] <Human CD117>

[0199] Antagonism:

[0200] Briquilimab / AMG191 / JSP191 (Amgen or Jasper therapeutics, WO2007 / 127317)

[0201] Barzolvolimab (Celldex, WO2022159737)

[0202] LMS359, GZQ167, LMJ451 (Clin Cancer Res(2018)24(17):4297-4308.Novatris)

[0203] AB85 / MGTA117 (Magenta therapeutics, WO2019084067, WO2020219748, WO2020219770 and WO2020219775)

[0204] Non-antagonistic

[0205] 104D2 (Biolegend)

[0206] DLY884, LPG166, LQS721, LMJ729, LPG167 (Clin Cancer Res(2018) 24(17):4297-4308.Novartis)

[0207] <Human CD184 (CXCR4)>

[0208] Antagonism:

[0209] Ulocuplumab (Bristol Meyers Squibb)

[0210] LY2510924 (Eli Lilly: Peptide)

[0211] LY2624587 (Eli Lilly)

[0212] ALX-0651 (Sanofi / Ablynx: Nanobody)

[0213] PF-06747143 (Pfizer)

[0214] <Human CD105>

[0215] TRC105(Tracon)

[0216] <Human CD34>

[0217] 8G12 (BD Biosciences)

[0218] 581(Biolegend)

[0219] S20016E(Biolegend)

[0220] <Human CD27>

[0221] Agonistic (non-antagonistic):

[0222] Boserolimab (Merck / Aduro)

[0223] Varlilumab (Celldex)

[0224] <Human CD133>

[0225] C-Mab43(Monoclonal Antibodies in Immunodiagnosis andImmunotherapy. October 2017. 231-235.)

[0226] HW350341.1(GenBank:HW350341.1)

[0227] Although there is no particular restriction on the method for combining the targeting molecule with the lipid nanoparticle, it is preferred that the targeting molecule is combined with any lipid component constituting the lipid nanoparticle. For example, when the lipid nanoparticle comprises an ionizable lipid, a sterol, a neutral lipid (e.g., a phospholipid, etc.), a lipid with a nonionic hydrophilic polymer (e.g., a lipid with polyethylene glycol, etc.), it is preferably combined with any one of the above-mentioned lipid components. For example, the targeting molecule can be combined with a lipid with a nonionic hydrophilic polymer (e.g., a lipid with polyethylene glycol, etc.), but is not particularly limited.

[0228] The number of targeting molecules of each lipid nanoparticle is not particularly limited, but generally speaking, for each lipid nanoparticle, one or more targeting molecules are preferred. In one embodiment, the number of targeting molecules of each lipid nanoparticle can be determined by measuring the concentration [A] of lipid nanoparticles by common method (for example, microfluidic resistive pulse sensing (MRPS) method, adjustable resistive pulse sensing (unable resistive pulse sensing, TRPS) method, nanoparticle tracking analysis (NTA), transmission electron microscope (TEM) etc.). The concentration [B] of the targeting molecule can be measured by common method (such as HPLC, BCA determination, Lowry determination, Bradford quantitative method (Bradford assay) etc.). The number of targeting molecules of each lipid nanoparticle can be calculated as [B] / [A]. The concentration [A] of lipid nanoparticles can also be calculated according to the volume average particle size of lipid nanoparticles, the molecular volume of each component and the molar ratio of each component.

[0229] <Sterols>

[0230] The lipid nanoparticles preferably contain a sterol.

[0231] In the present invention, since sterol is contained, membrane fluidity can be reduced and an effect of stabilizing lipid particles can be obtained.

[0232] The sterol is not particularly limited, and examples thereof include cholesterol, phytosterol (sitosterol, stigmasterol, fucosterol, spinasterol, brassicasterol, etc.), ergosterol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, etc. Among them, cholesterol is preferred.

[0233] The content of sterol is preferably 5 mol % to 80 mol %, more preferably 10 mol % to 80 mol %, still more preferably 10 mol % to 60 mol %, and further preferably 30 mol % to 50 mol %, relative to the total lipids.

[0234] <Ionizable lipids>

[0235] In the present invention, ionizable lipids are used. Ionizable lipids may be lipids having at least one biodegradable group. Ionizable lipids may be lipids having at least one ionizable amino group and at least one biodegradable group. Ionizable lipids are pH responsive cationic lipids. They are electrically neutral at physiological pH (such as in blood) and become cationic in acidic environments (such as endosomes). Examples of the above-mentioned biodegradable groups include groups represented by -O(CO)O-, -O(CO)-, or -(CO)O-.

[0236] <<Lipid represented by formula (4) or its salt>>

[0237] For example, a lipid represented by formula (4) or a salt thereof can be used as the ionizable lipid.

[0238] [Chemical formula 7]

[0239]

[0240] In this formula, X represents -NR 1 -or-O-,

[0241] R 1 represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a 21 -L 1 -R 22 -represented by a group, wherein R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, L 1 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula:

[0242] [Chemical formula 8]

[0243] And R 22 represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms,

[0244] R 2 and R 3 Each independently represents a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a 31 -L 2 -R 32 -represented by a group, wherein R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, L 2 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula:

[0245] [Chemical formula 9]

[0246] And R 32 represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms,

[0247] R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms which may be substituted,

[0248] R 4 With R 5 , R 10 With R 5 , R 5 With R 12 , R 4 With R 6 , R 5 With R 6 , R 6 With R 7 , R 6 With R 10 , R 12 With R 7 , and R 7 With R 8 The groups in any one or more pairs of may be linked to each other to form a 4- to 7-membered ring which may contain an O atom,

[0249] The substituents on the alkyl group having 1 to 18 carbon atoms which may be substituted are hydroxyl, carboxyl, -NR 45 R 46 An amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 , or -OR 44 A group represented by 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0250] The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are alkyl groups having 1 to 18 carbon atoms, hydroxyl groups, carboxyl groups, groups consisting of -NR 45 R 46 An amino group represented by -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 , or -OR 44 A group represented by 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and

[0251] a, b, c and d each independently represent an integer of 0 to 3, a+b is 1 or more, and c+d is 1 or more.

[0252] As R 1 A hydrocarbon group having 6 to 24 carbon atoms represented by R 2 and R 3 The hydrocarbon group with 3 to 24 carbon atoms represented by alkyl, alkenyl or alkynyl is preferred, and alkyl or alkenyl is more preferred. Alkyl with 6 to 24 carbon atoms and alkyl with 3 to 24 carbon atoms may be straight chain or branched chain, or may be chain or cyclic. Alkyl with 6 to 24 carbon atoms is preferably alkyl with 6 to 20 carbon atoms, and alkyl with 3 to 24 carbon atoms is more preferably alkyl with 6 to 20 carbon atoms. Specifically, examples thereof include hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, tetramethylhexadecyl (preferably 3,7,11,15-tetramethylhexadecyl), heptadecanyl, octadecyl, nonadecanyl, eicosyl and the like. The alkenyl group having 6 to 24 carbon atoms and the alkenyl group having 3 to 24 carbon atoms may be straight chain or branched chain, or may be chain-like or cyclic. The alkenyl group having 6 to 24 carbon atoms is preferably an alkenyl group having 6 to 20 carbon atoms, and the alkenyl group having 3 to 24 carbon atoms is more preferably an alkenyl group having 6 to 20 carbon atoms. Specifically, examples thereof include hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl (preferably (Z)-hexadec-9-enyl), hexadecadienyl, heptadecenyl (preferably (Z)-heptadec-8-enyl), heptadecadienyl (preferably (8Z, 11Z)-heptadecadienyl, octadecenyl (preferably (Z)-octadec-9-enyl), octadecadienyl (preferably (9Z, 12Z)-octadecadienyl, 12-nonadecenyl, eicosenyl (preferably (Z)-eicos-11-enyl), eicosadienyl (preferably (11Z, 14Z)-eicos-11,14-dienyl) and the like. The alkynyl group with 6 to 24 carbon atoms is preferably an alkynyl group with 6 to 20 carbon atoms, and the alkynyl group with 3 to 24 carbon atoms is more preferably an alkynyl group with 6 to 20 carbon atoms. Specifically, examples thereof include hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecenyl, octadecynyl, octadecynyl, etc. All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0253] By R 21 and R 31 The hydrocarbon group having 1 to 24 carbon atoms represented by is preferably an alkyl group having 10 to 24 carbon atoms, an alkenyl group having 10 to 24 carbon atoms, or an alkynyl group having 10 to 24 carbon atoms. The alkyl group having 10 to 24 carbon atoms may be straight chain or branched chain, or may be chain-like or cyclic. The alkyl group having 10 to 24 carbon atoms is preferably an alkyl group having 12 to 24 carbon atoms. Specifically, examples thereof include decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, tetramethylhexadecyl (preferably 3,7,11,15-tetramethylhexadecyl), heptadecyl, octadecyl, 2-butylhexyl, 2-butyloctyl, 1-pentylhexyl, 2-pentylheptyl, 3-pentyloctyl, 1-hexylheptyl, 1-hexylnonyl, 2-Hexyloctyl, 2-hexyldecyl, 3-hexylnonyl, 1-heptyloctyl, 2-heptylnonyl, 2-heptylundecyl, 3-heptyldecyl, 1-octylnonyl, 2-octyldecyl, 2-octyldodecyl, 3-octylundecyl, 2-nonylundecyl, 3-nonyldodecyl, 2-decyldodecyl, 2-decyltetradecyl, 3-decyltridecyl, 2-(4,4-dimethylpent-2-yl)-5,7,7-trimethyloctyl, etc. The alkenyl group having 10 to 24 carbon atoms may be linear or branched, or may be chain-like or cyclic. Specifically, examples thereof include decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (preferably (Z)-tridec-8-enyl), tetradecenyl (preferably tetradec-9-enyl), pentadecenyl (preferably (Z)-pentadeca-8-enyl), hexadecaenyl (preferably (Z)-hexadec-9-enyl), hexadecadienyl, heptadecenyl (preferably (Z)-heptadec-8-enyl), heptadecadienyl (preferably (8Z, 11Z)-heptadecadienyl (preferably (8Z, 11Z)-heptadecadienyl), octadecenyl (preferably (Z)-octadecadienyl), octadecadienyl (preferably (9Z, 12Z)-octadecadienyl) and the like. The alkynyl group having 10 to 24 carbon atoms may be straight chain or branched, or may be chain-like or cyclic. Specifically, examples thereof include decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecenyl, octadecynyl, etc. All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0254] By R 22 and R 32The divalent hydrocarbon linking group represented by is preferably an alkylene group having 1 to 18 carbon atoms or an alkenylene group having 2 to 18 carbon atoms. The alkylene group having 1 to 18 carbon atoms may be straight chain or branched chain, or may be chain-like or cyclic. The number of carbon atoms in the alkylene group is preferably 1 to 12, more preferably 1 to 10, and still more preferably 2 to 10. Specifically, examples thereof include methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene, 1,6-hexylene, 1,7-heptylene, 1,8-octylene, 1,9-nonylene, 1,10-decylene, 1,11-undecylene, 1,12-dodecylene, etc. The alkenylene group having 2 to 18 carbon atoms may be straight chain or branched chain, or may be chain-like or cyclic. The number of carbon atoms in the alkenylene group is preferably 1 to 12, and more preferably 2 to 10.

[0255] -O(CO)O-, -O(CO)- and -(CO)O- in L 1 In the preferred range, -O(CO)- and -(CO)O- are present in L 1 is within the more preferred range.

[0256] -O(CO)O-, -O(CO)- and -(CO)O- in L 2 In the preferred range, -O(CO)- and -(CO)O- are present in L 2 is within the more preferred range.

[0257] can be replaced and by R 4 , R 6 , R 9 , R 10 , R 11 and R 12 The alkyl group having 1 to 18 carbon atoms represented by alkyl may be straight chain or branched chain, or may be chain or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 12. Specifically, examples thereof include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc. In the case where the alkyl group has a substituent, as a substituent, a hydroxyl group, a carboxyl group, or a -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 , or -OR 44 The group represented by -O(CO)-R 42 or-(CO)OR 43 The group represented by is more preferred.

[0258] can be replaced and by R 5, R 7 and R 8 The alkyl group having 1 to 18 carbon atoms represented by alkyl may be straight chain or branched chain, or may be chain or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 12, and more preferably 1 to 8. Specifically, examples thereof include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc. In the case where the alkyl group has a substituent, as a substituent, a hydroxyl group, a carboxyl group, or a -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 , or -OR 44 The group represented by -O(CO)-R 42 、-(CO)OR 43 , or -OR 44 The group represented by is more preferred.

[0259] Examples of the 4- to 7-membered ring which may contain an O atom include an azetidine ring, a pyrrolidine ring, a piperidine ring, a morpholine ring, and an azepane ring. The 4- to 7-membered ring is preferably a 6-membered ring, and is preferably a piperidine ring or a morpholine ring.

[0260] In R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 In the case where the alkyl group having 1 to 18 carbon atoms which is represented and may be substituted has a substituted or unsubstituted aryl group as a substituent, the number of carbon atoms in the aryl group is preferably 6 to 22, more preferably 6 to 18, and still more preferably 6 to 10. Specifically, examples of the aryl group include phenyl, naphthyl, anthracenyl, phenanthryl, and the like. As a substituent on the aryl group, an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, a -NR 45 R 46 An amino group represented by -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 , or -OR 44 The group represented by is preferred, and a hydroxyl group or a carboxyl group is more preferred. Specifically, examples of the substituted aryl group include a hydroxyphenyl group, a carboxyphenyl group, and the like.

[0261] In R 4 , R 5 , R6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 In the case where the alkyl group having 1 to 18 carbon atoms which is represented and may be substituted has a substituted or unsubstituted heteroaryl group as a substituent, the number of carbon atoms in the heteroaryl group is preferably 1 to 12, and more preferably 1 to 6. Specifically, examples of the heteroaryl group include pyridyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, oxazolyl, and the like. As a substituent on the heteroaryl group, an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, a -NR 45 R 46 An amino group represented by -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 , or -OR 44 A group represented by is preferred, and a hydroxyl group or a carboxyl group is more preferred. Specifically, examples of the substituted or unsubstituted heteroaryl group include a hydroxypyridyl group, a carboxypyridyl group, a pyridonyl group and the like.

[0262] As R 41 , R 42 , R 43 , R 44 , R 45 and R 46The hydrocarbon group having 1 to 18 carbon atoms represented by alkyl, alkyl, alkenyl, or alkynyl having 2 to 18 carbon atoms is preferred, and alkyl or alkenyl having 1 to 18 carbon atoms is more preferred. The alkyl group having 1 to 18 carbon atoms may be straight or branched, or may be chain or cyclic. The number of carbon atoms in the alkyl group is preferably 3 to 18, and more preferably 5 to 18. Specifically, examples thereof include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, etc. The alkenyl group having 2 to 18 carbon atoms may be linear or branched, or may be chain-like or cyclic. The number of carbon atoms in the alkenyl group is preferably 3 to 18, and more preferably 5 to 18. Specifically, examples thereof include allyl, isoprenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably (Z)-2-nonenyl or (E)-2-nonenyl), decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (preferably (Z)-trideca-8-enyl), tetradecenyl (preferably tetradec-9-enyl), pentadecenyl (preferably (Z)-pentadeca-8-enyl), hexadecaenyl (preferably (Z)-hexadec-9-enyl), hexadecadienyl, heptadecenyl (preferably (Z)-heptadeca-8-enyl), heptadecaadienyl (preferably (8Z,11Z)-heptadeca-8,11-dienyl), octadecenyl (preferably (Z)-octadec-9-enyl), octadecadienyl (preferably (9Z,12Z)-octadecadienyl) and the like. The alkynyl group having 2 to 18 carbon atoms may be straight chain or branched chain, or may be chain-like or cyclic. The number of carbon atoms in the alkynyl group is preferably 3 to 18, and more preferably 5 to 18. Specifically, examples thereof include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecenyl, octadecynyl, and the like.

[0263] The X indicates -NR 1 -In the case of R 1 Preferably represents a hydrocarbon group having 6 to 24 carbon atoms or a 21 -L 1 -R 22 -represented group. In this case, preferably R 2 and R 3 One of them represents a hydrogen atom, and the other represents a hydrocarbon group having 6 to 24 carbon atoms or a 31 -L2 -R 32 -represented group.

[0264] In the case where X represents -O-, preferably R 2 and R 3 Each independently represents a hydrocarbon group having 6 to 24 carbon atoms or R 31 -L 2 -R 32 -represented group.

[0265] R is preferred 4 , R 6 , R 9 , R 10 , R 11 and R 12 Each represents a hydrogen atom.

[0266] R 5 Preferably, it is a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, a -O(CO)-R 42 or-(CO)OR 43 In R 5 In the case of an alkyl group, R 5 Can be used with R 4 , R 6 , R 10 and R 12 are connected to form a ring which may contain an O atom. 5 Preferably, it is an alkyl group having 1 to 18 carbon atoms, which may be -O(CO)-R 42 or-(CO)OR 43 An alkyl group having 1 to 18 carbon atoms which may be substituted by an alkyl group, an alkyl group having 1 to 12 carbon atoms which may be substituted by an aryl group, or an alkyl group having 1 to 8 carbon atoms which may be substituted by a hydroxyl group, and more preferably an alkyl group having 1 to 18 carbon atoms or an alkyl group having -O(CO)-R 42 or-(CO)OR 43 A substituted alkyl group having 1 to 18 carbon atoms.

[0267] R 7 and R 8 Preferably each independently represents a hydrogen atom, a hydrocarbon group having 1 to 18 carbon atoms, a 42 or-(CO)OR 43 substituted alkyl group having 1 to 18 carbon atoms, alkyl group having 1 to 8 carbon atoms which may be substituted by an aryl group, or alkyl group having 1 to 8 carbon atoms which may be substituted by a hydroxyl group.7 and R 8 They are linked to each other to form a 4- to 7-membered ring which may contain an O atom.

[0268] R 5 Not with R 7 or R 8 connected and not with R 7 or R 8 Form a ring.

[0269] a+b is preferably 1 or 2, and more preferably 1. c+d is preferably 1 or 2, and more preferably 1.

[0270] The compound represented by formula (4) is preferably a compound represented by formula (21).

[0271] [Chemical formula 10]

[0272]

[0273] In this formula, R 2 and R 3 Each independently represents a hydrocarbon group containing one or more unsaturated bonds and having 3 to 24 carbon atoms, or R 2 and R 3 Each independently represents R 31 -L 2 -R 32 - represented by a group, or R 2 and R 3 One of them means that R 31 -L 2 -R 32 - represents a group, and the other represents a hydrocarbon group having 3 to 24 carbon atoms,

[0274] R 31 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0275] L 2 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula:

[0276] [Chemical formula 11]

[0277]

[0278] And R 32 represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms,

[0279] R 5 Indicates that -O(CO)-R 42 or-(CO)OR 43Substituted alkyl having 1 to 18 carbon atoms, wherein R 42 and R 43 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0280] R 7 and R 8 each independently represents an alkyl group having 1 to 4 carbon atoms,

[0281] e means 2 or 3.

[0282] In formula (21), preferably R 2 and R 3 One of them is R 31 -L 2 -R 32 -, and the other is a hydrocarbon group having 3 to 24 carbon atoms. 2 Preferably it represents -O(CO)- or -(CO)O-.

[0283] The compound represented by formula (4) may form a salt.

[0284] Examples of the salt in the basic group include salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid and sulfuric acid; salts formed with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid and trifluoroacetic acid; and salts formed with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid and naphthalenesulfonic acid.

[0285] Examples of the salt in the acidic group include salts formed with alkali metals such as sodium and potassium; salts formed with alkaline earth metals such as calcium and magnesium; ammonium salts; salts formed with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenylethylamine, 1-diphenylhydroxymethylamine (1-ephenamine) and N,N'-dibenzylethylenediamine; and the like.

[0286] Among the above salts, for example, pharmacologically acceptable salts are preferred.

[0287] The lipid represented by formula (4) and the method for producing the lipid are described in WO2019 / 235635A and WO2021 / 095876A, the entire contents of which are incorporated herein by reference.

[0288] <<Lipid represented by formula (1) or its salt>>

[0289] As another example, a lipid represented by formula (1) or a salt thereof can be used as the ionizable lipid.

[0290] [Chemical formula 12]

[0291]

[0292] In this formula,

[0293] R 1 and R 2 Each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, wherein R 1 , R 2 and R 3 The hydrocarbon group represented by the invention may be one or more selected from -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 AND-OR 56 Substituents are substituted,

[0294] R 4 represents a hydrocarbon group having 1 to 8 carbon atoms,

[0295] R 5 and R 6 Each independently represents a hydrocarbon group having 1 to 8 carbon atoms or -R 8 -L 1 -R 9 , but R 5 and R 6 Except for the case where all of them are hydrocarbon groups having 1 to 8 carbon atoms,

[0296] R 7 Indicates -R 10 -L 2 -R 11 -L 3 -R 12 ,

[0297] R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms,

[0298] R 53 , R 54 , R 55 and R 56 each independently represents a hydrocarbon group having 1 to 24 carbon atoms,

[0299] By R 53 , R 54 , R 55 and R56 The hydrocarbon group represented by may be replaced by an aryl group having 6 to 20 carbon atoms or -SR 58 replace,

[0300] The above aromatic groups having 6 to 20 carbon atoms may be replaced by -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 、-OR 56 , or -(hydrocarbon group having 1 to 12 carbon atoms)-R 57 replace,

[0301] R 58 represents a hydrocarbon group having 1 to 12 carbon atoms, and

[0302] R 57 Indicates -OH, COOH, -NR 61 R 62 、-OC(O)OR 63 、-C(O)OR 64 、-OC(O)-R 65 , or -OR 66 ,

[0303] R 61 and R 62 each independently represents a hydrocarbon group having 1 to 8 carbon atoms,

[0304] R 63 , R 64 , R 65 and R 66 each independently represents a hydrocarbon group having 1 to 24 carbon atoms,

[0305] By R 63 , R 64 , R 65 and R 66 The hydrocarbon group represented by may be replaced by an aryl group having 6 to 20 carbon atoms or -SR 68 replace,

[0306] The above aromatic groups having 6 to 20 carbon atoms may be replaced by -OH, COOH, -NR 61 R 62 、-OC(O)OR 63 、-C(O)OR 64 、-OC(O)-R 65 、-OR 66 , or -(hydrocarbon group having 1 to 12 carbon atoms)-R 67 replace,

[0307] R 68 represents a hydrocarbon group having 1 to 12 carbon atoms, and

[0308] L 1 , L 2 and L 3 each independently represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-,

[0309] R 8 represents a hydrocarbon group having 1 to 12 carbon atoms,

[0310] R 9 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0311] R 10 represents a hydrocarbon group having 1 to 8 carbon atoms,

[0312] R 11 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0313] R 12 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0314] By R 9 and R 12 The hydrocarbon group represented by 53 、-C(O)OR 54 、-OC(O)-R 55 , or -SR 58 Substitution, where R 53 , R 54 , R 55 and R 58 is defined above, and

[0315] By R 11 The hydrocarbon group represented by -OC(O)OR 53 、-C(O)OR 54 , or -OC(O)-R 55 Substitution, where R 53 , R 54 and R 55 The definition of is as above.

[0316] The hydrocarbyl group having 1 to 24 carbon atoms, the hydrocarbyl group having 1 to 18 carbon atoms, the hydrocarbyl group having 1 to 12 carbon atoms, the hydrocarbyl group having 2 to 8 carbon atoms, and the hydrocarbyl group having 1 to 8 carbon atoms are each preferably an alkyl group, an alkenyl group, or an alkynyl group.

[0317] The alkyl group may be straight chain or branched chain, or may be chain-like or cyclic. Specifically, examples of the alkyl group include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, tetramethylhexadecyl (preferably 3,7,11,15-tetramethylhexadecyl), heptadecyl, octadecyl, 2-butylhexyl, 2-butyloctyl, 1-pentylhexyl, 2-pentylheptyl, 3-pentyloctyl, 1-hexylheptyl, 1-hexylnonyl, 2-hexyloctyl, 2-hexyldecyl, 3-hexylnonyl, 1-heptyloctyl, 2-heptylnonyl, 2-heptylundecyl, 3-heptyldecyl, 1-octylnonyl, 2-octyldecyl, 2-octyldodecyl, 3-octylundecyl, 2-nonylundecyl, 3-nonyldodecyl, 2-decyldodecyl, 2-decyltetradecyl, 3-decyltridecyl, 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl and the like.

[0318] The alkenyl group may be linear or branched, or may be chain-like or cyclic. Specifically, examples of the alkenyl group include allyl, isoprenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably (Z)-2-nonenyl or (E)-2-nonenyl), decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (preferably (Z)-trideca-8-enyl), tetradecenyl (preferably tetradec-9-enyl), pentadecenyl (preferably (Z)-pentadeca-8-enyl), hexadecaenyl (preferably (Z)-hexadec-9-enyl), hexadecadienyl, heptadecenyl (preferably (Z)-heptadeca-8-enyl), heptadecaadienyl (preferably (8Z,11Z)-heptadeca-8,11-dienyl), octadecenyl (preferably (Z)-octadec-9-enyl), octadecadienyl (preferably (9Z,12Z)-octadecadienyl) and the like.

[0319] Alkynyl can be straight chain or branched chain, or can be chain or cyclic. Specifically, examples of alkynyl include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecenyl, octadecynyl, etc.

[0320] All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0321] -(hydrocarbon group having 1 to 12 carbon atoms)-R 67The hydrocarbon group having 1 to 12 carbon atoms in is preferably an alkylene group having 1 to 12 carbon atoms or an alkenylene group having 2 to 12 carbon atoms. The alkylene group having 1 to 12 carbon atoms and the alkenylene group having 2 to 12 carbon atoms may be straight chain or branched chain, or may be chain or cyclic.

[0322] Specifically, examples thereof include methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene, 1,6-hexylene, 1,7-heptylene, 1,8-octylene, 1,9-nonylene, 1,10-decylene, 1,11-undecylene and the like.

[0323] The aryl group preferably has 6 to 20 carbon atoms, more preferably has 6 to 18 carbon atoms, and even more preferably has 6 to 10 carbon atoms. Specifically, examples of the aryl group include phenyl, naphthyl, anthryl, phenanthryl and the like.

[0324] R 1 and R 2 Each independently preferably represents a hydrocarbon group having 1 to 12 carbon atoms, more preferably represents a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably represents a hydrocarbon group having 1 to 3 carbon atoms.

[0325] R 3 Preferably represents a hydrocarbon group having 2 to 6 carbon atoms and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.

[0326] By R 1 , R 2 and R 3 The hydrocarbon groups represented may preferably be substituted by -OH.

[0327] L 1 and L 3 Each independently preferably represents -C(O)O- or -OC(O)-.

[0328] L 2 Preferably it represents -OC(O)O-, -C(O)O-, or -OC(O)-.

[0329] R 8 Preferably represents a hydrocarbon group having 1 to 10 carbon atoms and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms.

[0330] R 9 Preferably represents a hydrocarbon group having 1 to 20 carbon atoms and more preferably represents a hydrocarbon group having 1 to 16 carbon atoms.

[0331] R 11 Preferably represents a hydrocarbon group having 1 to 16 carbon atoms and more preferably represents a hydrocarbon group having 1 to 9 carbon atoms.

[0332] R 12 Preferably represents a hydrocarbon group having 1 to 20 carbon atoms and more preferably represents a hydrocarbon group having 1 to 16 carbon atoms.

[0333] By R 9 and R 12 The hydrocarbon group represented by 58 Here, R 58 Preferably it represents a hydrocarbon group having 1 to 8 carbon atoms.

[0334] By R 11 The hydrocarbon group represented by -C(O)OR 55 or -OC(O)-R 56 Substitution, where R 55 and R 56 Each independently represents a hydrocarbon group having 1 to 16 carbon atoms.

[0335] By R 55 and R 56 The hydrocarbon group represented by may be preferably replaced by an aryl group having 6 to 20 carbon atoms or -SR 58 Replace, and R 58 The definition of is as above.

[0336] The compound represented by formula (1) is preferably a compound represented by formula (1-1) as a first example.

[0337] [Chemical formula 13]

[0338]

[0339] In this formula,

[0340] R 1 and R 2 Each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, wherein R 1 , R 2 and R 3 The hydrocarbon group represented by -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 , or -OR 56 replace,

[0341] R 4 represents a hydrocarbon group having 1 to 8 carbon atoms,

[0342] R 5 and R 6 Each independently represents a hydrocarbon group having 1 to 8 carbon atoms or -R 8 -L 1 -R 9 , but R 5 and R 6 Except for the case where all of them are hydrocarbon groups having 1 to 8 carbon atoms,

[0343] L 1 represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-,

[0344] R 8 represents a hydrocarbon group having 1 to 12 carbon atoms,

[0345] R 9 represents a hydrocarbon group having 1 to 24 carbon atoms, wherein R 9 The hydrocarbon group represented by 53 、-C(O)OR 54 、-OC(O)-R 55 , or -SR 58 replace,

[0346] R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms,

[0347] R 53 , R 54 , R 55 and R 56 each independently represents a hydrocarbon group having 1 to 24 carbon atoms,

[0348] By R 53 , R 54 , R 55 and R 56 The hydrocarbon group represented by may be replaced by an aryl group having 6 to 20 carbon atoms or -SR 58 replace,

[0349] The above aromatic groups having 6 to 20 carbon atoms may be replaced by -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 、-OR 56 , or -(hydrocarbon group having 1 to 12 carbon atoms)-R 57 replace,

[0350] R 58represents a hydrocarbon group having 1 to 12 carbon atoms, and

[0351] R 57 Indicates -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 , or -OR 56 , R 13 represents a hydrocarbon group having 1 to 8 carbon atoms,

[0352] R 14 Indicates -R 15 -L 5 -R 16 , where R 15 represents a hydrocarbon group having 1 to 24 carbon atoms, L 5 represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-, and R 16 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0353] By R 15 The hydrocarbon group having 1 to 24 carbon atoms represented by -OC(O)OR 53 、-C(O)OR 54 , or -OC(O)-R 55 Substitution, where R 53 , R 54 and R 55 is defined above, and

[0354] By R 16 The hydrocarbon group having 1 to 24 carbon atoms represented by -OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 or -SR 58 Substitution, where R 53 , R 54 , R 55 and R 58 The definition of is as above.

[0355] In formula (1-1), R 1 and R 2 Each independently preferably represents a hydrocarbon group having 1 to 12 carbon atoms, more preferably represents a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably represents a hydrocarbon group having 1 to 3 carbon atoms.

[0356] R 3Preferably represents a hydrocarbon group having 2 to 6 carbon atoms and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.

[0357] By R 1 , R 2 and R 3 The hydrocarbon groups represented may preferably be substituted by -OH.

[0358] L 1 Preferably it represents -C(O)O- or -OC(O)-.

[0359] R 8 Preferably represents a hydrocarbon group having 1 to 10 carbon atoms and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms.

[0360] R 9 Preferably represents a hydrocarbon group having 1 to 18 carbon atoms, and is represented by R 9 The hydrocarbon group represented by may be replaced by an aryl group having 6 to 20 carbon atoms or -SR 58 replace.

[0361] R 14 Preferably represents -R 15 -L 5 -R 16 , where R 15 represents a hydrocarbon group having 1 to 18 carbon atoms, L 5 represents -OC(O)O-, and R 16 represents a hydrocarbon group having 1 to 18 carbon atoms.

[0362] By R 15 The hydrocarbon group having 1 to 18 carbon atoms represented by 55 or -OC(O)-R 56 Replace. 55 and R 56 Each independently represents a hydrocarbon group having 1 to 16 carbon atoms, and R 55 and R 56 The hydrocarbon group represented by may be replaced by an aryl group having 6 to 20 carbon atoms or -SR 58 Substitution, where R 58 The definition of is as above.

[0363] By R 16 The hydrocarbon group having 1 to 18 carbon atoms represented by 58 Substitution, where R 58 The definition of is as above.

[0364] The compound represented by formula (1) is preferably a compound represented by formula (1-2) as a second example.

[0365] [Chemical formula 14]

[0366]

[0367] In this formula,

[0368] R 1 and R 2 Each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, wherein R 1 , R 2 and R 3 The hydrocarbon group represented by -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 , or -OR 56 replace,

[0369] R 4 and R 8 each independently represents a hydrocarbon having 1 to 8 carbon atoms,

[0370] R 21 and R 22 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0371] R 23 and R 24 each independently represents a hydrocarbon group having 1 to 12 carbon atoms,

[0372] R 25 and R 26 each independently represents a hydrocarbon group having 1 to 24 carbon atoms,

[0373] L 21 and L 22 each independently represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-,

[0374] By R 25 and R 26 The hydrocarbon group represented by may be replaced by an aryl group having 6 to 20 carbon atoms, -OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 , or -SR 58 replace,

[0375] R 51 and R52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms,

[0376] R 53 , R 54 , R 55 and R 56 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0377] The above aromatic groups having 6 to 20 carbon atoms may be substituted by OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 、-OR 56 , or -(hydrocarbon group having 1 to 12 carbon atoms)-R 57 Replace, and

[0378] R 57 Indicates -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 , or -OR 56 , R 58 represents a hydrocarbon group having 1 to 12 carbon atoms.

[0379] In formula (1-2), R 1 and R 2 Each independently preferably represents a hydrocarbon group having 1 to 12 carbon atoms, more preferably represents a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably represents a hydrocarbon group having 1 to 3 carbon atoms. 1 and R 2 The hydrocarbon group represented may preferably be substituted with -OH, but is more preferably a hydrocarbon having no substituent.

[0380] R 3 Preferably represents a hydrocarbon group having 2 to 6 carbon atoms and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.

[0381] R 21 and R 22 Each independently preferably represents a hydrocarbon group having 1 to 12 carbon atoms, more preferably represents a hydrocarbon group having 1 to 8 carbon atoms, and even more preferably represents a hydrocarbon group having 1 to 6 carbon atoms.

[0382] R 23 and R 24Each independently preferably represents a hydrocarbon group having 1 to 10 carbon atoms and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms.

[0383] R 25 and R 26 Each independently preferably represents a hydrocarbon group having 1 to 20 carbon atoms, more preferably represents a hydrocarbon group having 1 to 16 carbon atoms, and even more preferably represents a hydrocarbon group having 1 to 12 carbon atoms.

[0384] L 21 and L 22 Each independently preferably represents -C(O)O- or -OC(O)-.

[0385] The compound represented by formula (1) is preferably a compound represented by formula (1-3) as a third example.

[0386] [Chemical formula 15]

[0387]

[0388] In this formula,

[0389] R 1 and R 2 Each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, wherein R 1 , R 2 and R 3 The hydrocarbon group represented by -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 , or -OR 56 replace,

[0390] R 4 and R 8 each independently represents a hydrocarbon group having 1 to 8 carbon atoms,

[0391] R 31 , R 32 , R 33 and R 34 each independently represents a hydrocarbon group having 1 to 12 carbon atoms,

[0392] R 35 , R 36 , R 37 and R 38 each independently represents a hydrocarbon group having 1 to 24 carbon atoms,

[0393] L 31 , L 32 , L 33 and L 34 each independently represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-,

[0394] By R 35 , R 36 , R 37 and R 38 The hydrocarbon group represented by may be replaced by an aryl group having 6 to 20 carbon atoms, -OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 , or SR 58 replace,

[0395] R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms,

[0396] R 53 , R 54 , R 55 and R 56 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0397] The above aromatic groups having 6 to 20 carbon atoms may be substituted by OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 、-OR 56 , or -(hydrocarbon group having 1 to 12 carbon atoms)-R 57 Replace, and

[0398] R 57 Indicates -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 , or -OR 56 , R 58 represents a hydrocarbon group having 1 to 12 carbon atoms.

[0399] In formula (1-3), R 1 and R 2Each independently preferably represents a hydrocarbon group having 1 to 12 carbon atoms, more preferably represents a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably represents a hydrocarbon group having 1 to 3 carbon atoms. 1 and R 2 The hydrocarbon group represented may preferably be substituted with -OH, but is more preferably a hydrocarbon having no substituent.

[0400] R 3 Preferably represents a hydrocarbon group having 2 to 6 carbon atoms and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.

[0401] R 31 , R 32 , R 33 and R 34 Each independently preferably represents a hydrocarbon group having 1 to 10 carbon atoms, more preferably represents a hydrocarbon group having 1 to 8 carbon atoms, and even more preferably represents a hydrocarbon group having 1 to 3 carbon atoms.

[0402] R 35 , R 36 , R 37 and R 38 Each independently preferably represents a hydrocarbon group having 1 to 20 carbon atoms, more preferably represents a hydrocarbon group having 1 to 16 carbon atoms, and even more preferably represents a hydrocarbon group having 1 to 12 carbon atoms. 35 , R 36 , R 37 and R 38 The hydrocarbon group represented by may be preferably replaced by an aryl group having 6 to 20 carbon atoms or an SR 58 More preferably, these may be replaced by -SR 58 replace.

[0403] R 35 , R 36 , R 37 and R 38 Each independently particularly preferably represents -SR 58 A substituted hydrocarbon group having 1 to 12 carbon atoms, or a hydrocarbon group having 1 to 12 carbon atoms.

[0404] L 31 , L 32 , L 33 and L 34 Each independently preferably represents -C(O)O-, or -OC(O)-.

[0405] R 58 Preferably represents a hydrocarbon group having 1 to 10 carbon atoms and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms.

[0406] The compounds according to embodiments of the present invention may form salts.

[0407] Examples of the salt in the basic group include salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid and sulfuric acid; salts formed with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid and trifluoroacetic acid; and salts formed with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid and naphthalenesulfonic acid.

[0408] Examples of the salt in the acidic group include salts formed with alkali metals such as sodium and potassium; salts formed with alkaline earth metals such as calcium and magnesium; ammonium salts; salts formed with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenylethylamine, 1-diphenylhydroxymethylamine and N,N'-dibenzylethylenediamine; and the like.

[0409] Among the above-mentioned salts, for example, pharmacologically acceptable salts are preferred.

[0410] The lipid represented by formula (1) and a method for producing the lipid are described in WO2022 / 230964A, the entire contents of which are incorporated herein by reference.

[0411] <<Lipid represented by formula (5) or its salt>>

[0412] For example, a lipid represented by formula (5) or a salt thereof can be used as the ionizable lipid.

[0413] [Chemical formula 16]

[0414]

[0415] Where R 51 and R 52 Each independently represents a hydrocarbon group having 1 to 21 carbon atoms, which may have a substituent A,

[0416] The substituent A represents a hydroxyl group, or -G 20 -CH(R 55 )(R 56 )、-N(R 58 )(R 59 ) or -G 20 -R 60 The group represented by

[0417] G 20 represents -O(CO)-, or -(CO)O-,

[0418] R 55 and R56 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms,

[0419] R 58 and R 59 Each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms, which may have a substituent B,

[0420] The substituent B is -N(R 61 )(R 62 ),

[0421] R 61 and R 62 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms,

[0422] R 60 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0423] L 10 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0424] G 30 Indication-S-(CO)-NR 64 ,

[0425] R 64 Indicated by -L 30 -G 20 -CH(R 55 )(R 56 ) represents a group,

[0426] a represents 0 or 1,

[0427] L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms,

[0428] G 10 Represents -O(CO)-, -(CO)O-, -O(CO)O-, or -N(C(O)R 63 )-,

[0429] R 63 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0430] L 20 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0431] b represents 0 or 1,

[0432] R 53 , R 54 and R 57Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms, which may have a substituent C,

[0433] The substituent C represents -(CO)OR 65 or -O(CO)-R 65 The group represented by

[0434] R 65 represents a hydrocarbon group having 1 to 18 carbon atoms or -L 40 -CH(R 66 )(R 67 ) represents a group,

[0435] L 40 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0436] R 66 and R 67 represents a hydrocarbon group or an alkoxy group having 1 to 10 carbon atoms.

[0437] The compound represented by formula (5) may be a compound represented by formula (5A):

[0438] [Chemical formula 17]

[0439]

[0440] Where R 51 and R 52 Each independently represents a hydrocarbon group having 1 to 21 carbon atoms, which may have a substituent A,

[0441] The substituent A represents a hydroxyl group, or -G 20 -CH(R 55 )(R 56 ) represents a group,

[0442] G 20 represents -O(CO)-, or -(CO)O-,

[0443] R 55 and R 56 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0444] L 10 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0445] G 10 represents -O(CO)-, or -(CO)O-,

[0446] R 63 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0447] R 53 , R 54 and R 57 Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms.

[0448] The compound represented by formula (5) may be a compound represented by formula (5B):

[0449] [Chemical formula 18]

[0450] Where R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms,

[0451]

[0452] L 10 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0453] G 10 represents -O(CO)O-,

[0454] L 20 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0455] R 53 , R 54 and R 57 Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms, which may have a substituent C,

[0456] The substituent C represents -O(CO)-R 65 The group represented by

[0457] R 65 represents a hydrocarbon group having 1 to 18 carbon atoms or -L 40 -CH(R 66 )(R 67 ) represents a group,

[0458] L 40 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0459] R 66 and R 67 represents an alkoxy group having 1 to 10 carbon atoms.

[0460] The compound represented by formula (5) may be a compound represented by formula (5C):

[0461] [Chemical formula 19]

[0462]

[0463] Where R51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms,

[0464] L 10 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0465] G 10 Represents -N(C(O)R 63 )-,

[0466] R 63 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0467] R 53 , R 54 and R 57 Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms, which may have a substituent C,

[0468] The substituent C represents -(CO)OR 65 The group represented by

[0469] R 65 Indicated by -L 40 -CH(R 66 )(R 67 ) represents a group,

[0470] L 40 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0471] R 66 and R 67 represents a hydrocarbon group having 1 to 10 carbon atoms.

[0472] The compound represented by formula (5) may be a compound represented by formula (5D):

[0473] [Chemical formula 20]

[0474]

[0475] Where R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms,

[0476] L 10 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0477] G 30 Indication-S-(CO)-NR 64 ,

[0478] R 64 Indicated by -L 30 -G20 -CH(R 55 )(R 56 ) represents a group,

[0479] L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms,

[0480] G 20 represents -(CO)O-,

[0481] R 55 and R 56 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms,

[0482] G 10 represents -(CO)O-,

[0483] R 53 , R 54 and R 57 Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms.

[0484] The hydrocarbon radical having 1 to 21 carbon atoms is preferably an alkyl radical having 1 to 21 carbon atoms, an alkenyl radical having 2 to 21 carbon atoms, or an alkynyl radical having 2 to 21 carbon atoms, more preferably an alkyl radical having 1 to 21 carbon atoms, or an alkenyl radical having 2 to 21 carbon atoms. The alkyl radical having 1 to 21 carbon atoms may be straight chain or branched chain, and may be chain-like or cyclic. The number of carbon atoms is preferably 3 to 21, and more preferably 5 to 21 carbon atoms. Examples include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, heptadecanyl and octadecyl. The alkenyl group having 2 to 18 carbon atoms may be linear or branched, and may be chain-like or cyclic. The number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18. Examples include allyl, isoprenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably (Z)-2-nonenyl or (E)-2-nonenyl), decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (preferably (Z)-tridec-8-enyl), tetradecenyl (preferably tetradec-9-enyl), pentadecenyl (preferably (Z)-pentadeca-8-enyl), hexadecaenyl (preferably (Z)-hexadec-9-enyl), hexadecadienyl, heptadecenyl (preferably (Z)-heptadeca-8-enyl), heptadecadienyl (preferably (8Z, 11Z)-heptadeca-8, 11-dienyl), octadecenyl (preferably (Z)-octadec-9-enyl), octadecadienyl (preferably (9Z, 12Z)-octadecadienyl, 12-dienyl). Alkynyl groups with 2 to 21 carbon atoms may be straight or branched, and may be chain-like or cyclic. The number of carbon atoms is preferably 3 to 21, and more preferably 5 to 21 carbon atoms. Examples include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecinyl, octadecynyl, etc. Examples of hydrocarbon groups with 1 to 18 carbon atoms include hydrocarbon groups with 1 to 18 carbon atoms in hydrocarbon groups with 1 to 21 carbon atoms.

[0485] As the cyclic hydrocarbon group, a cycloalkyl group having 3 to 10 carbon atoms, a cycloalkenyl group having 3 to 10 carbon atoms, a cycloalkynyl group having 3 to 10 carbon atoms and an aryl group having 6 to 10 carbon atoms are preferred.

[0486] The hydrocarbon radical with 1 to 6 carbon atoms is preferably an alkyl radical with 1 to 6 carbon atoms, an alkenyl radical with 2 to 6 carbon atoms, or an alkynyl radical with 2 to 6 carbon atoms, and more preferably an alkyl radical with 1 to 6 carbon atoms, or an alkenyl radical with 2 to 6 carbon atoms. The alkyl radical with 1 to 6 carbon atoms may be straight chain or branched chain, and may be chain-like or cyclic. Its specific examples include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl and hexyl. The alkenyl radical with 2 to 6 carbon atoms may be straight chain or branched chain, and may be chain-like or cyclic. Its specific examples include allyl, isoprenyl, pentenyl and hexenyl. The alkynyl radical with 2 to 6 carbon atoms may be straight chain or branched chain, and may be chain-like or cyclic. Its specific examples include propargyl, butynyl, pentynyl and hexynyl.

[0487] The hydrocarbon radical with 1 to 10 carbon atoms is preferably an alkyl radical with 1 to 10 carbon atoms, an alkenyl radical with 2 to 10 carbon atoms, or an alkynyl radical with 2 to 10 carbon atoms, and preferably an alkyl radical with 1 to 10 carbon atoms, or an alkenyl radical with 2 to 10 carbon atoms. The alkyl radical with 1 to 10 carbon atoms may be straight chain or branched chain, and may be chain-like or cyclic. The number of carbon atoms is preferably 3 to 10, and more preferably 5 to 10 carbon atoms. Specific examples include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl and decyl. The alkenyl radical with 2 to 10 carbon atoms may be straight chain or branched chain, and may be chain-like or cyclic. The number of carbon atoms is preferably 3 to 10, and more preferably 5 to 10. Specific examples include allyl, isopentadienyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably (Z)-2-nonenyl or (E)-2-nonenyl), and decenyl. Alkynyl groups having 2 to 10 carbon atoms may be straight or branched, and may be chain-like or cyclic. The number of carbon atoms is preferably 3 to 10, and more preferably 5 to 10 carbon atoms. Specific examples include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl.

[0488] The compound represented by formula (5) may form a salt.

[0489] Examples of the salt in the basic group include salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid and sulfuric acid; salts formed with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid and trifluoroacetic acid; and salts formed with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid and naphthalenesulfonic acid.

[0490] Among the above salts, for example, pharmacologically acceptable salts are preferred.

[0491] The lipid represented by formula (4) and the method for producing the lipid are described in WO2019 / 235635A and WO2021 / 095876A.

[0492] <<Examples of Ionizable Lipids>>

[0493] Examples of the ionizable lipids include the following lipids: Note that cKK-E12 (MD-1), C12-200, 306Oi10, YSK05 and 93-O17S are compounds not included in the above formula (5).

[0494] [Chemical formula 21]

[0495] MC3

[0496]

[0497] L-319

[0498]

[0499] ALC-0315

[0500]

[0501] SM-102

[0502]

[0503] Lipid 5

[0504]

[0505] Lipid 29

[0506]

[0507] ATX-100

[0508]

[0509] Lipid A9

[0510]

[0511] Lp01

[0512]

[0513] TCL053

[0514]

[0515] TCL065

[0516]

[0517] Lipid 9

[0518]

[0519] Lipid 19

[0520]

[0521] 13-B43

[0522]

[0523] Compound 1

[0524]

[0525] Compound 2

[0526]

[0527] Compound 3

[0528]

[0529] Compound 4

[0530]

[0531] cKK-E12

[0532]

[0533] C12-200

[0534]

[0535] 306Oi10

[0536]

[0537] 93-O17S

[0538]

[0539] YSK05

[0540]

[0541] CL4H6

[0542]

[0543] ssPalmOPhe

[0544]

[0545] Compound 5

[0546]

[0547] Compound 6

[0548]

[0549] Compound 7

[0550]

[0551] Compound 8

[0552]

[0553] Compound 9

[0554]

[0555] In the lipid composition of the present invention, the content of the ionizable lipid or its salt is preferably 10 mol % to 80 mol %, more preferably 20 mol % to 80 mol %, still more preferably 30 mol % to 70 mol %, further more preferably 40 mol % to 60 mol %, relative to the total lipid.

[0556] <Neutral lipids>

[0557] The lipid particles according to the present invention may contain neutral lipids.

[0558] The neutral lipid is preferably a zwitterionic lipid.

[0559] As zwitterionic lipids, phospholipids are preferred. Examples thereof include phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, etc. As phospholipids, phospholipids with a choline group such as phosphatidylcholine are preferred. Zwitterionic lipids can be used alone or in combination with a variety of different neutral lipids.

[0560] Phosphatidylcholine is not particularly limited, and examples thereof include soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), egg yolk lecithin (EPC), hydrogenated egg yolk lecithin (HEPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylcholine (DLPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and the like. Among them, dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC) and dilauroylphosphatidylcholine (DLPC) are preferred. Distearoylphosphatidylcholine (DSPC) is particularly preferred.

[0561] DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine

[0562] [Chemical formula 22]

[0563]

[0564] The phosphatidylethanolamine is not particularly limited, and examples thereof include dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylethanolamine (DLoPE), diphytanoylphosphatidylethanolamine (D(Phy)PE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), ditetradecylphosphatidylethanolamine, dihexadecylphosphatidylethanolamine, distetradecylphosphatidylethanolamine, diphytylphosphatidylethanolamine and the like.

[0565] The sphingomyelin is not particularly limited, and examples thereof include egg yolk-derived sphingomyelin, milk-derived sphingomyelin and the like.

[0566] In the lipid composition of the present invention, the amount of the mixed neutral lipid is preferably 1 to 30 mol %, more preferably 5 to 25 mol %, still more preferably 7 to 23 mol % relative to the total amount of the constituent lipid components.

[0567] <Lipid with nonionic hydrophilic polymer>

[0568] The lipid composition of the present invention may include a lipid having a nonionic hydrophilic polymer. The lipid having a nonionic hydrophilic polymer preferably contains an acyl group, and the carbon chain length of the acyl group is preferably 8 to 26.

[0569] The nonionic hydrophilic polymer is not particularly limited, and examples thereof include nonionic vinyl polymers, nonionic polyamino acids, nonionic polyesters, nonionic polyethers, nonionic natural polymers, nonionic modified natural polymers, and block polymers or graft copolymers having two or more of these polymers as structural units.

[0570] Among these nonionic hydrophilic polymers, nonionic polyethers, nonionic polyesters, nonionic polyamino acids, or nonionic synthetic polypeptides are preferred, nonionic polyethers or nonionic polyesters are more preferred, nonionic polyethers or nonionic monoalkoxy polyethers are even more preferred, and polyethylene glycol (hereinafter, polyethylene glycol is also referred to as PEG) is particularly preferred. That is, preferably, the lipid nanoparticles may contain PEG-bound lipids.

[0571] The lipid having a nonionic hydrophilic polymer is not particularly limited, and examples thereof include PEG-modified diacylphosphoethanolamine, diacylglycerol PEG derivatives, monoacylglycerol PEG derivatives, dialkylglycerol PEG derivatives, cholesterol PEG derivatives, ceramide PEG derivatives, and the like. Among them, PEG-modified diacylphosphoethanolamine and diacylglycerol PEG are preferred. The acyl group in PEG-modified diacylphosphoethanolamine and diacylglycerol PEG preferably has 14 or more carbon atoms, more preferably 16 or more carbon atoms.

[0572] The weight average molecular weight of the nonionic hydrophilic polymer is preferably 100 to 10,000, more preferably 500 to 5,000, and even more preferably 750 to 3,000.

[0573] The nonionic hydrophilic polymer chain may be branched, or may have a substituent such as a hydroxymethyl group.

[0574] Preferred examples of the lipid having a nonionic hydrophilic polymer include the following lipids.

[0575] DMG-mPEG2000: 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000

[0576] DPG-mPEG2000: 1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-2000

[0577] DSG-mPEG2000: 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol-2000

[0578] [Chemical formula 23]

[0579]

[0580] When a targeting molecule is bound to a lipid having a nonionic hydrophilic polymer, a lipid having a reactive group for binding to the targeting molecule (e.g., a maleimide group, a thiol group, an orthopyridyl disulfide (OPSS) group, an N-hydroxysuccinimide (NHS) group, an alkyne group, a dibenzocyclooctine (DBCO) group, an azide group, an amino group, a carboxyl group, etc.) can be used as all or part of the lipid having a nonionic hydrophilic polymer.

[0581] In the lipid composition of the present invention, the amount of lipid having a nonionic hydrophilic polymer is preferably 0.1 mol % to 10 mol %, more preferably 0.3 mol % to 8 mol %, further preferably 0.5 mol % to 5 mol %, and even more preferably 1 mol % to 3 mol %, relative to the total amount of lipid.

[0582] <Therapeutic Agents>

[0583] The lipid composition of the present invention contains a therapeutic agent. As a therapeutic agent, nucleic acids such as polynucleotides are preferred. Nucleic acids such as polynucleotides can be DNA or RNA, and can be plasmids, single-stranded DNA, double-stranded DNA, siRNA (small interfering RNA), miRNA (microRNA), mRNA, single-stranded guide RNA (sgRNA), antisense oligonucleotides (also referred to as ASO), ribozymes, aptamers, decoy nucleic acids, guide RNA (gRNA) used in genome editing, etc. It can also contain modified nucleic acids. When sgRNA and mRNA are used, sgRNA and mRNA can be included in the lipid composition individually or together. Preferably, sgRNA and mRNA can be included in the lipid composition together.

[0584] In the lipid composition of the present invention, the weight ratio of lipid to therapeutic agent is preferably 5 to 100, more preferably 5 to 70, still more preferably 5 to 40, and particularly preferably 5 to 35.

[0585] <Method for producing composition>

[0586] A method for producing the lipid composition of the present invention will be described.

[0587] The method for making lipid composition is not limited. For example, lipid composition can be made by the following method: all components of lipid granules or some oil-soluble components of lipid granules are dissolved in organic solvent etc. so as to form oil phase, water-soluble components of lipid granules are dissolved in water so as to form water phase, and oil phase and water phase are mixed together. Micro mixer can be used to mix, or emulsifier such as homogenizer, ultrasonic emulsifier or high pressure injection emulsifier can be used to emulsify.

[0588] Alternatively, the lipid composition can also be produced by evaporating a lipid-containing solution to dryness under reduced pressure using an evaporator, or spray-drying using a spray dryer to prepare a dried mixture containing lipids, and adding the mixture to an aqueous solvent and further emulsifying using the aforementioned emulsifier, etc.

[0589] One example of a method for producing a lipid particle containing a nucleic acid is a method comprising the following steps:

[0590] A step (a) of dissolving constituent components of lipid particles containing a compound according to an embodiment of the present invention in an organic solvent to obtain an oil phase;

[0591] A step (b) of mixing the oil phase obtained in step (a) with the aqueous phase containing nucleic acid;

[0592] A step (c) of diluting the mixed solution containing the oil phase and the aqueous phase obtained in step (b) to obtain a dispersion of the nucleic acid-containing lipid composition; and

[0593] A step (d) of removing the organic solvent from the dispersion of the nucleic acid-lipid composition obtained in step (c).

[0594] In step (a), the lipid component is dissolved in an organic solvent (alcohol such as ethanol, ester, etc.). The total lipid concentration is not particularly limited, but is generally 1 mmol / L to 100 mmol / L, preferably 3 mmol / L to 50 mmol / L, and more preferably 5 mmol / L to 30 mmol / L.

[0595] In step (b), the aqueous phase can be obtained by dissolving the nucleic acid (e.g., siRNA, antisense nucleic acid, mRNA, etc.) in water or a buffer. If necessary, components such as antioxidants can be added. The mixing ratio (volume ratio) of the aqueous phase: oil phase is preferably 5:1 to 1:1, and more preferably 4:1 to 2:1.

[0596] In step (b), the mixed solution may be diluted with water or a buffer (eg, phosphate buffered saline (PBS) etc.).

[0597] In step (c), as a method for removing the organic solvent from the dispersion of the lipid composition, a general method can be used without particular limitation. For example, the organic solvent can be removed by dialyzing the dispersion with phosphate buffered saline.

[0598] If necessary, the lipid composition may be fractionated. Although the fractionation method is not particularly limited, an extruder or the like may be used to reduce the particle size.

[0599] <Composition>

[0600] Compositions of the present invention can be lipid particles.Lipid particles mean the particles made of lipid, and include compositions with any structure selected from lipid aggregates (e.g., lipid nanoparticles), micelles, and liposomes in which lipids are gathered. However, the structure of lipid particles is not limited to these, as long as the composition contains lipid.

[0601] The form of lipid particles can be checked by electron microscopy, structural analysis using X-rays, etc. For example, by using a method of cryo-transmission electron microscopy (CryoTEM method), it is possible to check whether, for example, lipid particles (such as liposomes) have a structure consisting of a bimolecular lipid membrane structure (lamellar structure) and an internal water layer, or a structure consisting of a core having a high electron density and filled with constituent components (including lipids). Small-angle X-ray scattering (SAXS) analysis also makes it possible to check whether lipid particles have a bimolecular lipid membrane structure (lamellar structure).

[0602] When the lipid composition of the present invention is a particle, the particle size is not particularly limited, but is preferably 10 to 1,000 nm, more preferably 30 to 500 nm, and even more preferably 50 to 250 nm. The particle size of lipid particles can be measured by general methods (e.g., dynamic light scattering, laser diffraction, etc.).

[0603] When the lipid composition of the present invention is a particle, the zeta potential of the particle is not particularly limited, but is preferably -20 to +20 mV, and more preferably -10 to 10 mV. The zeta potential in the present invention is a value measured by the *** method obtained by diluting the lipid composition in a phosphate buffer solution, but the method is not limited thereto.

[0604] The pKa of the lipid composition of the present invention is not particularly limited, but is preferably 9 to 4, more preferably 8 to 5, and even more preferably 7.5 to 6. The pKa of the lipid composition in the present invention adopts a value measured by TNS assay, but is not limited thereto.

[0605] <Uses of lipid compositions>

[0606] As the example of the purposes of the lipid composition in the present invention, therapeutic agent (for example, nucleic acid) can be introduced into the cell by the lipid composition containing nucleic acid.That is to say, the lipid composition of the present invention can be used as the composition for introducing nucleic acid into the cell.

[0607] In addition, the lipid composition of the present invention can be used as a pharmaceutical composition for in vivo nucleic acid delivery.

[0608] In particular, in the present invention, the therapeutic agent can be delivered to hematopoietic stem / progenitor cells or mesenchymal stem cells. Therefore, examples of organs to which the therapeutic agent can be delivered include bone marrow, spleen, and the like.

[0609] In addition, when the lipid composition of the present invention contains nucleic acid with medical use, the lipid composition can be administered to a living body as a nucleic acid drug. When the lipid composition of the present invention is used as a nucleic acid drug, the lipid composition of the present invention can be administered to a living body alone, or the lipid composition can be mixed with a pharmaceutically acceptable carrier (for example, an administration medium such as saline or phosphate buffer) and administered to a living body. That is to say, the lipid composition of the present invention can further contain a pharmaceutically acceptable carrier.

[0610] The concentration of the lipid composition in a mixture with a pharmaceutically acceptable carrier is not particularly limited, and may generally be 0.05% to 90% by weight. In addition, other pharmaceutically acceptable additives such as pH regulating buffer and osmotic pressure regulator may be added to the nucleic acid drug containing the lipid composition of the present invention.

[0611] The route of administration for the lipid composition of the present invention is not particularly limited. The lipid composition can be administered by any method. The example of the method of administration includes oral administration and parenteral administration (intraarticular administration, intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, intravitreal administration, intraperitoneal administration, intramuscular administration, intravaginal administration, intravesical administration, intrathecal administration, pulmonary administration, rectal administration, colon administration, buccal administration, nasal administration, intracisternal administration, inhalation, etc.). Wherein, parenteral administration is preferred. As the method of administration, intravenous injection, subcutaneous injection, intradermal injection or intramuscular injection are preferred. Intravenous injection or intramuscular injection are particularly preferred. As administration, nucleic acid delivery can also be carried out by local administration in vivo. The lipid composition of the present invention can also be administered by direct injection into the diseased part.

[0612] The dosage form of the lipid granules according to the embodiments of the present invention is not particularly limited. For oral administration, by combining with suitable excipients, the lipid composition of the present invention can be used in the form of tablets, lozenges, capsules, pills, suspensions, syrups, etc. In addition, additives such as antioxidants, buffers, antibacterial agents, isotonic sterile injections, suspending agents, solubilizers, thickeners, stabilizers and preservatives can be appropriately incorporated into the preparations suitable for parenteral administration.

[0613] <Use of lipid nanoparticles as nucleic acid delivery vectors>

[0614] The lipid granules in the present invention can retain nucleic acid with a high encapsulation rate. Therefore, lipid granules are extremely useful as nucleic acid delivery vectors. According to the use of nucleic acid delivery vectors of the present invention, for example, by mixing the obtained lipid granules with nucleic acid etc. and transfecting in vitro or in vivo, nucleic acid etc. can be introduced into cells. In addition, the nucleic acid delivery vector of the present invention can also be used as the nucleic acid delivery vector in nucleic acid drugs. That is to say, the lipid granules according to the embodiments of the present invention can be used as the compositions for delivering nucleic acid in vitro or in vivo (preferably in vivo).

[0615] Next, the present invention will be described based on embodiments, but the present invention is not limited thereto.

[0616] Drugs with immunosuppressive effects or inhibitors of CD117 kinase activity

[0617] Antihistamines (preferred)

[0618] Examples of H1 Histamine Blockers

[0619] Acrivastine, alimamazine, amitriptyline, amoxapine, aripiprazole, azelastine, bilastine, bromodiphenhydramine, brompheniramine, buclizine, carbinoxamine, cetirizine, chlorphenidol, chlordiphenhydramine, chlorpheniramine, chlorpromazine, chlorprothixene, clopyramine, cinnarizine, clemastine, clomipramine, clozapine, cyclizine, cyproheptadine, desloratadine,

[0620] Dexbrompheniramine, dexchlorpheniramine, dimenhydrinate, dimethinidine, diphenhydramine, dothiophene,

[0621] Doxepin, antacid, ebastine, enbramine, fexofenadine, fluoxetine, hydroxyzine,

[0622] Imipramine, ketotifen, levocabastine, levocetirizine, levomepromazine, loratadine, maprotiline, meclizine, misalazine, mirtazapine, olanzapine, olopatadine, orphenadrine,

[0623] Percynandine, phenindamine, pheniramine, phenyltoloxamine, promethazine, mepyramine, quetiapine, rupatadine, setastine, settiline, trazodone, tripeliramine, triprolidine

[0624] Cetirizine and diphenhydramine are most preferred.

[0625] H2 histamine blockers

[0626] Cimetidine, famotidine, lafutidine, nizatidine, ranitidine, roxatidine, thiotidine

[0627] Mast cell stabilizers

[0628] Cromolyn sodium, nedocromil

[0629] Corticosteroids (preferred)

[0630] Prednisone (Deltasone, Orasone)

[0631] Prednisolone (Millipred)

[0632] Methylprednisolone (Medrol, Depo-Medrol, Solu-Medrol)

[0633] Betamethasone (Celestone)

[0634] Dexamethasone (Dexamethasone Intensol)

[0635] Budesonide (Entocort EC)

[0636] Triamcinolone acetonide (Aristospan Intra-Articular, Aristospan Intralesional, triamcinolone acetonide (Kenalog))

[0637] Examples of steroids

[0638] Progesterone steroids:

[0639] Fluoromesterolone, fluorometholone, medroxyprogesterone acetate, chlormadinone acetate, cyproterone acetate, medrogestone, medroxyprogesterone acetate, megestrol acetate, and norgestrel acetate

[0640] Hydrocortisone-type steroids:

[0641] Chloropredasol, Chloropredasol, Difluprednate, Fludrocortisone, Fluocinolone, Fluperone, Fluprednisolone, Loteprednol, Methylprednisolone, Prednicarbate, Prednisone, Prednisolone, Tixocortol, Triamcinolone,

[0642] Betamethasone-type steroids:

[0643] Alclometasone, betamethasone, beclomethasone, clobetasol, clobetasone, clocortolone, desoximetasone, dexamethasone, diflorasone, diflucortolone, fluclosonide, flumethasone, fluocortidine, flucortolone, fluprednidine, fluticasone, fluticasone furoate, halometasone, methylprednisone, mometasone, mometasone furoate, paramethasone, prednididine, rimexolone, ulbetasol (halobetasol),

[0644] Acetonides and related steroids:

[0645] Amcinolone, budesonide, ciclesonide, deflazacort, desonide, formocortal (flurandrenolone), fluclosonide (fluchloride), flurandrenolide (flurandrenolone, flurandrenolide), flunisolide, fluocinolone, fluocinolone acetate, halcinonide, triamcinolone acetonide,

[0646] Other steroids:

[0647] Cortisol, RU-28362 (6-methyl-11β,17β-dihydroxy-17α-(1-propynyl)androsta-1,4,6-trien-3-one)

[0648] CD117 kinase activity inhibitor (preferred)

[0649] Axitinib, cabozantinib, dasatinib, flumatinib, imatinib, imatinib, imatinib, masitinib, midostaurin, nilotinib, pazopanib, sorafenib, sunitinib, tocitanib, acetaminophen

[0650] Nonsteroidal anti-inflammatory drugs: NSAIDS

[0651] Salicylates, such as aspirin, diflunisal, salicylic acid and its salts, salsalate,

[0652] Propionic acid derivatives, such as ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, perlbiprofen, zaltoprofen, fenbufen, tiaprofenic acid, and carprofen

[0653] Acetic acid derivatives, such as indomethacin, acemetacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, fenclofenac, aceclofenac, bromfenac, fentiazine, nabumetone,

[0654] Enolic acid (oxicam) derivatives, such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, phenylbutazone,

[0655] Anthranilic acid derivatives, such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, etofenamate,

[0656] Selective COX-2 inhibitors, such as celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, firocoxib

[0657] Sulfonanilides, such as nimesulide

[0658] Other possible preprocessing

[0659] Janus kinase inhibitors

[0660] Tofacitinib (Xeljanz)

[0661] Calcineurin inhibitors

[0662] Cyclosporine (Neoral, Sandimmune, SangCya)

[0663] Tacrolimus (Astagraf XL, Envarsus XR, Prograf)

[0664] mTOR inhibitors

[0665] Sirolimus (Rapamune)

[0666] Everolimus (Afinitor, Zortress)

[0667] IMDH inhibitors

[0668] Azathioprine (Azasan, Imuran)

[0669] Leflunomide (Arava)

[0670] Mycophenolate mofetil (CellCept, Myfortic)

[0671] Biologics

[0672] Abatacept (Orencia)

[0673] Adalimumab (Humira)

[0674] Anakinra (Gineret)

[0675] Certolizumab (Cimzia)

[0676] Etanercept (Enbrel)

[0677] Golimumab (Simponi)

[0678] Infliximab (Remicade)

[0679] Ixekizumab (Taltz)

[0680] Natalizumab (Tysabri)

[0681] Rituximab (Rituxan)

[0682] Secukinumab (Cosentyx)

[0683] Tocilizumab (Actemra)

[0684] Ustekinumab (Stelara)

[0685] Vedolizumab (Entyvio)

[0686] Basiliximab (Simulect)

[0687] Daclizumab (Zinbryta)

[0688] Example

[0689] <Materials and Methods>

[0690] <sirna>

[0691] The following custom siRNAs were manufactured by Horizon.

[0692] siCD45 siRNA sequences

[0693] Sense strand: mCmUGGmCmUGAAmUmUmUmCAGAGmCAdTdT,

[0694] Antisense strand: UGCUCUGAAAUUmCAGCmCAGdTdT

[0695] Abbreviations for nucleotide monomers used in nucleic acid sequence representations. It will be understood that these monomers, when present in an oligonucleotide, are linked to each other via 5'-3'-phosphodiester bonds.

[0696] Abbreviation Nucleotide

[0697] A: adenosine-3'-phosphate, C: cytidine-3'-phosphate, G: guanosine-3'-phosphate, U: uridine-3'-phosphate,

[0698] mA: 2'-O-methyladenosine-3'-phosphate, mC: 2'-O-methylcytidine-3'-phosphate, mG: 2'-O-methylguanosine-3'-phosphate, mU: 2'-O-methyluridine-3'-phosphate,

[0699] dT: 2'-deoxythymidine-3'-phosphate, dTs: 2'-deoxythymidine-3'-phosphorothioate

[0700] <mrna>

[0701] Cre mRNA can be purchased from TriLink.

[0702] <Components of lipid composition>

[0703] [Chemical formula 24]

[0704] DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine

[0705]

[0706] [Chemical formula 25]

[0707] cholesterol

[0708]

[0709] DMG-mPEG2000: 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000

[0710] DPG-mPEG2000: 1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-2000

[0711] DSG-mPEG2000: 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol-2000

[0712] [Chemical formula 26]

[0713]

[0714] DSPE-PEG2000-Maleimide

[0715] <Chemical structure of ionizable lipid> [Chemical formula 27]

[0716] Compound 1

[0717]

[0718] Compound 2

[0719]

[0720] Compound 3

[0721]

[0722] Compound 4

[0723]

[0724] MC3

[0725]

[0726] L-319

[0727]

[0728] ALC-0315

[0729]

[0730] SM-102

[0731]

[0732] Lipid 5

[0733]

[0734] Lipid 29

[0735]

[0736] ATX-100

[0737]

[0738] Lipid A9

[0739]

[0740] Lp01

[0741]

[0742] TCL053

[0743]

[0744] cKK-E12

[0745]

[0746] C12-200

[0747]

[0748] 306Oi10

[0749]

[0750] 93-O17S

[0751]

[0752] YSK05

[0753]

[0754] CL4H6

[0755]

[0756] ssPalmOPhe

[0757]

[0758] Compound 5

[0759]

[0760] Compound 6

[0761]

[0762] Compound 7

[0763]

[0764] Compound 8

[0765]

[0766] Compound 9

[0767]

[0768] <Lipid Nanoparticle Preparation>

[0769] The siRNA was diluted with 10 mM citrate buffer (pH 3.0) (aqueous phase), while an appropriate amount of lipid was co-dissolved in 200 degrees of ethanol (ethanol phase). Nanoparticles prepared via a microfluidic device were synthesized in an aqueous phase to an ethanol phase at a 3:1 v / v ratio. The lipid nanoparticles were then dialyzed overnight in a 20 kDa MWCO cassette with PBS containing 10 mM EDTA at 4 ° C or room temperature.

[0770] <Conjugation of Antibodies to Lipid Nanoparticles>

[0771] Anti-CD117 antibody (clone 2B8, Bio X Cell) was reduced with 5 equivalents of TCEP (10 mM in PBS). Similarly, rat IgG2b isotype control (anti-keyhole limpet hemocyanin, Bio X Cel) was reduced with 5 equivalents of 10 mM TCEP. The antibody was reduced by incubation at 37°C for one hour with gentle shaking. After incubation, excess TCEP was removed with a Zeba 7kMWCO desalting column.

[0772] Mix the maleimide-lipid nanoparticle dispersion and the reducing antibody solution in a molar concentration range of the reducing antibody to maleimide of 1:100 to 1:5, and place it on a vertical cylindrical mixer at room temperature for 1 to 2 hours to conjugate the free thiols with the maleimide on the LNP. Thereafter, store the mixture at 4 °C until the purification step. <Gel filtration purification of antibody-conjugated lipid nanoparticles>

[0773] Load the reaction mixture containing the antibody-conjugated lipid nanoparticles onto a gel filtration qEV column and fractionate it using PBS as the mobile phase. Measure the protein concentration of each fraction to identify the fraction containing the target antibody-lipid nanoparticles. After collection, combine the antibody-lipid nanoparticle fractions, then concentrate them using an Amicon Ultra filter, and filter the concentrated antibody-lipid nanoparticles through a 2-μm syringe filter and store at 4 °C.

[0774] <Particle size measurement>

[0775] For lipid nanoparticles, obtain the particle size, PDI (polydispersity index), and ζ potential (i.e., zeta potential) using a Zetasizer (Malvern). For size measurement, dilute the LNP with PBS at a 1 / 200 v / v ratio and report the z-average. For ζ potential measurement, dilute the LNP with 0.1X PBS.

[0776] <Quantification of siRNA concentration and encapsulation>

[0777] Determine the siRNA concentration in the dialyzed particles via a modified Quant-iT RiboGreen RNA assay (Thermo Fisher). Prepare a nanoparticle dilution of siRNA at approximately 1 ng / μL in TE buffer (pH 8.5), and prepare a series from 2 ng / μL -l to 0.125 ng / μL -l -l ​siRNA standards within a range. 50 μL of each solution was added to a separate well of a 96-well black polystyrene plate. 50 μL of TE buffer or 50 μL of TE containing 2% Triton-X was added to each well. The plate was incubated at 37°C for 15 minutes with shaking at 350 rpm. After incubation, diluted RiboGreen reagent (100 μL per well) was added and the plate was incubated as before for 3 minutes. RiboGreen fluorescence was measured using a Tecan microplate reader according to the supplied protocol, and the nanoparticle siRNA concentration was determined using siRNA standards. It should be noted that two separate standards were prepared: one with Triton-X and one without Triton-X. The particles in TE buffer were used to determine the unencapsulated siRNA concentration and TE-TX, and the encapsulation efficiency was determined via the following formula:

[0778] [Chemical formula 28]

[0779]

[0780] <In vitro gene silencing>

[0781] EML cells were cultured in IMDM culture medium supplemented with 20% HI-FBS, PenStrep and 200ng / mL mouse stem cell factor (mSCF, Peprotech Inc.). In a cell culture medium of 100 μL volume, cells were plated in 96-well U-shaped bottom plates at 50,000 cells / well. siRNA lipid nanoparticles were added to cells at various concentrations and incubated for 40-48 hours. In each experiment, three technical replicates of each transfection condition were used. After incubation, the cell culture medium was removed and the cells were washed with PBS.

[0782] After washing, 100 μL QuickExtract TM Total RNA was extracted from EML cells using an RNA extraction kit (Lucigen, catalog number QER090150). For qPCR, 2 μl of RNA extract was added to a 384-well plate containing 0.5 μl B2M TaqMan probe (Applied Biosystems catalog number Mm00437762_m1) or 0.5 μl CD45 TaqMan probe (Applied Biosystems catalog number Mm01293577_m1) and 7 μl Luna TM The PCR products were expressed in the master mix of the Universal Probe One-Step RT-qPCR Kit (NEB Catalog No. E3006). Real-time PCR was performed in a Light Cycler 480 (Roche). Unless otherwise specified, each duplex was tested in two or three independent transfections, and each transfection was assayed in duplicate.

[0783] To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with the same concentration of siRNA against luciferase or mock-transfected cells. IC50 was calculated using Graphpad Prism software.

[0784] <Bone marrow flow cytometry>

[0785] Mice were killed and femurs and tibias were collected. Femurs and tibias were cut in half, and then bone was centrifuged in Eppendorf tubes at 13,000xg for 45 seconds to collect bone marrow cells. The resulting precipitation was resuspended in 1mL of PBS, filtered through a 70 μm coarse filter, and washed with 10mL of PBS. After centrifugation, 1mL of RBC lysis buffer (Qiagen) was used to lyse BM cells on ice for 10 minutes under gentle shaking. After RBC lysis, bone marrow cells were transferred to 96-well U-shaped bottom plates for flow cytometry staining. Staining markers include lineage markers (CD3, Gr-1, CD11b, CD45R / B220, mTer-119), CD117, Sca1 and CD45.

[0786] Samples were analyzed on a BD LSR Fortessa II and data were analyzed using FlowJo.

[0787] Example 1: In vitro RNA delivery to CD117+ cells

[0788] The ability of anti-murine CD117 antibody-modified lipid nanoparticles to deliver mRNA to EML cells (ATCC CRL-11691, a murine CD117-positive stem cell factor-dependent lymphohematopoietic progenitor cell line) was tested in vitro compared to non-antibody-modified lipid nanoparticles.

[0789] EML cells were cultured in IMDM medium in the presence of 200 ng / mL mouse stem cell factor (mSCF1, R&D Systems). Cells were plated at 50,000 cells / well in 96-well plates in 100 μL volume of cell culture medium containing various concentrations of siRNA-LNPs, with three technical replicates in each experiment. After 40 hours, the cell culture medium was removed and the cells were washed with PBS.

[0790] Using QuickExtract TM Total RNA was extracted from EML cells using an RNA extraction kit (Lucigen, catalog number QER090150). 2 μl of RNA extract was added to a 384-well plate containing 0.5 μl of B2M TaqMan probe (Applied Biosystems catalog number Mm00437762_m1) or 0.5 μl of CD45 TaqMan probe (Applied Biosystems catalog number Mm01293577_m1) and 7 μl of Luna TM The PCR products were expressed in the master mix of the Universal Probe One-Step RT-qPCR Kit (NEB Catalog No. E3006). Real-time PCR was performed in a Light Cycler 480 (Roche). Unless otherwise specified, each duplex was tested in two or three independent transfections, and each transfection was assayed in duplicate.

[0791] To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with the same concentration of siRNA against luciferase or mock-transfected cells. IC50 was calculated using Graphpad Prism software.

[0792] Results are shown in Figure 1 middle. Figure 1 Shown is an in vitro evaluation of CD45 gene silencing on EML cells using LNPs conjugated to aCD117.

[0793] [Table 1]

[0794]

[0795] Example 2: In vitro promotion of ionizable lipids

[0796] In order to test the in vitro scalability of this anti-CD117 lipid nanoparticle, various ionizable lipids were formulated into LNPs containing siRNA for CD45 and then conjugated with anti-mouse CD117 antibody or rat IgG2b isotype control antibody. After conjugation, the lipid nanoparticles were then added to EML cells at a dose of 50ng siRNA / well. 40 hours after transfection, the cells were washed with PBS and then resuspended and lysed in QuickExtract RNA solution. Using the ΔΔCt method, qPCR was used to determine the knockdown of CD45 in treated cells.

[0797] Results are shown in Figure 2 middle. Figure 2 It was shown that knockdown of CD45 via aCD117-receptor interaction is generalizable to other ionizable lipids.

[0798] From the data it can be shown that conjugation of anti-CD117 resulted in efficient delivery of all ionizable lipids tested. It should be understood that delivery is mediated by anti-CD117 antibody-receptor interaction, given that conjugation of these lipid nanoparticles with isotype control antibodies did not show silencing.

[0799] [Table 2]

[0800]

[0801] Example 3: In vivo RNA delivery to bone marrow CD117+ hematopoietic stem and progenitor cells (DMG vs. DSG)

[0802] The ability of anti-CD117 antibody-modified lipid nanoparticles to deliver RNA in vivo to HSPCs was evaluated in C57BL / 6 mice. Lipid nanoparticles containing siCD45 and PEG-lipids of different alkyl chain lengths were prepared. DMG (C14) and DSG (C18) of different alkyl chain lengths were included to test the effect of LNP circulation time on delivery to bone marrow cells. After preparation, LNP was conjugated with mouse anti-CD117, and after conjugation, unreacted antibodies were removed by size exclusion chromatography.

[0803] Lipid nanoparticles were administered to mice at a dose of 1 mg / kg (20 ug) siRNA via iv tail vein injection. After 72 hours, mice were sacrificed and bones (femur + tibia) were processed into single cell suspensions and then stained for flow cytometry analysis. To analyze delivery to HSPCs, bone marrow cells were gated with standard mouse HSPC markers to identify cells that were negative for lineage markers and double positive for Sca1 and c-Kit (also known as LSK cells). Within the LSK population, the MFI of CD45 was analyzed to determine silencing.

[0804] Results are shown in Figure 3 middle. Figure 3 CD45 expression levels in bone marrow LSK cells quantified by flow cytometry are shown. Both DMG and DSG preparations showed significant CD45 knockdown in bone marrow LSK cells. Compared with the silencing observed in preparations containing DMG (30%), the use of DSG in the preparation showed a higher level of silencing (60%). Lipid nanoparticles modified with anti-CD117 antibodies enable effective delivery of RNA to HSPCs in vivo. These results indicate that lipid nanoparticles with longer circulation times are more advantageous for delivery.

[0805] Table 4: Formulations tested in mice

[0806]

[0807] In order to further evaluate the delivery of mRNA to bone marrow CD117+ hematopoietic stem cells and progenitor cells, anti-CD117 conjugated LNPs were prepared with Cre recombinase mRNA and then administered to Ai14 reporter mice. These mice contain loxP-flanked termination boxes that prevent transcription of fluorescent TdTomato protein. During Cre recombination, cells will express TdTomato, allowing functional mRNA delivery to be analyzed at the cellular level by flow cytometry. Anti-CD117 LNPs or PBS containing Cre mRNA were injected into mice via the tail vein. Forty-eight hours after injection, mice were killed, bones (femurs and tibias) were harvested and processed into single cell suspensions for flow cytometric analysis. TdTomato fluorescence was evaluated in HSPC (defined as Lin-Sca1+c-Kit+) and long-acting hematopoietic stem cells (or LT-HSC, defined as Lin-Sca1+cKit+CD34-CD135-). LT-HSC is capable of self-renewal and is the most biologically significant cell when considering stem cell therapy.

[0808] Figure 4 In vivo Cre mRNA delivery in bone marrow HSPCs using the Ai14 mouse model was shown. LNPs conjugated to aCD117 showed very high levels (>90%) of mRNA delivery to bone marrow HSPCs and LT-HSCs. Figure 4 As shown, aCD117-LNPs were able to achieve high levels of mRNA delivery and Cre recombination (approximately 90%) to both HSPCs and LT-HSCs. In addition, approximately 50% of all bone marrow cells expressed TdTomato.

[0809] Example 4: In vivo RNA delivery to bone marrow CD117+ hematopoietic stem and progenitor cells 2 (DPG(C16) vs. DSG(C18))

[0810] To further evaluate the effect of PEG lipid alkyl chain length on aCD117-LNP delivery efficacy, we compared DPG-PEG (C16) with DMG-PEG (C14) and DSG-PEG (C18) formulations. LNPs were formulated with siRNA for CD45 and different PEG lipids. In addition, LNPs were fluorescently labeled with the lipophilic dye DiR to track the uptake differences of these formulations. LNPs were administered to mice via the tail vein at a dose of 1 mg / kg. 72 hours after administration, bone marrow was collected and then processed into single cell suspensions. Uptake and silencing in bone marrow HSPCs were analyzed by flow cytometry.

[0811] Results are shown in Figure 5 middle. Figure 5 Shown (left) LNP uptake in HSPCs or LT-HSCs correlates with longer alkyl chain lengths. DSG-PEG (C18) showed the highest level of uptake in the target cell population. (right) Functional gene silencing levels in HSPCs at different alkyl chain lengths.

[0812] Table 5: Formulations tested in mice

[0813]

[0814] Example 5: In vivo promotion of ionizable lipids

[0815] In order to test the scalability of this anti-CD117 lipid nanoparticle in vivo, various ionizable lipids were formulated into LNPs containing siRNA for CD45 and then conjugated with anti-mouse CD117 antibodies. LNPs were administered to mice via the tail vein at a dose of 1 mg / kg. 72 hours after administration, bone marrow was collected and then processed into a single cell suspension. CD45 silencing in bone marrow HSPC was analyzed by flow cytometry. The preparations shown in Table 6 were prepared and tested.

[0816] Results are shown in Figure 7 middle. Figure 7 Functional knockdown of CD45 in HSPC is shown. LNPs were injected at a lower dose of 0.3 mg / kg RNA to evaluate the effect of ligand density on RNA delivery. Both uptake and silencing show the optimal ligand density for RNA delivery using anti-CD117 conjugated lipid nanoparticles. From the data, it can be shown that the conjugation of anti-CD117 results in the effective delivery of all tested ionizable lipids in vivo.

[0817] Table 6: Formulations tested in mice

[0818]

[0819] Example 6: In vivo (or in vitro) antibody density optimization

[0820] In order to optimize the antibody density on the LNP surface, Ab-LNP (Table 7) was prepared with various molar ratios of maleimide and antibody during conjugation. Using more antibody during the conjugation reaction will result in more Ab on the LNP surface. In addition, Ab-LNP was also labeled with lipophilic dye DiR to track the uptake of these nanoparticles in vivo. These fluorescently labeled LNPs containing siRNA for CD45 were injected intravenously to mice at 0.3 mg / kg. 72 hours after injection, bone marrow was collected and both uptake and CD45 knockdown in HSPC (Lin-Sca1+cKit+ cells) were evaluated by flow cytometry.

[0821] Results are shown in Figure 7 middle. Figure 7 Shown (left) uptake in HSPC (as measured by % of LSK cells of DiR+). (right) Functional knockdown of CD45 in HSPC. LNPs were injected at a lower dose of 0.3 mg / kg RNA to evaluate the effect of ligand density on RNA delivery. Both uptake and silencing show the optimal ligand density for RNA delivery using anti-CD117 conjugated lipid nanoparticles.

[0822] Table 7: Formulations tested in mice

[0823] LNP# Ionizable lipids Phospholipids cholesterol PEG-lipid 1 PEG-lipid 2 LNP27 ALC-0315 DSPC cholesterol DSG-PEG2000 DSPE-PEG2000-Maleimide LNP28 ALC-0315 DSPC cholesterol DSG-PEG2000 DSPE-PEG2000-Maleimide LNP29 ALC-0315 DSPC cholesterol DSG-PEG2000 DSPE-PEG2000-Maleimide

[0824] LNP# Composition (mol%) Antibody Maleimide:Ab Ratio LNP27 50:10:38:1.5:0.5 Anti-mouse CD117 1:100 LNP28 50:10:38:1.5:0.5 Anti-mouse CD117 1:20 LNP29 50:10:38:1.5:0.5 Anti-mouse CD117 1:10

[0825] Using the optimal ligand density, the in vivo dose response of these Ab-LNPs was evaluated by administering 0.1 mg / kg, 0.3 mg / kg and 1 mg / kg doses to mice. 72 hours after LNP administration, mice were killed, bone marrow was collected, and processed into single cell suspensions. For dose curve analysis, nanoparticle uptake and silencing were evaluated in various cell populations (including HSPC, LT-HSC, mature immune cells and CD117- cells).

[0826] Results are shown in Figure 8 middle. Figure 8 Shown (top) LNP uptake in various cell populations of the bone marrow (as determined by % DiR positive). There was a dose-dependent uptake in HSPCs and LT-HSCs. In addition, LNPs mixed with free Ab (unconjugated) and isotype control LNPs (Iso-LNPs) did not show any uptake by HSPCs or LT-HSCs, and (bottom) dose response of functional siCD45 knockdown using Ab-LNP formulations.

[0827] Example 7: In vivo RNA delivery to murine bone marrow HSPCs using various antibodies

[0828] We also wanted to investigate the use of other antibodies that could potentially be used for in vivo delivery to HSPCs. We selected a small panel of other receptors expressed on HSPCs (CD49d, CD44, and IL-6R) and conjugated LNPs to antibodies against these receptors. In addition, we investigated another clone of CD117 (clone ACK2). Of the targets screened, only CD117 was effective for LNP uptake and RNA delivery ( Fig. 9 , left). Other antibodies may not be suitable for targeted delivery to HSPCs due to factors such as the clone of the antibody used or receptor-dependent factors (such as expression level, internalization rate, or off-target tissue expression level that may serve as antigen sinking). Interestingly, CD117 demonstrated clonal differences in Ab-LNP performance, indicating that the selection of antibodies against specific cell targets greatly affects its effectiveness in targeted delivery using antibody-modified lipid nanoparticles. Clone 2B8 is a non-antagonistic clone, while clone ACK2 is reported to be antagonistic; however, in the case of Ab-LNPs conjugated to clones or any other antibodies, no depletion of bone marrow HSPCs was observed after administration of Ab-LNPs ( Fig. 9 ,right).

[0829] Example 8: In vitro RNA delivery to human primary HSPCs using non-antagonistic antibodies

[0830] We formulated LNPs with anti-human CD117 antibody clone LMJ729 (non-antagonistic antibody) and assessed cell viability in vitro using primary human bone marrow CD34+ cells. Fig.10

[0831] Table 8: LNP formulations encapsulating Cas9 mRNA and sgRNA targeting the B2M gene

[0832]

[0833] Example 9: In vitro RNA delivery to human primary HSPCs using various receptor-antibody combinations

[0834] To investigate whether other antibodies could potentially be used for HSPC delivery, another panel of antibodies was tested. Firefly luciferase mRNA was encapsulated into LNPs conjugated with anti-human CD117 clone 104D2 (non-antagonist), anti-human CD184 (CXCR4) clone 12G5, anti-human CD105 (endoglin) clone 43A3, anti-human CD34 clone 581, or their isotype controls as non-targeting controls. TM The luminescence values ​​were quantified by luciferase assay system (Promega), and the transfection efficiency of these LNPs was tested in vitro using human primary bone marrow CD34+ HSPCs.

[0835] like Fig.11 As shown, all targeted LNP formulations tested showed luciferase expression in vitro at a dose of 100 ng mRNA per 5,000 cells, whereas LNPs conjugated to an isotype control were unable to effectively transfect these cells.

[0836] Table 9: LNP formulations encapsulating firefly luciferase mRNA

[0837]

[0838] Example 10: In vitro RNA delivery to mouse primary bone marrow or mesenchymal stem cells (MSCs) using anti-CD105-LNPs

[0839] To test that our targeted LNPs can also be used for other stem cells, the RNA delivery efficiency of anti-CD105 LNPs was also tested in vitro in primary murine bone marrow mesenchymal stem cells (MSCs). siRNA against murine integrin β1 (Itgb1) was encapsulated in anti-CD105 antibody-modified LNPs and unmodified LNPs (as described in **). Frozen primary murine bone marrow MSCs were obtained from CellBiologics and cultured in RPMI medium containing 10% FBS. Then, MSCs were transferred to 96-well plates at a density of 10,000-15,000 cells / well, and the medium was replaced with 90 μl of serum-free RPMI medium. MSCs were transfected with 10 μl of LNP solution encapsulating Itgb1 siRNA duplexes (final concentration of 100 nM). Four hours after transfection, the cell culture medium was replaced with serum-containing medium. After 24 hours of incubation, the treated cells were harvested and the remaining Itgb1 mRNA levels were measured by RT-qPCR under each condition. The results are shown in Fig.12 middle.

[0840] Table 10: LNP formulations encapsulating siRNA targeting Itgb1

[0841]

[0842] Example 11: In vitro RNA delivery to primary human HSPCs using various ionizable lipids (2)

[0843] We formulated targeted Ab-LNPs with firefly luciferase mRNA with various ionizable lipids and used Steady-Glo TM Luminescence was assessed 24 h after transfection using the luciferase assay system (Promega). Fig.13 As shown, all formulations tested showed luminescence higher than the PBS control at a dose of 100 ng mRNA per 5,000 cells.

[0844] Table 11: LNP formulations encapsulating firefly luciferase mRNA

[0845]

[0846] Example 12: In vivo Cre recombinase-mediated gene editing of bone marrow HSPCs leads to long-term myeloid and lymphoid genetic transformation

[0847] Next, we turned to evaluate the delivery efficacy of anti-CD117 LNPs containing mRNA. To this end, we utilized a transgenic Ai14 mouse model that contains a LoxP-flanked stop cassette that prevents transcription of the fluorescent protein TdTomato. Upon Cre recombination (via delivery of Cre mRNA), this stop cassette is excised and the cells then constitutively express TdTomato ( Fig.14 a). Then, we determined the gene editing levels in HSPCs after treatment with our Ab-LNPs using flow cytometry. In addition, we followed mature immune cells in peripheral blood long term after LNP administration to observe TdTomato expression levels in edited progeny at 2, 4, 6, 8, and 14 weeks ( Fig.14 b).

[0848] We first implemented the -1 Dose response of anti-CD117 LNP (anti-mouse CD117 clone 2B8, ALC-0315) at 0.3 mg kg -1 With a single dose of (6 μg) mRNA, we observed efficient delivery of approximately 75% TdTomato+ cells in both HSPC and LT-HSC populations 48 hours after administration. When the dose was increased to 20 μg of mRNA, nearly all (approximately 90%) HSPCs and LT-HSCs were transfected ( Fig.14 d) At 1 mg kg - 1 Unconjugated LNPs at Cre mRNA doses showed about 25% TdTomato expression, indicating that conventional non-targeted LNP formulations have the ability to transfect HSPCs at low levels. By incorporating HSPC targeting ligands, the delivery level is greatly improved. As shown by analyzing peripheral blood populations (erythrocytes, myeloid cells, B cells, and T cells), the stem cell properties of transfected HSPCs are maintained in both the short and long term. At week 2, CD11b+ myeloid cells composed of granulocytes and monocytes were already 90% TdTomato+. Since B and T cells are cells with a longer lifespan, the TdTomato expression levels of these cell types naturally lag behind those of myeloid cells at week 2 (B cells are about 32% and T cells are about 3.3%), increasing to about 70% TdTomato+B cells and about 50% TdTomato+T cells ( Fig.14 e). T cell subsets (CD4 and CD8) were analyzed at week 4 after LNP administration, when the level of TdTomato expression in T cells became more evident. At each time point, the TdTomato population was higher in CD4 T cells compared to CD8 T cells ( Fig.14 f). At week 14, erythrocytes also showed almost 100% TdTomato expression ( Fig.14 g). Overall, nearly all HSPCs were transfected, which in turn produced high levels of corrected progeny in all immune cell populations analyzed.

[0849] Example 13: PEG lipids

[0850] Ab-LNP formulations containing Cre mRNA and different PEG-lipids were administered at 0.3 mg kg -1 The dose of 100 μg / mL was injected into Ai14 mice. 48 hours after injection, the mice were sacrificed and the right hind limbs were collected for flow cytometry analysis. TdTomato expression was evaluated in bone marrow LSK cells. The results are shown in Fig.15 The data were analyzed by one-way ANOVA and Tukey's multiple comparison test (*P<0.05, **P<0.01).

[0851] Example 14: Dexamethasone pretreatment improves clinical signs after LNP administration

[0852] Mice were pretreated with PBS or 9 mg / kg dexamethasone (intraperitoneal injection) 1 hour before administration of lipid nanoparticles (LNP). Clinical examination was performed 6 hours after LNP administration. The results are shown in Table 13.

[0853] Table 12: LNP formulations tested in dexamethasone pre-treated mice

[0854]

[0855] Table 13: Clinical examination 6 hours after LNP administration (1 mg / kg mRNA encoding firefly luciferase)

[0856] < / mrna> < / sirna>

Claims

1. A lipid composition comprising (A) a therapeutic agent and (B) a lipid nanoparticle conjugated to a targeting molecule, wherein the lipid nanoparticles comprise ionizable lipids, and The targeting molecule specifically binds to a marker of hematopoietic stem / progenitor cells or mesenchymal stem cells.

2. A lipid composition according to claim 1, wherein the lipid nanoparticle comprises a PEG-lipid conjugated to the targeting molecule.

3. A lipid composition according to claim 1, wherein the ionizable lipid has at least one ionizable amino group and at least one biodegradable group, and wherein the biodegradable group is represented by -O(CO)O-, -O(CO)-, -(CO)O- or SS.

4. The lipid composition according to claim 1, wherein the ionizable lipid is a compound represented by formula (4): [Chemical formula 1] Where X represents NR 1 -or-O-, R 1 represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a 21 -L 1 -R 22 - represents a group, R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 means -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [Chemical formula 2] R 22 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms, R 2 and R 3 Each independently represents a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a 31 -L 2 -R 32 - represents a group, R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 means -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [Chemical formula 3] R 32 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms, R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms, R 4 With R 5 , R 10 With R 5 , R 5 With R 12 , R 4 With R 6 , R 5 With R 6 , R 6 With R 7 , R 6 With R 10 , R 12 With R 7 , and R 7 With R 8 Any one or more groups in may be linked together to form a 4- to 7-membered ring which may contain an O atom, The substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms represents a hydroxyl group, a carboxyl group, a NR 45 R 46 amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 , or -OR 44 A group represented by 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group represent alkyl groups having 1 to 18 carbon atoms, hydroxyl groups, carboxyl groups, groups consisting of -NR 45 R 46 An amino group represented by -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 , or -OR 44 represents a group, and R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, a, b, c and d each independently represent an integer of 0 to 3, wherein a+b is 1 or more, and c+d is 1 or more.

5. The lipid composition according to claim 1, wherein the ionizable lipid is a compound represented by formula (1): [Chemical formula 4] In the formula, R 1 and R 2 Each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, wherein R 1 , R 2 and R 3 The hydrocarbon group represented by may be one or more selected from -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 AND-OR 56 Substituents are substituted, R 4 represents a hydrocarbon group having 1 to 8 carbon atoms, R 5 and R 6 Each independently represents a hydrocarbon group having 1 to 8 carbon atoms or -R 8 -L 1 -R 9 , but R 5 and R 6 Except for the case where all of them are hydrocarbon groups having 1 to 8 carbon atoms, R 7 Indicates -R 10 -L 2 -R 11 -L 3 -R 12 , R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 53 , R 54 , R 55 and R 56 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, By R 53 , R 54 , R 55 and R 56 The hydrocarbon group represented by may be replaced by an aryl group having 6 to 20 carbon atoms or -SR 58 The above aromatic groups having 6 to 20 carbon atoms may be substituted by -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 、-OR 56 , or -(hydrocarbon group having 1 to 12 carbon atoms)-R 57 replace, R 58 represents a hydrocarbon group having 1 to 12 carbon atoms, and R 57 Indicates -OH, COOH, -NR 61 R 62 、-OC(O)OR 63 、-C(O)OR 64 、-OC(O)-R 65 , or -OR 66 , R 61 and R 62 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 63 , R 64 , R 65 and R 66 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, By R 63 , R 64 , R 65 and R 66 The hydrocarbon group represented by may be replaced by an aryl group having 6 to 20 carbon atoms or -SR 68 The above aromatic groups having 6 to 20 carbon atoms may be substituted by -OH, COOH, -NR 61 R 62 、-OC(O)OR 63 、-C(O)OR 64 、-OC(O)-R 65 、-OR 66 , or -(hydrocarbon group having 1 to 12 carbon atoms)-R 67 replace, R 68 represents a hydrocarbon group having 1 to 12 carbon atoms, and L 1 , L 2 and L 3 each independently represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-, R 8 represents a hydrocarbon group having 1 to 12 carbon atoms, R 9 represents a hydrocarbon group having 1 to 24 carbon atoms, R 10 represents a hydrocarbon group having 1 to 8 carbon atoms, R 11 represents a hydrocarbon group having 1 to 24 carbon atoms, R 12 represents a hydrocarbon group having 1 to 24 carbon atoms, By R 9 and R 12 The hydrocarbon group represented by 53 、-C(O)OR 54 、-OC(O)-R 55 , or -SR 58 Substitution, where R 53 , R 54 , R 55 and R 58 is defined above, and By R 11 The hydrocarbon group represented by -OC(O)OR 53 、-C(O)OR 54 , or -OC(O)-R 55 Substitution, where R 53 , R 54 and R 55 The definition of is as above.

6. The lipid composition according to claim 1, wherein the ionizable lipid is a compound represented by the following formula (5): [Chemical formula 5] Where R 51 and R 52 Each independently represents a hydrocarbon group having 1 to 21 carbon atoms, which may have a substituent A, The substituent A represents a hydroxyl group, or -G 20 -CH(R 55 )(R 56 )、-N(R 58 )(R 59 ) or -G 20 -R 60 The group represented by G 20 represents -O(CO)-, or -(CO)O-, R 55 and R 56 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R 58 and R 59 Each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms, which may have a substituent B, The substituent B is -N(R 61 )(R 62 ), R 61 and R 62 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R 60 represents a hydrocarbon group having 1 to 18 carbon atoms, L 10 represents a hydrocarbon group having 1 to 18 carbon atoms, G 30 Indication-S-(CO)-NR 64 , R 64 Indicated by -L 30 -G 20 -CH(R 55 )(R 56 ) represents a group, a represents 0 or 1, L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G 10 Represents -O(CO)-, -(CO)O-, -O(CO)O-, or -N(C(O)R 63 )-, R 63 represents a hydrocarbon group having 1 to 18 carbon atoms, L 20 represents a hydrocarbon group having 1 to 6 carbon atoms, b represents 0 or 1, R 53 , R 54 and R 57 Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms, which may have a substituent C, The substituent C represents -(CO)OR 65 or -O(CO)-R 65 The group represented by R 65 represents a hydrocarbon group having 1 to 18 carbon atoms or -L 40 -CH(R 66 )(R 67 ) represents a group, L 40 represents a hydrocarbon group having 1 to 6 carbon atoms, R 66 and R 67 represents a hydrocarbon group or an alkoxy group having 1 to 10 carbon atoms.

7. The lipid composition according to claim 1, wherein the ionizable lipid is at least one selected from the compounds represented by the following formula: [Chemical formula 6] MC3 L-319 ALC-0315 SM-102 Lipid 5 Lipid 29 ATX-100 Lipid A9 Lp01 TCL053 Compound 1 Compound 2 Compound 3 Compound 4 cKK-E12 C12-200 306Oi10 93-O17S YSK05 CL4H6 ssPalmOPhe Compound 5 Compound 6 Compound 7 Compound 8 Compound 9 8. The lipid composition of claim 1, wherein the lipid nanoparticles comprise sterols.

9. The lipid composition of claim 1, wherein the lipid nanoparticles comprise phospholipids.

10. The lipid composition of claim 1, wherein the therapeutic agent comprises a polynucleotide.

11. The lipid composition of claim 9, wherein the polynucleotide is DNA or RNA.

12. The lipid composition of claim 9, wherein the polynucleotide is mRNA, sgRNA or siRNA.

13. The lipid composition of claim 1, wherein the targeting molecule is at least one selected from a nucleic acid, a peptide, an antibody and a small molecule.

14. The lipid composition of claim 12, wherein the targeting molecule is an antibody.

15. The lipid composition of claim 1, wherein the marker of hematopoietic stem / progenitor cells is CD34, CD105, CD117, or CD184 (CXCR4).

16. The lipid composition of claim 1, wherein the marker of hematopoietic stem / progenitor cells is CD117.

17. The lipid composition of claim 1, wherein the marker of mesenchymal stem cells is CD105.

18. A method of delivering a therapeutic agent to cells expressing markers of hematopoietic stem / progenitor cells or mesenchymal stem cells, comprising administering the lipid composition of claim 1 to an individual.

19. The method of claim 18, further comprising administering to the individual a therapeutically effective amount of an inflammation reducing agent prior to administering to the individual the lipid composition of claim 1.

20. The method of claim 19, wherein the inflammation reducing agent is selected from (a) corticosteroids, (b) antihistamines, (c) acetaminophen, (d) NSAIDS, (e) kinase inhibitors having CD117 kinase activity inhibitory activity, or (f) other immunosuppressants.

21. A method for reducing adverse reactions associated with administration of anti-CD117 antibody-modified LNPs, the method comprising administering to a subject a therapeutically effective amount of an inflammation reducing agent prior to administration of the CD117 antibody-modified LNPs.

22. The method of claim 21, wherein the inflammation reducing agent is selected from (a) corticosteroids, (b) antihistamines, (c) acetaminophen, (d) NSAIDS, (e) kinase inhibitors having CD117 kinase activity inhibitory activity, or (f) other immunosuppressants.

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