Lipid Compositions and Methods for Delivering Therapeutic Agents - Patent application

Abstract

The present invention provides a lipid composition capable of delivering nucleic acids such as RNA to hematopoietic stem / progenitor cells or mesenchymal stem cells, and a method for delivering a therapeutic agent to cells using the lipid composition. The present invention provides a lipid composition comprising (A) a therapeutic agent and (B) lipid nanoparticles bound to a targeting molecule, wherein the therapeutic agent is encapsulated in the lipid nanoparticles, the lipid nanoparticles comprise an ionizable lipid, and the targeting molecule specifically binds to a marker for hematopoietic stem / progenitor cells or mesenchymal stem cells.

Description

[Technical Field]

[0001] The present invention relates to lipid compositions comprising a therapeutic agent and lipid nanoparticles (LNPs), and methods of delivering a therapeutic agent to cells using said lipid compositions. [Background technology]

[0002] Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are important targets for gene therapy. Current gene therapy protocols involve the collection of HSCs or MSCs from donors / patients, in vitro culture, transduction with retroviral vectors, and retransplantation into bone marrow-conditioned patients. In addition to technical complexity and production costs, drawbacks of this approach include the need for culture in the presence of multiple cytokines, which may affect HSC pluripotency and engraftment. Furthermore, the need for myeloablative treatments, such as total body irradiation (TBI) or lethal chemotherapy containing busulfan / cyclophosphamide (BU / CY), in patients with non-malignant diseases poses additional risks.

[0003] Furthermore, from the perspective of allogeneic transplantation, donor HSCs or MSCs must avoid immune rejection by the recipient. Recognition of HLA incompatibility by the immune system is a major barrier to allogeneic hematopoietic stem cell transplantation. Therefore, although an HLA-genotype-identical sibling donor is the gold standard for transplantation purposes, only 30% of patients have such a donor. For the remaining 70% of patients, alternative sources of stem cells are matched unrelated adult volunteer donors, half-matched donors, or umbilical cord blood units.

[0004] On the other hand, CD117 is known to be expressed in hematopoietic stem cells. Non-Patent Document 1 describes the usefulness of anti-CD117 antibody-modified nanoparticles and the combination of hematopoietic stem / progenitor cell mobilization and anti-CD117 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. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. WO2019 / 213308 [Patent Document 2] International Publication No. WO2015 / 153805 [Non-patent literature]

[0006] [Non-Patent Document 1] Paula Cannon et al., HUMAN GENE THERAPY, VOLUME 32, NUMBERS 1 and 2 (pages 31-43) DOI: 10.1089 / hum.2020.263 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention addresses these needs 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. [Means for solving the problem]

[0008] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that a therapeutic agent can be efficiently delivered to hematopoietic stem / progenitor cells by administering lipid nanoparticles to cells, which are conjugated with a targeting molecule that specifically binds to a marker for hematopoietic stem / progenitor cells and which encapsulate a therapeutic agent. The present invention was completed based on the above findings.

[0009] According to the present invention, the following inventions are provided. <1> 1. A lipid composition comprising: (A) a therapeutic agent; and (B) a lipid nanoparticle conjugated to a targeting molecule, the therapeutic agent is encapsulated in lipid nanoparticles; the lipid nanoparticles comprise an ionizable lipid; A lipid composition, wherein the targeting molecule specifically binds to a marker of hematopoietic stem / progenitor cells or mesenchymal stem cells. <2> The lipid nanoparticles comprise PEG lipids conjugated to a targeting molecule. <1> The lipid composition described in <3> The ionizable lipid has at least one ionizable amino group and at least one biodegradable group, and the biodegradable group is represented by -O(CO)O-, -O(CO)-, -(CO)O-, or SS; <1> The lipid composition described in <4> The ionizable lipid is a compound represented by the following formula (4): <1> The lipid composition described in [ka] During the ceremony, X is -NR 1 - or -O-, R 1 is a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R 21 -L 1 -R 22 -, and R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [ka] indicates R 22 represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R 2 and R 3 are each independently a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R 31 -L 2 -R 32 -, and R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [ka] indicates R 32 represents a divalent linking group, 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 and R 5 , R 10 and R 5 , R 5 and R 12 , R 4 and R 6 , R 5 and R 6 , R 6 and R 7 , R 6 and R 10 , R 12 and R 7 , and R 7 and R 8 any one or more pairs of may be linked to each other to form a 4- to 7-membered ring optionally containing an O atom, The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, -NR 45 R 46an amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 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, The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, -NR 45 R 46 Amino group represented by -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 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, provided that a+b is 1 or more, and c+d is 1 or more. <5> The ionizable lipid is a compound represented by formula (1): <1> The lipid composition described in [ka] During the ceremony, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, and R 1 , R 2 and R 3 The hydrocarbon group represented by is -OH, COOH, -NR 51 R 52 , -OC(O)OR53 , -C(O)OR 54 , -OC(O)-R 55 , and OR 56 and optionally substituted with one or more substituents selected from R 4 represents a hydrocarbon group having 1 to 8 carbon atoms, R 5 and R 6 are each independently a hydrocarbon group having 1 to 8 carbon atoms, or R 8 -L 1 -R 9 where R 5 and R 6 are both hydrocarbon groups having 1 to 8 carbon atoms, R 7 -R 10 -L 2 -R 11 -L 3 -R 12 indicates, 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, R 53 ,R 54 ,R 55 , and R 56 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 58 may be substituted with The above aryl group having 6 to 20 carbon atoms includes -OH, -COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 57 may be substituted with R 58 represents a hydrocarbon group having 1 to 12 carbon atoms, R57 -OH, COOH, -NR 61 R 62 , -OC(O)OR 63 , -C(O)OR 64 , -OC(O)-R 65 , or -OR 66 Shows. 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, R 63 ,R 64 ,R 65 , and R 66 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 68 may be substituted with The above aryl group having 6 to 20 carbon atoms includes -OH, -COOH, -NR 61 R 62 , -OC(O)OR 63 , -C(O)OR 64 , -OC(O)-R 65 , -OR 66 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 67 may be substituted with R 68 represents a hydrocarbon group having 1 to 12 carbon atoms, 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, R12 represents a hydrocarbon group having 1 to 24 carbon atoms, R 9 , and R 12 The hydrocarbon group represented by is an aryl group, -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or SR 58 and R 53 , R 54 , R 55 , and R 58 is defined as above, R 11 The hydrocarbon group represented by is -OC(O)OR 53 , -C(O)OR 54 , or -OC(O)-R 55 and R 53 , R 54 , and R 55 The definition of is as above. <6> The ionizable lipid is a compound represented by formula (5): <1> The lipid composition described in [ka] In the formula, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, The above substituent A is a hydroxyl group, -G 20 -CH(R 55 )(R 56 ), -N(R 58 )(R 59 ), or -G 20 -R 60 represents a 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 59each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have a substituent B, Substituent B is -N(R 61 )(R 62 ) and 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 is -S-(CO)-NR 64 - indicates R 64 L 30 -G 20 -CH(R 55 )(R 56 ) represents a group represented by a represents 0 or 1, L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G 10 is -O(CO)-, -(CO)O-, -O(CO)O- or -N(C(O)R 63 )- indicates 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 is —(CO)O —R 65 , or -O(CO) -R 65 represents a group represented by R 65 is a hydrocarbon group having 1 to 18 carbon atoms or -L 40 -CH(R 66 )(R 67) represents a group represented by L 40 represents a hydrocarbon group having 1 to 6 carbon atoms, R 66 and R 67 represents a hydrocarbon group or alkoxy group having 1 to 10 carbon atoms. <7> The ionizable lipid is at least one selected from the compounds represented by the following formula: <1> The lipid composition described in [ka] JPEG2025535815000008.jpg217170JPEG2025535815000009.jpg230170JPEG20255358150 00010.jpg243170JPEG2025535815000011.jpg244170JPEG2025535815000012.jpg237170 <8> the lipid nanoparticles contain a sterol; <1> The lipid composition described in <9> the lipid nanoparticles comprise phospholipids; <1> The lipid composition described in <10> the therapeutic agent comprises a polynucleotide; <1> The lipid composition described in <11> the polynucleotide is DNA or RNA; <9> The lipid composition described in <12> the polynucleotide is an mRNA, sgRNA, or siRNA; <9> The lipid composition described in <13> The targeting molecule is at least one selected from a nucleic acid, a peptide, an antibody, and a small molecule; <1> The lipid composition described in <14> the targeting molecule is an antibody; <12> The lipid composition described in <15> Hematopoietic stem / progenitor cell markers are CD34, CD105, CD117, or CD184 (CXCR4); <1> The lipid composition described in <16> The marker for hematopoietic stem / progenitor cells is CD117. <1> The lipid composition described in <17> The marker for mesenchymal stem cells is CD105. <1> The lipid composition described in <18> <1> A method for delivering a therapeutic agent to cells expressing markers for hematopoietic stem / progenitor cells or mesenchymal stem cells, comprising administering to a subject the lipid composition described in claim 1. <19> <1> 20. The method of claim 18, further comprising administering to the subject a therapeutically effective amount of an inflammation-reducing agent prior to administering to the subject the lipid composition of claim 18. <20> 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. <19> The method described below. <21> A method for reducing side effects associated with administration of anti-CD117 antibody-modified LNPs, comprising administering to a subject a therapeutically effective amount of an inflammation-reducing agent prior to administering the CD117 antibody-modified LNPs. <22> 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> The method described below. Also provided herein are methods for delivering therapeutic agents to hematopoietic stem / progenitor cells (HSPCs) or mesenchymal stem cells in culture. [Effects of the Invention]

[0010] The lipid compositions and methods of the present invention enable efficient delivery of therapeutic agents to hematopoietic stem / progenitor cells or mesenchymal stem cells. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows the in vitro evaluation of CD45 gene silencing on EML cells using aCD117-conjugated LNPs. [Figure 2] FIG. 2 shows knockdown of CD45 by interaction of ionizable lipids with the CD117 receptor. [Figure 3] FIG. 3 shows CD45 expression levels in bone marrow LSK cells quantified by flow cytometry. [Figure 4] Figure 4 shows in vivo Cre mRNA delivery to bone marrow HSPCs using the Ai14 mouse model. [Figure 5] Figure 5 shows LNP uptake in HSPCs or LT-HSCs (left) and the level of functional gene silencing in HSPCs with different alkyl chain lengths (right). [Figure 6] FIG. 6 shows uptake in HSPCs measured as % of LSK cells that are DiR+ (left) and functional knockdown of CD45 in HSPCs (right). [Figure 7] Figure 7 shows LNP uptake in various cell populations of bone marrow as measured by DiR positivity (top row), and the dose response of functional siCD45 knockdown by Ab-LNP formulations (bottom row). [Figure 8] FIG. 8 shows (top) LNP uptake in various cell populations of bone marrow as measured by DiR positivity, and (bottom) dose response of functional siCD45 knockdown by Ab-LNP formulations. [Figure 9] Figure 9 (left) shows the screening of surrogate markers for in vivo HSPC delivery at a dose of 1 mg kg-1 siCD45 (n = 3 mice in the anti-CD49d condition, n = 2 mice in all other conditions, one-way ANOVA with Dunnett's multiple comparison test). Data are expressed as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001). (right) shows that treatment with ligand-modified LNPs at a dose of 1 mg kg-1 siCD45 does not result in HSC depletion or clearance. Statistics were calculated by one-way ANOVA with Dunnett's multiple comparison test against the PBS control group (n = 3 mice in the anti-CD49 group, n = 2 mice in all other groups). Significant differences were defined as P < 0.05. [Figure 10] FIG. 10 shows in vitro RNA delivery to human primary HSPCs using a non-antagonistic antibody (Clone LMJ729). [Figure 11]FIG. 11 shows luciferase expression in human primary bone marrow CD34+ cells, quantified using the SteadyGlo Luciferase Assay System after treatment with LNP or PBS at a dose of 100 ng / 5,000 cells for 24 hours. [Figure 12] Figure 12 shows Itgb1 mRNA levels in primary mouse bone marrow mesenchymal stem cells quantified using RT-qPCR 24 hours after LNP treatment with 100 nM siRNA. Residual Itgb1 mRNA levels were normalized to the housekeeping gene B2m. [Figure 13] FIG. 13 shows luciferase expression in human primary bone marrow CD34+ cells, quantified using the SteadyGlo Luciferase Assay System after treatment with LNP or PBS at a dose of 100 ng / 5,000 cells for 24 hours. [Figure 14] Figure 14 shows that anti-CD117 LNPs encapsulating Cre mRNA exhibit high levels of editing in vivo. (a) Schematic diagram of the Ai14 transgenic mouse LoxP-flanked STOP cassette, which blocks TdTomato transcription. Upon delivery of Cre recombinase via Cre mRNA, the STOP cassette is removed, and cells express TdTomato. (b) Timeline of the experimental workflow for bone marrow and blood analysis. Panels a and b were generated using BioRender.com. (c) Representative flow cytometry scatter plots of TdTomato expression in LT-HSCs with varying doses of CremRNA. (d) Quantification of the dose-response in HSPCs and LT-HSCs (n = 4 mice in the 1 mg kg-1 Ab-LNP group, n = 3 mice in the other groups). Statistics were performed using two-way ANOVA with 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). [Figure 15]FIG. 15 shows the effect of PEG lipid alkyl length on mRNA delivery to HSPCs in the bone marrow. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. In this specification, the symbol "to" indicates a range that includes the numerical values ​​before and after it as the minimum and maximum values, respectively. The present invention provides a lipid composition comprising (A) a therapeutic agent and (B) a lipid nanoparticle conjugated to a targeting molecule, the therapeutic agent is encapsulated in lipid nanoparticles; the lipid nanoparticles comprise an ionizable lipid; The present invention relates to a lipid composition, wherein the targeting molecule is a molecule that specifically binds to a marker for hematopoietic stem / progenitor cells or mesenchymal stem cells.

[0013] In one embodiment, the lipid compositions of the present invention are used in combination with an inflammation-reducing agent selected from (a) corticosteroids, (b) antihistamines, (c) acetaminophen, (d) NSAIDS, (e) kinase inhibitors that inhibit CD117 kinase activity, or (f) other immunosuppressants. The present invention further relates to a method for delivering a therapeutic agent to cells expressing a marker for hematopoietic stem / progenitor cells or mesenchymal stem cells, comprising administering to a subject the lipid composition of the present invention. Preferably, the method for delivering a therapeutic agent to cells according to the present invention may further comprise administering to the subject a therapeutically effective amount of an inflammation-reducing agent prior to administering to the subject the lipid composition of the present invention. In the present invention, lipid nanoparticles conjugated to targeting molecules are used. The present invention further relates to a method for reducing side effects associated with administration of anti-CD117 antibody-modified LNPs, comprising administering a therapeutically effective amount of an inflammation-reducing agent to a subject prior to administering the CD117 antibody-modified LNPs. 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.

[0014] <Targeting molecule> The targeting molecule is a molecule that specifically binds to a marker for hematopoietic stem / progenitor cells or mesenchymal stem cells. By using a molecule that specifically binds to a marker for hematopoietic stem / progenitor cells or mesenchymal stem cells as a targeting molecule, it becomes possible to efficiently deliver a therapeutic agent encapsulated in a lipid nanoparticle to hematopoietic stem / progenitor cells or mesenchymal stem cells. The targeting molecule is not particularly limited as long as it specifically binds to a marker of hematopoietic stem / progenitor cells or mesenchymal stem cells, and examples thereof include molecules that bind to the cell surface or the extracellular matrix. Examples of molecules that bind to the cell surface include molecules that bind to membrane proteins such as receptors or channels exposed on the cell surface. It is preferable to use a molecule that binds to a marker of hematopoietic stem / progenitor cells or mesenchymal stem cells. It is preferable to use a non-antagonistic molecule as the targeting molecule. For example, at least one selected from carbohydrates, nucleic acids, peptides, proteins, antibodies, antibody fragments, antigen-binding domains, immunoglobulins or immunoglobulin fragments, and small molecules can be used as the targeting molecule. The targeting molecule is preferably an antibody.

[0015] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen or epitope. An antibody may be a natural or recombinantly derived intact immunoglobulin or an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. Antibodies of the present invention may exist in various 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 portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, VHH, Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0016] Markers of hematopoietic stem / progenitor cells 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), and CD11 4 (CSF3 receptor, G-CSF receptor), CD115 (CSF1 receptor), CD116 (GM-CSF receptor), CD117 (c-Kit / SCF receptor), CD121a (IL-1R), CD1 23(IL-3R), CD124(IL-4R), CD125(IL-5Rα), CD126(IL-6R), CD128(IL-8Rα), CD129(IL-9R), CD133(Prominin Examples of targeting molecules include CD117, CD118, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126, CD127, CD128, CD129 ... Hematopoietic stem / progenitor cells express c-Kit (CD117, a dimeric transmembrane receptor tyrosine kinase). Signaling involving CD117 is essential for many hematopoietic stem / progenitor cell functions, including homing, proliferation, adhesion, maintenance, and survival. CD117 is also expressed on other cell types, including cancer cells.

[0017] An example of a marker for mesenchymal stem cells is CD105. Examples of antibody clones for each marker are shown below. <Mouse CD117> Antagonistic: ACK2 (BioXCell) Non-antagonistic: 2B8 (BioXCell) <Human CD117> Antagonistic: Briquilimab / AMG191 / JSP191 (Amgen or Jasper therapeutics, WO2007 / 127317) Barzolvolimab (Celldex, WO2022159737) LMS359, GZQ167, LMJ451 (Clin Cancer Res(2018)24(17):4297-4308. Novatris) AB85 / MGTA117 (Magenta therapeutics, WO2019084067, WO2020219748, WO2020219770 and WO2020219775) Non-antagonistic: 104D2 (Biolegend) DLY884, LPG166, LQS721, LMJ729, LPG167, (Clin Cancer Res(2018)24(17):4297-4308. Novartis) <Human CD184 (CXCR4)> Antagonistic: Ulocuplumab (Bristol Meyers Squibb) LY2510924 (Eli Lilly: peptide) LY2624587 (Eli Lilly) ALX-0651 (Sanofi / Ablynx: nanobody) PF-06747143 (Pfizer) <Human CD105> TRC105 (Tracon) <Human CD34> 8G12 (BD Biosciences) 581 (Biolegend) S20016E (Biolegend) <Human CD27> Agonistic (non-antagonistic): Boselolimab (Merck / Aduro) Varlilumab (Celldex) <Human CD133> C-Mab43(Monoclonal Antibodies in Immunodiagnosis and Immunotherapy.Oct2017.231-235.) HW350341.1(GenBank:HW350341.1)

[0018] The method of binding a targeting molecule to a lipid nanoparticle is not particularly limited, but it is preferable to bind the targeting molecule to any lipid component constituting the lipid nanoparticle. For example, when the lipid nanoparticle contains an ionizable lipid, a sterol, a neutral lipid (e.g., a phospholipid), or a lipid having a non-ionic hydrophilic polymer (e.g., a lipid bound with polyethylene glycol), it is preferable to bind the targeting molecule to any of the above lipid components. As an example, the targeting molecule can be bound to a lipid having a non-ionic hydrophilic polymer (e.g., a lipid bound with polyethylene glycol), but this is not particularly limited. The number of targeting molecules per lipid nanoparticle is not particularly limited, but generally, one or more targeting molecules per lipid nanoparticle is preferred. In one embodiment, the number of targeting molecules per lipid nanoparticle can be determined by measuring the concentration [A] of the lipid nanoparticle using a common method (e.g., microfluidic resistive pulse sensing (MRPS) method, tunable resistive pulse sensing (TRPS) method, nanoparticle tracking analysis (NTA), transmission electron microscope (TEM), etc.). The concentration [B] of the targeting molecule can be measured using a common method such as HPLC, BCA assay, Lowry assay, Bradford assay, etc. The number of targeting molecules per lipid nanoparticle can be calculated as [B] / [A]. The concentration [A] of the lipid nanoparticle can also be calculated from the volume average particle size of the lipid nanoparticle, the molecular volume of each component, and the molar ratio of each component.

[0019] <Sterol> The lipid nanoparticles preferably comprise a sterol. In the present invention, the inclusion of sterol can reduce membrane fluidity and provide a stabilizing effect on lipid particles. The sterol is not particularly limited, but examples thereof include cholesterol, phytosterols (sitosterol, stigmasterol, fucosterol, spinasterol, brassicasterol, etc.), ergosterol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, etc. Among these, cholesterol is preferred. The sterol content is preferably 5 mol% to 80 mol%, more preferably 10 mol% to 80 mol%, more preferably 10 mol% to 60 mol%, and even more preferably 30 mol% to 50 mol%, based on the total lipids.

[0020] <Ionizable lipids> In the present invention, ionizable lipids are used. The ionizable lipids may be lipids having at least one biodegradable group. The ionizable lipids may be lipids having at least one ionizable amino group and at least one biodegradable group. The ionizable lipids are pH-responsive cationic lipids. They are electrically neutral at physiological pH, such as in blood, and change to cations in an acidic environment, such as an endosome. Examples of the biodegradable group include groups represented by -O(CO)O-, -O(CO)-, and -(CO)O-.

[0021] <<Lipid represented by formula (4) or a salt thereof>> As the ionizable lipid, for example, a lipid represented by the following formula (4) or a salt thereof may be used. [ka] During the ceremony, X is -NR 1 - or -O-, R 1is a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R 21 -L 1 -R 22 -, and R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [ka] indicates R 22 represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R 2 and R 3 are each independently a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R 31 -L 2 -R 32 -, and R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [ka] indicates R 32 represents a divalent linking group, 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 and R 5 , R 10 and R 5 , R 5 and R 12 , R 4 and R 6 , R 5 and R 6 , R 6 and R 7 , R6 and R 10 , R 12 and R 7 , and R 7 and R 8 any one or more pairs of may be linked to each other to form a 4- to 7-membered ring optionally containing an O atom, The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, -NR 45 R 46 an amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 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, The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, -NR 45 R 46 Amino group represented by -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 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, provided that a+b is 1 or more, and c+d is 1 or more.

[0022] R 1 and R 2 and R 3The hydrocarbon group having 3 to 24 carbon atoms in the formula (I) is preferably an alkyl group, an alkenyl group, or an alkynyl group, and more preferably an alkyl group or an alkenyl group. The alkyl group having 6 to 24 carbon atoms and the alkyl group having 3 to 24 carbon atoms may be linear or branched, and may be chain-like or cyclic. The alkyl group having 6 to 24 carbon atoms is preferably an alkyl group having 6 to 20 carbon atoms, and more preferably an alkyl group having 3 to 24 carbon atoms. Specific examples 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), heptadecyl, octadecyl, nonadecyl, and icosyl. The alkenyl groups having 6 to 24 carbon atoms and the alkenyl groups having 3 to 24 carbon atoms may be linear or branched, and may be linear or cyclic. The alkenyl groups having 6 to 24 carbon atoms are preferably alkenyl groups having 6 to 20 carbon atoms, and the alkenyl groups having 3 to 24 carbon atoms are more preferably alkenyl groups having 6 to 20 carbon atoms. Specifically, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group, a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadeca-8-enyl group), a heptadecadienyl group (preferably a (8Z, (11Z)-heptadeca-8,11-dienyl group), octadecenyl group (preferably, (Z)-octadec-9-enyl group), octadecadienyl group (preferably, (9Z,12Z)-octadeca-9,12-dienyl group), nonadecenyl group, icosenyl group (preferably, (Z)-icos-11-enyl group), icosadienyl group (preferably, (11,14)-icosa-11,14-dienyl group), and the like.The alkynyl group having 6 to 24 carbon atoms is preferably an alkynyl group having 6 to 20 carbon atoms, and the alkynyl group having 3 to 24 carbon atoms is more preferably an alkynyl group having 6 to 20 carbon atoms. Specific examples include a hexynyl group, a heptynyl group, an octynyl group, a nonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, and an octadecynyl group. Each of the above alkenyl groups preferably has one or two double bonds, and each of the alkynyl groups preferably has one or two triple bonds.

[0023] R 21 and R 31The hydrocarbon group having 1 to 24 carbon atoms 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 linear or branched, and may be linear 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 a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (preferably a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (preferably a 3,7,11,15-tetramethylhexadecyl group), a heptadecyl group, an octadecyl group, a 2-butylhexyl group, a 2-butyloctyl group, a 1-pentylhexyl group, a 2-pentylheptyl group, a 3-pentyloctyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, Examples include a 2-hexyloctyl group, a 2-hexyldecyl group, a 3-hexylnonyl group, a 1-heptyloctyl group, a 2-heptylnonyl group, a 2-heptylundecyl group, a 3-heptyldecyl group, a 1-octylnonyl group, a 2-octyldecyl group, a 2-octyldodecyl group, a 3-octylundecyl group, a 2-nonylundecyl group, a 3-nonyldodecyl group, a 2-decyldodecyl group, a 2-decyltetradecyl group, a 3-decyltridecyl group, and a 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group. The alkenyl group having 10 to 24 carbon atoms may be linear or branched, open-chain or cyclic.Specifically, a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably, a (Z)-tridec-8-enyl group), a tetradecenyl group (preferably, a tetradec-9-enyl group), a pentadecenyl group (preferably, a (Z)-pentadecen-8-enyl group), a hexadecenyl group (preferably, a (Z)-hexadec-9 ... Examples of the alkynyl group include a dienyl group, a heptadecenyl group (preferably a (Z)-heptadecen-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadeca-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadecen-9-enyl group), and an octadecadienyl group (preferably a (9Z,12Z)-octadeca-9,12-dienyl group). The alkynyl group having 10 to 24 carbon atoms may be linear or branched, and may be linear or cyclic. Specific examples include a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, and an octadecynyl group. Preferably, all of the above alkenyl groups have one or two double bonds, and preferably, all of the alkynyl groups have one or two triple bonds.

[0024] R 22 and R 32 Regarding the above, the divalent linking group and hydrocarbon linking group having 1 to 18 carbon atoms 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 linear or branched, and may be chain-like or cyclic. It preferably has 1 to 12 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 2 to 10 carbon atoms. Specific examples include a methylene group, ethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, heptamethylene group, octamethylene group, nonamethylene group, decamethylene group, undecamethylene group, and dodecamethylene group. The alkenylene group having 2 to 18 carbon atoms may be linear or branched, and may be chain-like or cyclic. It preferably has 1 to 12 carbon atoms, and more preferably 2 to 10 carbon atoms. L 1The preferred range of is -O(CO)O-, -O(CO)-, or -(CO)O-, and -O(CO)- or -(CO)O- is more preferred. L 2 The preferred range of is -O(CO)O-, -O(CO)-, or -(CO)O-, and -O(CO)- or -(CO)O- is more preferred.

[0025] R 4 , R 6 , R 9 , R 10 , R 11 , and R 12 The alkyl group having 1 to 18 carbon atoms in the optionally substituted alkyl group having 1 to 18 carbon atoms may be linear or branched, and may be linear or cyclic. The number of carbon atoms is preferably 1 to 12. Specific examples include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, and a dodecyl group. When the alkyl group has a substituent, the substituent may be a hydroxyl group, a carboxyl group, -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 A group represented by the formula: -O(CO)-R is preferred. 42 OR-(CO)OR 43 A group represented by the following formula is more preferred.

[0026] R 5 , R 7 , and R 8The alkyl group having 1 to 18 carbon atoms in the optionally substituted alkyl group having 1 to 18 carbon atoms may be linear or branched, chain-like or cyclic. The number of carbon atoms is preferably 1 to 12, and more preferably 1 to 8. Specific examples include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, and a dodecyl group. When the alkyl group has a substituent, the substituent may be a hydroxyl group, a carboxyl group, -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 A group represented by the formula: -O(CO)-R is preferred. 42 , -(CO)OR 43 A group represented by the following formula is more preferred. 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, and a 6-membered ring is preferred, with a piperidine ring and a morpholine ring being more preferred.

[0027] R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 Regarding the above, when the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is a substituted or unsubstituted aryl group, the aryl group preferably has 6 to 22 carbon atoms, more preferably 6 to 18 carbon atoms, and even more preferably 6 to 10 carbon atoms. Specific examples include a phenyl group, a naphthyl group, an anthracenyl group, and a phenanthrenyl group. Examples of the substituent on the aryl group include an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, -NR 45 R 46 Amino group represented by -O(CO)OR 41 , -O(CO)-R 42, -(CO)OR 43 , or -OR 44 A group represented by the formula: is preferred, and a hydroxyl group or a carboxyl group is more preferred. Specific examples of the substituted aryl group include a hydroxyphenyl group and a carboxyphenyl group. R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 In the case where the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is a substituted or unsubstituted heteroaryl group, the heteroaryl group preferably has 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Specific examples include a pyridyl group, a pyrazolyl group, an imidazolyl group, a benzimidazolyl group, a thiazolyl group, and an oxazolyl group. Examples of the substituent on the heteroaryl group include an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, -NR 45 R 46 Amino group represented by -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 A group represented by the formula: is preferred, and a hydroxyl group or a carboxyl group is more preferred. Specific examples of the substituted or unsubstituted heteroaryl group include a hydroxypyridyl group, a carboxypyridyl group, and a pyridonyl group.

[0028] R 41 , R 42 , R 43 , R 44 , R 45 and R 46The hydrocarbon group having 1 to 18 carbon atoms in the above formula is preferably an alkyl group having 1 to 18 carbon atoms, an alkenyl group having 2 to 18 carbon atoms, or an alkynyl group having 2 to 18 carbon atoms, and more preferably an alkyl group having 1 to 18 carbon atoms or an alkenyl group having 2 to 18 carbon atoms. The alkyl group having 1 to 18 carbon atoms may be linear or branched, and may be linear or cyclic. The number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18. Specific 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, heptadecyl, and octadecyl. The alkenyl group having 2 to 18 carbon atoms may be linear or branched, open-chain, or cyclic. The number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18. Specifically, an allyl group, a prenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (preferably, a (Z)-2-nonenyl group or an (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably, a (Z)-trideca-8-enyl group), a tetradecenyl group (preferably, a tetradec-9-enyl group), a pentadecenyl group (preferably, a (Z)-pentadeca-8-enyl group), Examples of the alkynyl group include a hexadecenyl group (preferably a (Z)-hexadecan-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadecan-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadecan-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadecan-9-enyl group), and an octadecadienyl group (preferably a (9Z,12Z)-octadecan-9,12-dienyl group). The alkynyl group having 2 to 18 carbon atoms may be linear or branched, open-chain or cyclic. The alkynyl group preferably has 3 to 18 carbon atoms, and more preferably has 5 to 18 carbon atoms.Specific examples include a propargyl group, butynyl group, pentynyl group, hexynyl group, heptynyl group, octynyl group, nonynyl group, decynyl group, undecynyl group, dodecynyl group, tetradecynyl group, pentadecynyl group, hexadecynyl group, heptadecynyl group, and octadecynyl group.

[0029] X is -NR 1 - When indicating R 1 is a hydrocarbon group having 6 to 24 carbon atoms, or R 21 -L 1 -R 22 In this case, R 2 and R 3 is a hydrogen atom; 2 and R 3 The other is a hydrocarbon group having 6 to 24 carbon atoms, or R 31 -L 2 -R 32 A group represented by - is preferred. When X represents -O-, R 2 and R 3 are each independently a hydrocarbon group having 6 to 24 carbon atoms, or R 31 -L 2 -R 32 A group represented by - is preferred. R 4 , R 6 , R 9 , R 10 , R 11 , and R 12 is preferably a hydrogen atom.

[0030] R 5 represents a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, -O(CO)-R 42 OR-(CO)OR 43 Preferably, R is an alkyl group having 1 to 18 carbon atoms which may be substituted with an alkyl group having 1 to 18 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 18 carbon atoms which may be substituted with a hydroxyl group. When R is an alkyl group, 4 , R 6 , R 10 and R 12and may be linked together to form a ring which may contain an O atom. Among these, alkyl groups having 1 to 18 carbon atoms, -O(CO)-R 42 OR-(CO)OR 43 The alkyl group may be substituted with an alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 12 carbon atoms, or an alkyl group having 1 to 8 carbon atoms, which may be substituted with a hydroxyl group. 42 OR-(CO)OR 43 It is more preferably an alkyl group having 1 to 18 carbon atoms which may be substituted with.

[0031] R 7 and R 8 are each independently a hydrogen atom, a hydrocarbon group having 1 to 18 carbon atoms, or -O(CO)-R 42 OR-(CO)OR 43 an alkyl group having 1 to 18 carbon atoms which may be substituted with an alkyl group having 1 to 8 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 8 carbon atoms which may be substituted with a hydroxyl group, or R 7 and R 8 are preferably linked to each other to form a 4- to 7-membered ring which may contain an O atom. R 5 and R 7 or R 8 are not linked to each other and do not form a ring. a+b is preferably 1 or 2, and more preferably 1. c+d is preferably 1 or 2, and more preferably 1.

[0032] The compound represented by formula (4) is preferably a compound represented by formula (21). [ka] During the ceremony, R 2 and R 3 are each independently a hydrocarbon group having 3 to 24 carbon atoms and containing one or more unsaturated bonds, R2 and R 3 are each independently R 31 -L 2 -R 32 -, or 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, R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, L 2 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [ka] indicates, R 32 represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R 5 is -O(CO)-R 42 OR-(CO)OR 43 represents an alkyl group having 1 to 18 carbon atoms which may be substituted by R 42 , and R 43 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, R 7 and R 8 each independently represents an alkyl group having 1 to 4 carbon atoms, e indicates 2 or 3. In formula (21), preferably, R 2 and R 3 One of them is R 31 -L 2 -R 32 In formula (21), preferably, L 2 represents -O(CO)- or -(CO)O-.

[0033] The compound represented by formula (4) may form a salt. Salts of basic groups include, for example, salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid, and sulfuric acid; salts 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 with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid. Salts of acidic groups include, for example, salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; and salts 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-phenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine. Of the above salts, preferred salts include pharmacologically acceptable salts. The lipid represented by formula (4) and a method for producing the same are described in WO2019 / 235635 and WO2021 / 095876.

[0034] <<Lipid represented by formula (1) or a salt thereof>> As another example, the ionizable lipid may be a lipid represented by the following formula (1) or a salt thereof: [ka] During the ceremony, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, and R 1 , R 2 and R 3 The hydrocarbon group represented by is -OH, COOH, -NR 51 R 52 , -OC(O)OR53 , -C(O)OR 54 , -OC(O)-R 55 , and -OR 56 and optionally substituted with one or more substituents selected from R 4 represents a hydrocarbon group having 1 to 8 carbon atoms, R 5 and R 6 are each independently a hydrocarbon group having 1 to 8 carbon atoms, or -R 8 -L 1 -R 9 where R 5 and R 6 are both hydrocarbon groups having 1 to 8 carbon atoms, R 7 -R 10 -L 2 -R 11 -L 3 -R 12 indicates, 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, R 53 ,R 54 ,R 55 , and R 56 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 58 may be substituted with The above aryl group having 6 to 20 carbon atoms includes -OH, -COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 57 may be substituted with R 58 represents a hydrocarbon group having 1 to 12 carbon atoms, R57 -OH, COOH, -NR 61 R 62 , -OC(O)OR 63 , -C(O)OR 64 , -OC(O)-R 65 , -OR 66 Shows. 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, R 63 ,R 64 ,R 65 , and R 66 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 68 may be substituted with The above aryl group having 6 to 20 carbon atoms includes -OH, -COOH, -NR 61 R 62 , -OC(O)OR 63 , -C(O)OR 64 , -OC(O)-R 65 , -OR 66 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 67 may be substituted with R 68 represents a hydrocarbon group having 1 to 12 carbon atoms, 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 12represents a hydrocarbon group having 1 to 24 carbon atoms, R 9 , and R 12 The hydrocarbon group represented by is an aryl group, -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -SR 58 and R 53 , R 54 , R 55 , and R 58 is defined as above, R 11 The hydrocarbon group represented by is -OC(O)OR 53 , -C(O)OR 54 , or -OC(O)-R 55 and R 53 , R 54 , and R 55 The definition of is as above.

[0035] The hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon group having 1 to 18 carbon atoms, the hydrocarbon group having 1 to 12 carbon atoms, the hydrocarbon group having 2 to 8 carbon atoms, and the hydrocarbon group having 1 to 8 carbon atoms are preferably an alkyl group, an alkenyl group, or an alkynyl group, respectively. The alkyl group may be linear or branched, and may be linear or cyclic. Specific examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (preferably a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (preferably a 3,7,11,15-tetramethylhexadecyl group), a heptadecyl group, an octadecyl group, a 2-butylhexyl group, and a 2-butyloctyl group. , 1-pentylhexyl group, 2-pentylheptyl group, 3-pentyloctyl group, 1-hexylheptyl group, 1-hexylnonyl group, 2-hexyloctyl group, 2-hexyldecyl group, 3-hexylnonyl group, 1-heptyloctyl group, 2-heptylnonyl group, 2-heptylundecyl group, 3-heptyldecyl group, 1-octylnonyl group, 2-octyldecyl group, 2-octyldodecyl group, 3-octylundecyl group, 2-nonylundecyl group, 3-nonyldodecyl group, 2-decyldodecyl group, 2-decyltetradecyl group, 3-decyltridecyl group, 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group, and the like.

[0036] The alkenyl group may be linear or branched, linear or cyclic. Specifically, it includes an allyl group, a prenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (preferably, a (Z)-2-nonenyl group or an (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably, a (Z)-trideca-8-enyl group), a tetradecenyl group (preferably, a tetradec-9-enyl group), and a pentadecenyl group (preferably, a (Z)-pentadeca-8-enyl group). , a hexadecenyl group (preferably a (Z)-hexadecan-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadecan-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadecan-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadecan-9-enyl group), an octadecadienyl group (preferably a (9Z,12Z)-octadecan-9,12-dienyl group), and the like. The alkynyl group may be linear or branched, open-chain or cyclic, and specific examples thereof include a propargyl group, a butynyl group, a pentynyl group, a hexynyl group, a heptynyl group, an octynyl group, a nonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, and an octadecynyl group.

[0037] Preferably, all of the above alkenyl groups have one or two double bonds, and preferably, all of the alkynyl groups have one or two triple bonds. -(C1-12 hydrocarbon group)-R 67 The hydrocarbon group having 1 to 12 carbon atoms 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 linear or branched, and may be linear or cyclic. Specific examples include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, an octamethylene group, a nonamethylene group, a decamethylene group, and an undecamethylene group.

[0038] The aryl group preferably has 6 to 20 carbon atoms, more preferably 6 to 18, and even more preferably 6 to 10. Specific examples include a phenyl group, a naphthyl group, an anthracenyl group, and a phenanthrenyl group. R 1 and R 2 are each independently preferably a hydrocarbon group having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. R 3 represents a hydrocarbon group preferably having 2 to 6 carbon atoms, and more preferably a hydrocarbon group having 2 to 4 carbon atoms. R 1 , R 2 and R 3 The hydrocarbon group represented by may preferably be substituted with —OH.

[0039] L 1 , and L 3 are each independently preferably -C(O)O- or -OC(O)-. L 2 preferably represents —OC(O)O—, —C(O)O—, or —OC(O)—. R 8 represents a hydrocarbon group preferably having 1 to 10 carbon atoms, and more preferably a hydrocarbon group having 1 to 8 carbon atoms. R 9 represents a hydrocarbon group preferably having 1 to 20 carbon atoms, and more preferably a hydrocarbon group having 1 to 16 carbon atoms. R 11 represents a hydrocarbon group preferably having 1 to 16 carbon atoms, and more preferably a hydrocarbon group having 1 to 9 carbon atoms. R 12 represents a hydrocarbon group preferably having 1 to 20 carbon atoms, and more preferably a hydrocarbon group having 1 to 16 carbon atoms.

[0040] R 9 , and R 12 The hydrocarbon group represented by is preferably an aryl group or -SR 58 where R 58 represents a hydrocarbon group preferably having 1 to 8 carbon atoms. R 11 The hydrocarbon group represented by is preferably —C(O)OR 55 , or -OC(O)-R 56 where R 55 , and R 56 each independently represents a hydrocarbon group having 1 to 16 carbon atoms; R 55 , and R 56 The hydrocarbon group represented by is preferably an aryl group having 6 to 20 carbon atoms or -SR 58 and R 58 The definition of is as above.

[0041] The compound represented by formula (1) is preferably, as a first example, a compound represented by the following formula (1-1). [ka] During the ceremony, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, and R 1 , R 2 and R 3 The hydrocarbon group represented by is -OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -OR 56 may be substituted with R 4 represents a hydrocarbon group having 1 to 8 carbon atoms, R 5 and R 6are each independently a hydrocarbon group having 1 to 8 carbon atoms, or -R 8 -L 1 -R 9 where R 5 and R 6 are both hydrocarbon groups having 1 to 8 carbon atoms, L 1 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, and R 9 The hydrocarbon group represented by is an aryl group, -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -SR 58 may be substituted with 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, R 53 ,R 54 ,R 55 , and R 56 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 58 may be substituted with The above aryl group having 6 to 20 carbon atoms includes -OH, -COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 57 may be substituted with R 58 represents a hydrocarbon group having 1 to 12 carbon atoms, R 57-OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 Shows. R 13 represents a hydrocarbon group having 1 to 8 carbon atoms, R 14 -R 15 -L 5 -R 16 indicates R 15 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 5 indicates -OC(O)O-, -C(O)O-, -OC(O)-, or -O-, and R 16 represents a hydrocarbon group having 1 to 24 carbon atoms, R 15 The hydrocarbon group having 1 to 24 carbon atoms is -OC(O)OR 53 , -C(O)OR 54 , or -OC(O)-R 55 and R 53 , R 54 , and R 55 is defined as above, R 16 The hydrocarbon group having 1 to 24 carbon atoms represented by is an aryl group having 6 to 20 carbon atoms, -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -SR 58 and R 53 , R 54 , R 55 , and R 58 The definition of is as above.

[0042] In formula (1-1), R 1 and R 2 are each independently preferably a hydrocarbon group having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. R 3represents a hydrocarbon group preferably having 2 to 6 carbon atoms, and more preferably a hydrocarbon group having 2 to 4 carbon atoms. R 1 , R 2 and R 3 The hydrocarbon group represented by may preferably be substituted with —OH. L 1 preferably represents —C(O)O— or —OC(O)—. R 8 represents a hydrocarbon group preferably having 1 to 10 carbon atoms, and more preferably a hydrocarbon group having 1 to 8 carbon atoms. R 9 preferably represents a hydrocarbon group having 1 to 18 carbon atoms, and R 9 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms, or -SR 58 may be substituted with R 14 is preferably -R 15 -L 5 -R 16 indicates R 15 represents a hydrocarbon group having 1 to 18 carbon atoms, and L 5 indicates -OC(O)O-, and R 16 represents a hydrocarbon group having 1 to 18 carbon atoms. R 15 The hydrocarbon group having 1 to 18 carbon atoms represented by is preferably —C(O)OR 55 , or -OC(O)-R 56 may be substituted with R 55 , and R 56 each independently represents a hydrocarbon group having 1 to 16 carbon atoms; R 55 , and R 56 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 58 and R 58 The definition of is as above. R 16 The hydrocarbon group having 1 to 18 carbon atoms represented by is preferably an aryl group or -SR 58 and R 58 The definition of is as above.

[0043] A second example of the compound represented by formula (1) is preferably a compound represented by the following formula (1-2). [ka] During the ceremony, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, and R 1 , R 2 and R 3 The hydrocarbon group represented by is -OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -OR 56 may be substituted with R 4 and R 8 each independently represents a hydrocarbon having 1 to 8 carbon atoms, R 21 and R 22 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, R 23 and R 24 each independently represents a hydrocarbon group having 1 to 12 carbon atoms, R 25 and R 26 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, L 21 and L 22 each independently represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-; R 25 and R 26 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms, -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -SR 58 may be substituted with R 51 and R 52each 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 18 carbon atoms, The above aryl group having 6 to 20 carbon atoms is OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 57 and optionally substituted by R 57 -OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 Shows. R 58 represents a hydrocarbon group having 1 to 12 carbon atoms.

[0044] In formula (1-2), R 1 and R 2 R each independently represents a hydrocarbon group preferably having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. 1 and R 2 The hydrocarbon group represented by may be substituted with -OH, but is more preferably a hydrocarbon group without any substituents. R 3 represents a hydrocarbon group preferably having 2 to 6 carbon atoms, and more preferably a hydrocarbon group having 2 to 4 carbon atoms. R 21 and R 22 are each independently preferably a hydrocarbon group having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 8 carbon atoms, and even more preferably a hydrocarbon group having 1 to 6 carbon atoms. R 23 and R24 are each independently preferably a hydrocarbon group having 1 to 10 carbon atoms, more preferably a hydrocarbon group having 1 to 8 carbon atoms. R 25 and R 26 are each independently preferably a hydrocarbon group having 1 to 20 carbon atoms, more preferably a hydrocarbon group having 1 to 16 carbon atoms, and even more preferably a hydrocarbon group having 1 to 12 carbon atoms. L 21 and L 22 are each independently preferably -C(O)O- or -OC(O)-.

[0045] A third example of the compound represented by formula (1) is preferably a compound represented by the following formula (1-3). [ka] During the ceremony, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, and R 1 , R 2 and R 3 The hydrocarbon group represented by is -OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -OR 56 may be substituted with R 4 and R 8 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 31 , R 32 , R 33 , and R 34 each independently represents a hydrocarbon group having 1 to 12 carbon atoms, R 35 , R 36 , R 37 , and R 38 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, L 31 , L 32 , L 33 , and L 34 each independently represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-; R 35 , R 36 , R 37 , and R 38 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms, -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or SR 58 may be substituted with 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 18 carbon atoms, The above aryl group having 6 to 20 carbon atoms is OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 57 and optionally substituted by R 57 -OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 Shows. R 58 represents a hydrocarbon group having 1 to 12 carbon atoms.

[0046] In formula (1-3), R 1 and R 2R each independently represents a hydrocarbon group preferably having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. 1 and R 2 The hydrocarbon group represented by may be substituted with -OH, but is more preferably a hydrocarbon group without any substituents. R 3 represents a hydrocarbon group preferably having 2 to 6 carbon atoms, and more preferably a hydrocarbon group having 2 to 4 carbon atoms. R 31 , R 32 , R 33 , and R 34 are each independently preferably a hydrocarbon group having 1 to 10 carbon atoms, more preferably a hydrocarbon group having 1 to 8 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. R 35 , R 36 , R 37 , and R 38 R each independently represents a hydrocarbon group having preferably 1 to 20 carbon atoms, more preferably a hydrocarbon group having 1 to 16 carbon atoms, and even more preferably a hydrocarbon group having 1 to 12 carbon atoms. 35 , R 36 , R 37 , and R 38 The hydrocarbon group represented by is preferably an aryl group having 6 to 20 carbon atoms, or SR 58 More preferably, it is substituted with -SR 58 may be substituted with. R 35 , R 36 , R 37 , and R 38 are each independently particularly preferably -SR 58 or represents a hydrocarbon group having 1 to 12 carbon atoms substituted with L 31 , L 32 , L 33 , and L 34 are each independently preferably -C(O)O- or -OC(O)-. R 58represents a hydrocarbon group preferably having 1 to 10 carbon atoms, and more preferably a hydrocarbon group having 1 to 8 carbon atoms.

[0047] The compound of the present invention may form a salt. Examples of salts of basic groups include salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid, and sulfuric acid; salts 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 with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid. Salts of acidic groups include, for example, salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; and salts 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-phenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine. Of the above salts, preferred salts include pharmacologically acceptable salts. The lipid represented by formula (1) and its production method are described in WO2019 / 235635 and WO2022 / 230964, the contents of which are incorporated herein by reference in their entirety.

[0048] <<Lipid represented by formula (5) or a salt thereof>> As the ionizable lipid, for example, a lipid represented by formula (5) or a salt thereof may be used. [ka] In the formula, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, The above substituent A is a hydroxyl group, -G 20 -CH(R 55 )(R 56 ), -N(R 58 )(R 59 ), or -G 20 -R 60 represents a 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, Substituent B is -N(R 61 )(R 62 ) and 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 is -S-(CO)-NR 64 - indicates R 64 L 30 -G 20 -CH(R 55 )(R 56 ) represents a group represented by a represents 0 or 1, L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G 10 is -O(CO)-, -(CO)O-, -O(CO)O- or -N(C(O)R 63 )- indicates R 63 represents a hydrocarbon group having 1 to 18 carbon atoms, L 20represents 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 is —(CO)O —R 65 , or -O(CO) -R 65 represents a group represented by R 65 is a hydrocarbon group having 1 to 18 carbon atoms or -L 40 -CH(R 66 )(R 67 ) represents a group represented by L 40 represents a hydrocarbon group having 1 to 6 carbon atoms, R 66 and R 67 represents a hydrocarbon group or alkoxy group having 1 to 10 carbon atoms.

[0049] The compound represented by formula (5) may preferably be a compound represented by formula (5A). [ka] In the formula, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, The above substituent A is a hydroxyl group or -G 20 -CH(R 55 )(R 56 ) represents a group represented by G 20 represents -O(CO)- or -(CO)O-, R 55 and R 56 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, L 10 represents a hydrocarbon group having 1 to 18 carbon atoms, G 10 represents -O(CO)- or -(CO)O-, R 63represents a hydrocarbon group having 1 to 18 carbon atoms, R 53 , R 54 and R 57 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms.

[0050] The compound represented by formula (5) may preferably be a compound represented by formula (5B). [ka] In the formula, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms, L 10 represents a hydrocarbon group having 1 to 18 carbon atoms, G 10 represents -O(CO)O-, L 20 represents a hydrocarbon group having 1 to 6 carbon atoms, 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 is —O(CO) —R 65 represents a group represented by R 65 is a hydrocarbon group having 1 to 18 carbon atoms or -L 40 -CH(R 66 )(R 67 ) represents a group represented by L 40 represents a hydrocarbon group having 1 to 6 carbon atoms, R 66 and R 67 represents an alkoxy group having 1 to 10 carbon atoms.

[0051] The compound represented by formula (5) may preferably be a compound represented by formula (5C). [ka] In the formula, R 51 and R 52each independently represents a hydrocarbon group having 1 to 21 carbon atoms, L 10 represents a hydrocarbon group having 1 to 18 carbon atoms, G 10 is -N(C(O)R 63 )- indicates R 63 represents a hydrocarbon group having 1 to 18 carbon atoms, 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 is —(CO)O —R 65 represents a group represented by R 65 -L 40 -CH(R 66 )(R 67 ) represents a group represented by L 40 represents a hydrocarbon group having 1 to 6 carbon atoms, R 66 and R 67 represents a hydrocarbon group having 1 to 10 carbon atoms.

[0052] The compound represented by formula (5) may preferably be a compound represented by formula (5D). [ka] In the formula, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms, L 10 represents a hydrocarbon group having 1 to 18 carbon atoms, G 30 is -S-(CO)-NR 64 - indicates R 64 L 30 -G 20 -CH(R 55 )(R 56 ) represents a group represented by L 30 represents a hydrocarbon group having 1 to 18 carbon atoms, G20 represents -(CO)O-, R 55 and R 56 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, G 10 represents -(CO)O-, R 53 , R 54 and R 57 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms.

[0053] The hydrocarbon group having 1 to 21 carbon atoms is preferably an alkyl group having 1 to 21 carbon atoms, an alkenyl group having 2 to 21 carbon atoms, or an alkynyl group having 2 to 21 carbon atoms, and more preferably an alkyl group having 1 to 21 carbon atoms or an alkenyl group having 2 to 21 carbon atoms. The alkyl group having 1 to 21 carbon atoms may be linear or branched, and may be linear or cyclic. The number of carbon atoms is preferably 3 to 21, and more preferably 5 to 21. Specific 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, heptadecyl, and octadecyl. The alkenyl group having 2 to 18 carbon atoms may be linear or branched, open-chain, or cyclic. The number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18. Specifically, an allyl group, a prenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (preferably, a (Z)-2-nonenyl group or an (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably, a (Z)-trideca-8-enyl group), a tetradecenyl group (preferably, a tetradec-9-enyl group), a pentadecenyl group (preferably, a (Z)-pentadeca-8-enyl group), Examples of the alkynyl group include a hexadecenyl group (preferably a (Z)-hexadecanyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadecanyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadecanyl-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadecanyl group), and an octadecadienyl group (preferably a (9Z,12Z)-octadecanyl-9,12-dienyl group). The alkynyl group having 2 to 21 carbon atoms may be linear or branched, open-chain or cyclic. The alkynyl group preferably has 3 to 21 carbon atoms, and more preferably has 5 to 21 carbon atoms.Specific examples include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, octadecynyl, etc. Examples of hydrocarbon groups having 1 to 18 carbon atoms include hydrocarbon groups having 1 to 21 carbon atoms, and those having 1 to 18 carbon atoms.

[0054] The cyclic hydrocarbon group is preferably 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, or an aryl group having 6 to 10 carbon atoms. The hydrocarbon group having 1 to 6 carbon atoms is preferably an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms, and more preferably an alkyl group having 1 to 6 carbon atoms or an alkenyl group having 2 to 6 carbon atoms. The alkyl group having 1 to 6 carbon atoms may be linear or branched, and may be chain-like or cyclic. Specific examples include a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, and a hexyl group. The alkenyl group having 2 to 6 carbon atoms may be linear or branched, and may be chain-like or cyclic. Specific examples include an allyl group, a prenyl group, a pentenyl group, and a hexenyl group. The alkynyl group having 2 to 6 carbon atoms may be linear or branched, and may be chain-like or cyclic. Specific examples include a propargyl group, a butynyl group, a pentynyl group, and a hexynyl group.

[0055] The hydrocarbon group having 1 to 10 carbon atoms is preferably an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms, and more preferably an alkyl group having 1 to 10 carbon atoms or an alkenyl group having 2 to 10 carbon atoms. The alkyl group having 1 to 10 carbon atoms may be linear or branched, and may be chain-like or cyclic. It preferably has 3 to 10 carbon atoms, and more preferably has 5 to 10 carbon atoms. Specific examples include a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, and a decyl group. The alkenyl group having 2 to 10 carbon atoms may be linear or branched, and may be chain-like or cyclic. It preferably has 3 to 10 carbon atoms, and more preferably has 5 to 10 carbon atoms. Specific examples include allyl, prenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably (Z)-2-nonenyl or (E)-2-nonenyl), and decenyl. The alkynyl group having 2 to 10 carbon atoms may be linear or branched, and may be linear or cyclic. It preferably has 3 to 10 carbon atoms, and more preferably has 5 to 10 carbon atoms. Specific examples include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, and decynyl. The compound represented by formula (5) may form a salt. Salts of basic groups include, for example, salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid, and sulfuric acid; salts 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 with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid. Of the above salts, preferred salts include pharmacologically acceptable salts. The lipid represented by formula (4) and a method for producing the same are described in WO2019 / 235635 and WO2021 / 095876.

[0056] <<Examples of ionizable lipids>> Examples of ionizable lipids include the following lipids: cKK-E12(MD-1), C12-200, 306Oi10, YSK05, and 93-O17S are compounds not included in the above formula (5). [ka] JPEG2025535815000028.jpg217170JPEG2025535815000029.jpg217170JPEG2025535815000030.jpg204170 JPEG2025535815000031.jpg242170JPEG2025535815000032.jpg244170JPEG2025535815000033.jpg237170

[0057] In the lipid composition of the present invention, the content of the ionizable lipid or a salt thereof is preferably 10 mol% to 80 mol% relative to the total lipid amount, more preferably 20 mol% to 80 mol%, even more preferably 30 mol% to 70 mol%, and particularly preferably 40 mol% to 60 mol%.

[0058] <Neutral lipids> The lipid composition of the present invention may comprise a neutral lipid. The neutral lipid is preferably a zwitterionic lipid. The zwitterionic lipid is preferably a phospholipid. Specific examples include phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin. The phospholipid is preferably a phospholipid having a choline group, such as phosphatidylcholine. The zwitterionic lipid may be used alone or in combination with multiple different neutral lipids. Phosphatidylcholines include, but are not limited to, 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), etc. Among these, dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), and dilauroylphosphatidylcholine (DLPC) are preferred, with distearoylphosphatidylcholine (DSPC) being particularly preferred. DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine [ka]

[0059] Examples of phosphatidylethanolamines include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylethanolamine (DLoPE), diphytanoylphosphatidylethanolamine (D(Phy)PE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), ditetradecylphosphatidylethanolamine, dihexadecylphosphatidylethanolamine, dioctadecylphosphatidylethanolamine, and diphytanylphosphatidylethanolamine. Examples of sphingomyelin include, but are not limited to, egg yolk-derived sphingomyelin and milk-derived sphingomyelin. In the lipid composition of the present invention, the content of neutral lipids is preferably 1 to 30 mol %, more preferably 5 to 25 mol %, and even more preferably 7 to 23 mol %, based on the total amount of constituent lipid components.

[0060] <Lipids with non-ionic hydrophilic polymers> The lipid composition of the present invention may contain 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. Examples of nonionic hydrophilic polymers include, but are not limited to, 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 constituent units. 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 also referred to as PEG) is particularly preferred. That is, the lipid nanoparticles preferably contain lipids to which PEG is bound. The lipid having a nonionic hydrophilic polymer is not particularly limited, but includes PEG-modified phosphoethanolamine, diacylglycerol PEG derivative, monoacylglycerol PEG derivative, dialkylglycerol PEG derivative, cholesterol PEG derivative, ceramide PEG derivative, etc. Among these, monoacylglycerol PEG or diacylglycerol PEG is preferred. The acyl group in the PEG-modified diacylphosphoethanolamine and diacylglycerol PEG preferably has 14 or more carbon atoms, more preferably 16 or more carbon atoms.

[0061] The weight average molecular weight of the nonionic hydrophilic polymer is preferably from 100 to 10,000, more preferably from 500 to 5,000, and even more preferably from 750 to 3,000. The non-ionic hydrophilic polymer may be branched and may have a substituent such as a hydroxymethyl group. Preferred examples of lipids having a nonionic hydrophilic polymer include the following lipids: DMG-mPEG2000: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 DPG-mPEG2000: 1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-2000 DSG-mPEG2000: 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 [ka]

[0062] When a targeting molecule is bound to a lipid having a non-ionic hydrophilic polymer, a lipid having a reactive group for binding a targeting molecule (e.g., a maleimide group, a thiol group, an orthopyridyl disulfide (OPSS) group, an N-hydroxysuccinimide (NHS) group, an alkyne group, a dibenzocyclooctyne (DBCO) group, an azide group, an amino group, a carboxyl group, etc.) can be used as all or part of the lipid having a non-ionic hydrophilic polymer. In the lipid composition of the present invention, the content of lipids having nonionic hydrophilic polymers is preferably 0.1 to 10 mol %, more preferably 0.3 to 8 mol %, even more preferably 0.5 to 5 mol %, and particularly preferably 1 to 3 mol %, relative to the total amount of lipids.

[0063] <Therapeutic agent> The lipid composition of the present invention contains a therapeutic agent. The therapeutic agent is preferably a nucleic acid such as a polynucleotide. The nucleic acid such as a polynucleotide may be either DNA or RNA, and may include a plasmid, single-stranded DNA, double-stranded DNA, siRNA (small interfering RNA), miRNA (microRNA), mRNA, single guide RNA (sgRNA), antisense oligonucleotide (also known as ASO), ribozyme, aptamer, decoy nucleic acid, guide RNA (gRNA) used in genome editing, etc., and any of these may be included. Modified nucleic acids may also be included. When using sgRNA and mRNA, the sgRNA and mRNA may be contained separately or together in the lipid composition. Preferably, the sgRNA and mRNA may be contained together in the lipid composition. In the lipid composition of the present invention, the weight ratio of lipid to therapeutic agent is preferably 5-100, more preferably 5-70, even more preferably 5-40, and particularly preferably 5-35.

[0064] <Method of producing the composition> A method for producing the lipid composition of the present invention will now be described. The method for producing the lipid composition is not limited.For example, the lipid composition can be produced by dissolving all of the constituent components of the lipid particles or a part of the oil-soluble components of the lipid particles in an organic solvent or the like to form an oil phase, dissolving the water-soluble components of the lipid particles in water to form an aqueous phase, and then mixing the oil phase and the aqueous phase.A micromixer may be used for mixing, or emulsification may be performed using an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure injection emulsifier, or the like.

[0065] Alternatively, a lipid composition can be produced by subjecting a lipid-containing solution to vacuum drying using an evaporator or spray drying using a spray dryer under reduced pressure to prepare a dried mixture containing lipids, adding this mixture to an aqueous solvent, and further emulsifying it using the emulsifier described above or the like. An example of a method for producing lipid particles containing nucleic acids is Step (a) of dissolving components of lipid particles containing a compound according to an embodiment of the present invention in an organic solvent to obtain an oil phase; a step (b) of mixing the oil phase obtained in the step (a) with an aqueous phase containing nucleic acids; (c) diluting the mixture containing the oil phase and the aqueous phase obtained in step (b) to obtain a dispersion of a lipid composition containing nucleic acids; and a step (d) of removing the organic solvent from the dispersion of the nucleic acid-lipid composition obtained in the step (c); The method includes the steps of:

[0066] In step (a), lipid components are dissolved in an organic solvent (an alcohol such as ethanol, or an ester). 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. In step (b), the aqueous phase can be obtained by dissolving nucleic acids (e.g., siRNA, antisense oligonucleotides, mRNA, etc.) in water or a buffer solution. Components such as antioxidants can be added as needed. The mixing ratio (volume ratio) of the aqueous phase to the oil phase is preferably 5:1 to 1:1, more preferably 4:1 to 2:1. In step (b), the mixture can be diluted with water or a buffer solution (such as phosphate buffered saline (PBS)). In step (c), the method for removing the organic solvent from the dispersion of the lipid composition is not particularly limited, and a general method can be used. For example, the organic solvent can be removed by dialysis using phosphate-buffered saline. The lipid composition may be subjected to sizing as needed. The sizing method is not particularly limited, but the particle size can be reduced using an extruder or the like.

[0067] <Composition> The lipid composition of the present invention may be a lipid particle. The lipid particle means a particle composed of lipids, and includes compositions having any structure selected from lipid aggregates (e.g., lipid nanoparticles), micelles, and liposomes, but the structure of the lipid particle is not limited thereto as long as it is a composition containing lipids. The morphology of the lipid composition can be confirmed by electron microscopy or structural analysis using X-rays. For example, cryo-transmission electron microscopy (CryoTEM) can be used to determine whether the lipid particles have a lipid bilayer structure (lamellar structure) and an inner water layer, like liposomes, or whether the particles have a core with high electron density and are packed with lipids and other components. Small-angle X-ray scattering (SAXS) measurements can also be used to determine whether the lipid particles have a lipid bilayer structure (lamellar structure).

[0068] When the lipid composition of the present invention is in the form of particles, the particle size of the particles is not particularly limited, but is preferably 10 to 1000 nm, more preferably 30 to 500 nm, and even more preferably 50 to 250 nm. The particle size of the lipid particles can be measured by a general method (for example, dynamic light scattering, laser diffraction, etc.). When the lipid composition of the present invention is in the form of particles, the zeta potential of the particles is not particularly limited, but is preferably −20 to +20 mV, more preferably −10 to 10 mV. Note that the zeta potential in the present invention is a value measured by diluting the lipid composition in a phosphate buffer solution and using the **** method, but is not limited to this. 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 of the present invention is a value measured by TNS assay, but is not limited to this.

[0069] <Use of lipid composition> As an example of the use of the lipid composition of the present invention, a therapeutic agent (e.g., nucleic acid) can be introduced into a cell by introducing a lipid composition containing nucleic acid into the cell. That is, the lipid composition of the present invention can be used as a composition for introducing nucleic acid into a cell. The lipid composition of the present invention can also be used as a pharmaceutical composition for in vivo nucleic acid delivery. In the present invention, therapeutic agents can be delivered particularly to hematopoietic stem / progenitor cells or mesenchymal stem cells, and therefore, examples of organs to which therapeutic agents can be delivered include bone marrow and spleen. In addition, when the lipid composition of the present invention contains a nucleic acid having pharmaceutical uses, 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 mixed with a pharmaceutically acceptable carrier (e.g., an administration medium such as physiological saline or phosphate buffer).That is, the lipid composition of the present invention may further contain a pharmaceutically acceptable carrier.

[0070] The concentration of the lipid composition in the mixture with a pharmaceutically acceptable carrier is not particularly limited and can generally be 0.05% by mass to 90% by mass. In addition, other pharmaceutically acceptable additives, such as pH adjusting buffers and osmotic pressure adjusting agents, may be added to the nucleic acid drug containing the lipid composition of the present invention. The administration route of the lipid composition of the present invention is not particularly limited, and can be administered by any method. Examples of administration methods include oral administration and parenteral administration (intra-articular administration, intravenous administration, intra-arterial administration, subcutaneous administration, intradermal administration, intravitreal administration, intraperitoneal administration, intramuscular administration, intravaginal administration, intravesical administration, intrathecal administration, pulmonary administration, rectal administration, colonic administration, buccal administration, nasal administration, intracisternal administration, inhalation, etc.). Parenteral administration is preferred, and intravenous injection, subcutaneous injection, intradermal injection, or intramuscular injection is preferred, with intravenous injection or intramuscular injection being particularly preferred. Nucleic acid delivery can also be achieved by local administration in vivo. The lipid composition of the present invention can also be administered by direct injection to the diseased site. The dosage form of the lipid composition of the present invention is not particularly limited, but when administered orally, the lipid composition of the present invention can be combined with an appropriate excipient and used in the form of tablets, troches, capsules, pills, suspensions, syrups, etc. Furthermore, formulations suitable for parenteral administration can contain additives such as antioxidants, buffers, bacteriostatic agents, and isotonic sterile injections, suspending agents, solubilizers, thickeners, stabilizers, or preservatives, as appropriate.

[0071] <Use of lipid nanoparticles as nucleic acid delivery carriers> The lipid nanoparticles of the present invention are capable of retaining nucleic acids at a high encapsulation rate. Therefore, the lipid particles are very useful as nucleic acid delivery carriers. With the nucleic acid delivery carrier utilizing the present invention, for example, the resulting composition can be mixed with nucleic acids and transfected in vitro or in vivo, thereby introducing nucleic acids into cells. Furthermore, the nucleic acid delivery carrier utilizing the present invention is also useful as a nucleic acid delivery carrier for nucleic acid medicines. That is, the lipid nanoparticles of the present invention are useful as compositions for nucleic acid delivery in vitro or in vivo (preferably in vivo). The present invention will now be described with reference to examples, but the present invention is not limited to these examples.

[0072] Immunosuppressive drugs or CD117 kinase inhibitors Antihistamines (preferred) Examples of H1 histamine blockers Acrivastine, Alimemazine, Amitriptyline, Amoxapine, Aripiprazole, Azelastine, Bilastine, Bromodiphenhydramine, Brompheniramine, Buclizine, Carbinoxamine, Cetirizine, Chlophedianol, Chlordiphenhydramine, Chlorpheniramine, Chlorpromazine, Chlorprothixene, Chloropyramine, Cinnarizine, Clemastine, Clomipramine, Clozapine, Cyclizine, Cyproheptadine, Desloratadine, Dexbrompheniramine, Dexchlorpheniramine, Dimenhydrinate, Dimethindene, Diphenhydramine Dosulepin, doxepin, doxylamine, ebastine, embramine, fexofenadine, fluoxetine, hydroxyzine, imipramine, ketotifen, levocabastine, levocetirizine, levomepromazine, loratadine, maprotiline, meclizine, mianserin, mirtazapine, olanzapine, olopatadine, orphenadrine, pericyazine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, cetastine, setiptiline, trazodone, tripelennamine. Cetirizine and diphenhydramine are most preferred. H2 histamine blockers Cimetidine, famotidine, lafutidine, nizatidine, ranitidine, roxatidine, tiotidine Mast cell stabilizers Cromolyn sodium, nedocromil Corticosteroids (preferred) Prednisone (Deltasone, Orazon) Prednisolone (Miripred) Methylprednisolone (Medrol, Depo-Medrol, Solu-Medrol) Betamethasone (Celestone) Dexamethasone (Dexamethasone Intensol) Budesonide (Entocort EC) Triamcinolone acetonide (Aristospan intra-articular, Aristospan intra-articular, Kenalog) Examples of steroids Progesterone-type steroids: Fulgestone, fluorometholone, medrysone (hydroxymethylprogesterone), prebediolon acetate, chlormadinone acetate, cyproterone acetate, medrogestone, medroxyprogesterone acetate, megestrol acetate, segesterone acetate. Hydrocortisone-type steroids: Chloroprednisone, cloprednol, difluprednate, fludrocortisone, fluocinolone, fluperolone, fluprednisolone, loteprednol, methylprednisolone, prednicarbate, prednisone, prednisolone, tixocortol, triamcinolone. Methasone-type steroids: Alclometasone, betamethasone, beclomethasone, clobetasol, clobetasone, clocortolone, desoximetasone, dexamethasone, diflorasone, difluocortolone, fluchlororone, flumethasone, fluocortin, fluocortolone, fluprednidene, fluticasone, fluticasone furoate, halometasone, meprednisone, mometasone, mometasone furoate, paramethasone, prednylidene, rimexolone, urobetasol (halobetasol), Acetonides and related steroids: Amcinonide, budesonide, ciclesonide, deflazacort, desonide, formocortal (fluoroformirone), fluchlorone acetonide (flucloronide), fludroxycortide (flurandrenolon, flurandrenolide), flunisolide, fluocinolone acetonide, fluocinonide, halcinonide, triamcinolone acetonide. Other steroids: Cortivazol, RU-28362 (6-methyl-11β,17β-dihydroxy-17α-(1-propynyl)androsta-1,4,6-trien-3-one) CD117 kinase activity inhibitors (preferred): Axitinib, cabozatinib, dasatinib, flumatinib, imatinib, imetelstat, masatinib, midostaurin, nilotinib, pazopanib, sorafenib, sunitinib, toceranib, Acetaminophen Nonsteroidal anti-inflammatory drugs (NSAIDs) Salicylates such as aspirin, diflunisal, salicylic acid and its salts, and salsalate; Propionic acid derivatives such as ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, perbiprofen, zaltoprofen, fenbufen, tiaprofenic acid, and carprofen Acetic acid derivatives such as indomethacin, acemetacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, fenclofenac, aceclofenac, bromfenac, fentiazac, and nabumetone; Enolic acid (oxicam) derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, and phenylbutazone; Anthranilic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, etofenamate; Selective COX-2 inhibitors such as celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, and firocoxib; Sulfonanilides such as nimesulide.

[0073] Other possible pretreatments Janus kinase inhibitors Tofacitinib (Xeljanz) Calcineurin inhibitors Cyclosporine (Neoral, Sandimmune, Sangutia) Tacrolimus (Astagraf XL, Envalsus XR, Prograf) mTOR inhibitors Sirolimus (Rapamune) Everolimus (Afinitor, Zotres) IMDH inhibitors Azathioprine (Azasan, Imuran) Leflunomide (Arava) Mycophenolate (Cellcept, Myfortic) Biologics Abatacept (Orencia) Adalimumab (Humira) Anakinra (Kineret) Certolizumab (Cimzia) Etanercept (Enbrel) Golimumab (Simpony) Infliximab (Remicade) Ixekizumab (Tartz) Natalizumab (Tysabri) Rituximab (Rituxan) Secukinumab (Cosentyx) Tocilizumab (Actemra) ustekinumab (Stelara) Vedolizumab (Entyvio) Basiliximab (Simulect) Daclizumab (Zimbryta) [Example]

[0074] Materials and Methods <sirna> The following custom siRNAs were manufactured by Horizon. siCD45 siRNA sequence Sense strand: mCmUGGmCmUGAAmUmUmUmCAGAGmCAdTdT Antisense strand: UGCUCUGAAAUUmCAGCmCAGdTdT The abbreviations for nucleotide monomers used in representing nucleic acid sequences. When present in an oligonucleotide, these monomers are understood to be linked together by 5'-3'-phosphodiester bonds. Abbreviations Nucleotides A: adenosine 3'-phosphate C: cytidine-3'-phosphate G: Guanosine-3'-phosphate U: uridine-3'-phosphate mA: 2'-O-methyladenosine-3'-phosphate mC: 2'-O-methylcytidine-3'-phosphate mG: 2'-O-methylguanosine-3'-phosphate mU: 2'-O-methyluridine-3'-phosphate dT: 2'-deoxythymidine-3'-phosphate dTs: 2'-deoxythymidine-3'-phosphorothioate

[0075] <mrna> Cre mRNA can be purchased from TriLink.

[0076] <Constituents of lipid composition> [ka] [ka] DMG-mPEG2000: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 DPG-mPEG2000: 1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-2000 DSG-mPEG2000: 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 [ka]

[0077] <Chemical structure of ionizable lipids> [ka] JPEG2025535815000040.jpg212170JPEG2025535815000041.jpg223170JPEG2025535815000042.jpg24217 0JPEG2025535815000043.jpg185170JPEG2025535815000044.jpg244170JPEG2025535815000045.jpg58170

[0078] <Production of lipid nanoparticles> The siRNA was diluted in 10 mM citrate buffer (pH 3.0) (aqueous phase) while an appropriate amount of lipid was co-dissolved in 200-proof ethanol (ethanol phase). Nanoparticles prepared via a microfluidic device were synthesized at a ratio of 3:1 (v / v) of aqueous phase to ethanol phase. The lipid nanoparticles were then dialyzed against PBS containing 10 mM EDTA in a 20 kDa MWCO cassette overnight at 4 °C or room temperature.

[0079] <Binding of antibodies to lipid nanoparticles> Anti-CD117 antibody (clone 2B8, Bio X Cell) was reduced with 5 equivalents of TCEP (tris(2-carboxyethyl)phosphine) (10 mM in PBS). Similarly, rat IgG2b isotype control (anti-keyhole limpet hemocyanin, Bio X Cell) was reduced with 5 equivalents of 10 mM TCEP. The antibody was reduced by incubating at 37°C for 1 hour with gentle shaking. After incubation, excess TCEP was removed using a Zeba 7k MWCO desalting column. The maleimide-modified lipid nanoparticle dispersion was mixed with a reduced antibody solution at a molar ratio of 1:100 to 1:5. The resulting mixture was placed on an end-over-end mixer at room temperature for 1-2 hours to allow free thiols to bind to the maleimides on the LNPs. The mixture was then stored at 4°C until further purification.

[0080] <Gel filtration purification of antibody-bound lipid nanoparticles> The reaction mixture containing the antibody-conjugated lipid nanoparticles was loaded onto a gel filtration qEV column and fractionated using PBS as the mobile phase. The protein concentration of each fraction was measured to identify the fraction containing the antibody-conjugated lipid nanoparticles of interest. After collection, the antibody-lipid nanoparticle fractions were pooled and concentrated using an Amicon Ultra filter. The concentrated antibody-lipid nanoparticles were filtered through a 2 μm syringe filter and stored at 4°C.

[0081] <Particle size measurement> The particle size, PDI (polydispersity index), and zeta potential of the lipid nanoparticles were measured using a Zetasizer (Malvern). For size measurement, the lipid nanoparticles were diluted in PBS at a 1 / 200 v / v ratio, and the z-average value was reported. For zeta potential measurement, the lipid nanoparticles were diluted in PBS at a 1 / 200 (v / v ratio).

[0082] <Quantification and encapsulation of siRNA> The siRNA concentration in the dialyzed particles was measured using a modified QuantiT RiboGreen RNA quantification (Thermo Fisher). Nanoparticles of siRNA at ~1 ng μl in TE buffer (pH 8.5) were diluted, and siRNA standards were prepared in the range of 2 ng / μl to 0.125 ng / μl. 50 μL of each solution was added to separate wells of a 96-well black polystyrene plate. To each well, either 50 μL of TE buffer or 50 μL of 2% Triton-X in TE was added. The plate was incubated at 37 °C for 15 minutes with shaking at 350 rpm. After incubation, diluted RiboGreen reagent was added (100 μL per well), and the plate was incubated for 3 minutes as above. Ribogreen fluorescence was measured using a Tecan plate reader according to the attached protocol, and the nanoparticle siRNA concentration was measured using the siRNA standard. Two separate standards were made. One was the presence or absence of Triton-X. Particles in TE buffer were used to measure the unencapsulated siRNA concentration and TE-TX, and the encapsulation efficiency was determined by the following formula.

Chemical formula

[0083] <In vitro gene silencing> EML cells were cultured in IMDM medium supplemented with 20% HI-FBS, PenStrep (penicillin-streptomycin solution), and 200 ng / mL mouse stem cell factor (mSCF, Peprotech Inc.). Cells were plated in 100 μL of cell culture medium at 50,000 cells / well in a 96-well U-bottom plate. siRNA lipid nanoparticles were added to the cells at various concentrations and incubated for 40–48 hours. Three experiments (triplicates) were performed for each transfection condition. After incubation, the cell culture medium was removed, and the cells were washed with PBS. After washing, total RNA was extracted from EML cells using a 100 μL QuickExtract™ RNA Extraction Kit (Lucigen, Cat. No. QER090150). For qPCR, 2 μL of RNA extract was added per well of a 384-well plate to a master mix containing 0.5 μL of B2M TaqMan Probe (Applied Biosystems Cat. No. Mm00437762_m1) or 0.5 μL of CD45 TaqMan Probe (Applied Biosystems Cat. No. Mm01293577_m1) and 7 μL of Luna® Universal Probe One-Step RT-qPCR Kit (NEB Cat. No. E3006). Real-time PCR was performed in a Light Cycler 480 (Roche). Each duplix experiment was tested in two or three independent transfections, and each transfection was assayed in duplicate unless otherwise stated. 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 values ​​were determined using Graphpad Prism software.

[0084] <Bone marrow flow cytometry> Mice were sacrificed, and femurs and tibias were harvested. Bone marrow cells were collected by cutting the femurs and tibias in half and then centrifuging the bones in an Eppendorf tube at 13,000 × g for 45 seconds. The resulting pellet was resuspended in 1 mL of PBS, filtered through a 70 μm strainer, and washed with 10 mL of PBS. After centrifugation, bone marrow cells were lysed in 1 mL of RBC lysis buffer (Qiagen) for 10 minutes on ice with gentle shaking. After RBC lysis, bone marrow cells were transferred to a 96-well U-bottom plate for flow cytometry staining. Staining markers used were lineage markers (CD3, Gr-1, CD11b, CD45R / B220, mTer-119), CD117, Sca1, and CD45. Samples were analyzed on a BD LSR Fortessa II and data were analyzed in FlowJo.

[0085] Example 1: In vitro RNA delivery to CD117+ cells 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 in comparison with lipid nanoparticles without antibody modification. 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 of cell medium containing various concentrations of siRNA-LNP. Each experiment was performed in triplicate. After 40 hours, the cell medium was removed and the cells were washed with PBS. Total RNA was extracted from EML cells using the QuickExtract™ RNA Extraction Kit (Lucigen, Cat. No. QER090150). 2 μl of RNA extract was added to a master mix containing 0.5 μl of B2M TaqMan Probe (Applied Biosystems Cat. No. Mm00437762_m1) or 0.5 μl of CD45 TaqMan probe (Applied Biosystems Cat. No. Mm01293577_m1) and 7 μl of Luna® Universal Probe One-Step RT-qPCR Kit (NEB Cat. No. E3006) per well of a 384-well plate. Real-time PCR was performed on a Light Cycler 480 (Roche). Each duplicate experiment was tested in two or three independent transfections, and each transfection was assayed in duplicate unless otherwise noted. 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 values ​​were determined using Graphpad Prism software. The results are shown in Figure 1. Figure 1 shows in vitro evaluation of CD45 gene silencing in EML cells using aCD117-conjugated LNPs.

[0086] [Table 1]

[0087] Example 2: In vitro generalization of ionizable lipids To test the general applicability of these anti-CD117 lipid nanoparticles in vitro, various ionizable lipids were formulated into lipid nanoparticles containing siRNA against CD45, and the lipid nanoparticles were conjugated to either a murine CD117 antibody or a rat IgG2b isotype control antibody. After conjugation, the lipid nanoparticles were added to EML cells at 50 ng of siRNA per well. Forty hours after transfection, the cells were washed with PBS, resuspended, and dissolved in QuickExtract RNA solution. CD45 knockdown in the treated cells was assayed using qPCR using the ΔΔCt method. The results are shown in Figure 2. Figure 2 demonstrates that knockdown of CD45 by aCD117-receptor interaction can be generalized to other ionizable lipids. The data demonstrated that conjugation with anti-CD117 antibodies resulted in effective delivery of all ionizable lipids tested. Lipid nanoparticles conjugated with an isotype control antibody did not result in silencing of gene expression, indicating that delivery is mediated through the interaction of the anti-CD117 antibody with a receptor.

[0088] [Table 2]

[0089] Example 3: In vivo RNA delivery to bone marrow CD117+ hematopoietic stem / progenitor cells (DMG and DSG) The ability of anti-CD117 antibody-modified lipid nanoparticles to deliver RNA to HSPCs in vivo was evaluated in C57BL / 6 mice. Lipid nanoparticles containing siCD45 and PEG lipids of various alkyl chain lengths were formulated. To examine the effect of lipid nanoparticle circulation time on delivery to bone marrow cells, different alkyl chain lengths, DMG (C14) and DSG (C18), were included. After formulation, the lipid nanoparticles were conjugated to a mouse anti-CD117 antibody. After conjugation, unreacted antibody was removed by size exclusion chromatography. Lipid nanoparticles were administered to mice via intravenous tail vein injection at a dose of 1 mg / kg (20 μg) siRNA. After 72 hours, mice were sacrificed, and bones (femurs + tibias) were processed into single-cell suspensions and 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 lineage marker-negative and double-positive for Sca1 and c-Kit (also known as LSK cells). Within this LSK population, MFI of CD45 was analyzed to determine silencing. The results are shown in Figure 3, which shows CD45 expression levels in bone marrow LSK cells quantified by flow cytometry. Both DMG and DSG formulations demonstrated silencing in bone marrow LSK cells. The use of DSG in the formulation demonstrated higher silencing levels (60%) compared to the silencing observed with the DMG-containing formulation (30%). Lipid nanoparticles modified with anti-CD117 antibody enabled effective RNA delivery to HSPCs in vivo. These results indicate that lipid nanoparticles with longer circulation times are more suitable for delivery.

[0090] [Table 4]

[0091] To further evaluate mRNA delivery to bone marrow CD117+ hematopoietic stem and progenitor cells, we formulated Cre recombinase mRNA into anti-CD117-conjugated LNPs and administered them to Ai14 reporter mice. These mice contain a LoxP-flanked STOP cassette that blocks transcription of the fluorescent TdTomato protein. Upon Cre recombination, cells express TdTomato, allowing analysis of functional mRNA delivery at the cellular level by flow cytometry. Mice were injected via the tail vein with CremRNA-containing anti-CD117 LNPs or PBS. 48 hours after injection, mice were sacrificed, and bones (femurs and tibias) were harvested and processed into single-cell suspensions for flow cytometry analysis. TdTomato fluorescence was assessed in HSPCs (defined as Lin-Sca1+c-Kit+) and long-term hematopoietic stem cells (or LT-hematopoietic stem cells, defined as Lin-Sca1+c-Kit+CD34-CD135-). LT-hematopoietic stem cells are capable of self-renewal and are the most important biological cells when considering stem cell therapy. Figure 4 demonstrates CremRNA delivery to bone marrow HSPCs in vivo using the Ai14 mouse model. LNPs conjugated to aCD117 demonstrate very high levels (>90%) of mRNA delivery to bone marrow HSPCs and LT-HSCs. As shown in Figure 4, aCD117-LNPs can achieve high levels of mRNA delivery and Cre recombination (approximately 90%) to both HSPCs and LT-HSCs. Furthermore, approximately 50% of all bone marrow cells expressed TdTomato.

[0092] Example 4: In vivo RNA delivery to bone marrow CD117+ hematopoietic stem and progenitor cells 2 (DPG(C16) and DSG(C18)) To further evaluate the effect of alkyl chain length of the PEG lipid on the delivery efficacy of aCD117-LNP, we compared DPG-PEG (C16), DMG-PEG (C14), and DSG-PEG (C18) formulations. LNPs were formulated with different PEG lipids containing siRNA against CD45. Furthermore, to track the differences in uptake between these formulations, LNPs were fluorescently labeled with the lipophilic dye DiR. LNPs were administered to mice via the tail vein at a dose of 1 mg / kg. 72 hours after administration, bone marrow was harvested and processed into single-cell suspensions. Uptake and silencing in bone marrow HSPCs were analyzed by flow cytometry. The results are shown in Figure 5. Figure 5 shows that (left) LNP uptake in HSPCs or LT-HSCs correlates with longer alkyl chain lengths. DSG-PEG (C18) has the highest level of uptake in the target cell population. (right) Levels of functional gene silencing in HSPCs with different alkyl chain lengths.

[0093] [Table 5]

[0094] Example 5: In vivo generalization of ionizable lipids To investigate the in vivo versatility of these anti-CD117 lipid nanoparticles, various ionizable lipids were formulated into LNPs containing siRNA against CD45 and conjugated with an antibody against mouse CD117. LNPs were administered to mice via the tail vein at a dose of 1 mg / kg. 72 hours after administration, bone marrow was harvested and processed into a single-cell suspension. CD45 silencing in bone marrow HSPCs was analyzed by flow cytometry. The formulations listed in Table 6 were prepared and tested. The results are shown in Figure 7, which demonstrates functional knockdown of CD45 in HSPCs. LNPs were injected at a low dose of 0.3 mg / kg RNA to assess the effect of ligand density on RNA delivery. Both uptake and silencing demonstrated optimal ligand density for RNA delivery using anti-CD117-conjugated lipid nanoparticles. These data demonstrate that conjugating anti-CD117 enables effective in vivo delivery using all ionizable lipids tested.

[0095] [Table 6]

[0096] Example 6: Optimization of antibody density in vivo (or in vitro) To optimize antibody density on the LNP surface, Ab-LNPs were prepared using different molar ratios of maleimide to antibody during conjugation (Table 7). Using more antibody during the conjugation reaction resulted in more Abs present on the LNP surface. Furthermore, Ab-LNPs were labeled with the lipophilic dye DiR to track nanoparticle uptake in vivo. These fluorescently labeled LNPs containing siRNA against CD45 were injected intravenously into mice at 0.3 mg / kg. 72 hours after injection, bone marrow was harvested, and both CD45 uptake and knockdown in HSPCs (Lin-Sca1+cKit+ cells) were assessed by flow cytometry. The results are shown in Figure 7. Figure 7 shows (left) uptake in HSPCs as measured by the percentage of LSK cells that are DiR+ and (right) functional knockdown of CD45 in HSPCs. LNPs were injected at a low dose of 0.3 mg / kg RNA to assess the effect of ligand density on RNA delivery. Both uptake and silencing indicate an optimal ligand density for RNA delivery using anti-CD117-conjugated lipid nanoparticles.

[0097] [Table 7]

[0098] Using optimal ligand densities, we administered these Ab-LNPs to mice at 0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg to evaluate the in vivo dose response. 72 hours after LNP administration, mice were sacrificed, and bone marrow was harvested and processed into single-cell suspensions. For dose-curve analysis, we assessed nanoparticle uptake and silencing in various cell populations, including HSPCs, LT-HSCs, mature immune cells, and CD117 cells. The results are shown in Figure 8. Figure 8 (top panel) shows the uptake of LNPs in various cell populations in bone marrow as measured by the DiR positivity rate. Dose-dependent uptake was observed in HSPCs and LT-HSCs. Furthermore, LNPs mixed with free Ab (non-adjuvant) and isotype control LNPs (Iso-LNPs) showed no uptake in HSPCs or LT-HSCs. (bottom panel) Dose-response of functional siCD45 knockdown by Ab-LNP formulation.

[0099] Example 7: In vivo RNA delivery to mouse bone marrow HSPCs using various antibodies We also wanted to explore 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, IL-6R) and conjugated LNPs with antibodies against those receptors. Additionally, we also examined another CD117 clone (clone ACK2). Of the targets screened, only CD117 was effective for LNP uptake and RNA delivery (Figure 9, left). Other antibodies may not be suitable for targeted delivery to HSPCs due to factors such as the antibody clone used and receptor-dependent factors such as expression level, internalization rate, and expression levels in non-target tissues that may act as antigen sinks. Interestingly, CD117 showed inter-clone differences in Ab-LNP performance, indicating that the choice of antibody against a specific cellular target significantly impacts the usefulness of targeted delivery using antibody-modified lipid nanoparticles. Clone 2B8 is a non-antagonistic clone, whereas clone ACK2 is reported to be antagonistic. However, no depletion of bone marrow HSPCs was observed after Ab-LNP administration with either clone or Ab-LNP conjugated to any other antibody ( Figure 9 , right).

[0100] Example 8: In vitro RNA delivery to human primary HSPCs using non-antagonistic antibodies LNPs were formulated with the non-antagonistic anti-human CD117 antibody clone LMJ729, and cell viability was assessed in vitro using human primary bone marrow CD34+ cells. Table 8 and Figure 10

[0101] [Table 8]

[0102] Example 9: In vitro RNA delivery to human primary HSPCs using various receptor-antibody combinations To investigate whether other antibodies could be used for HSPC delivery, another antibody panel was tested. Firefly luciferase mRNA was encapsulated in LNPs conjugated with anti-human CD117 clone 104D2 (non-antagonistic), 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. The transfection efficiency of these LNPs was assessed using Steady-Glo. TM The antibody was tested in vitro using human primary bone marrow CD34+ HSPCs by quantifying luminescence using the Luciferase Assay System (Promega). As shown in Figure 11, all targeted LNP formulations tested demonstrated in vitro luciferase expression at a dose of 100 ng mRNA per 5,000 cells, but LNPs conjugated with an isotype control were not effective at transfecting these cells.

[0103] [Table 9]

[0104] Example 10: In vitro RNA delivery into mouse primary bone marrow or mesenchymal stem cells (MSCs) using anti-CD105-LNPs To verify that our targeted LNPs are also useful for other stem cells, we tested the RNA delivery efficiency of anti-CD105 LNPs in vitro using primary mouse bone marrow mesenchymal stem cells (MSCs). siRNA against murine integrin β1 (Itgb1) was encapsulated in anti-CD105 antibody-modified LNPs and unmodified LNPs as previously described. Frozen primary mouse bone marrow MSCs were obtained from Cell Biologics and cultured in RPMI medium containing 10% FBS. MSCs were then transferred to a 96-well plate at a density of 10,000–15,000 cells per well, and the culture medium was replaced with 90 μL of serum-free RPMI medium. MSCs were transfected with 10 μL of LNP solution containing 100 nM Itgb1 siRNA duplex. 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 in each condition were measured by RT-qPCR. The results are shown in Figure 12.

[0105] [Table 10]

[0106] Example 11: In vitro RNA delivery to human primary HSPCs using various ionizable lipids (2) We formulated Ab-LNPs targeting firefly luciferase mRNA with various ionizable lipids and used them in Steady-Glo TM Luminescence was assessed 24 hours after transfection using the Luciferase Assay System (Promega). As shown in Figure 13, all tested formulations showed higher luminescence than the PBS control at a dose of 100 ng mRNA per 5,000 cells.

[0107] [Table 11]

[0108] Example 12: Cre recombinase-mediated gene editing of bone marrow HSPCs in vivo results in long-term myeloid-lymphoid genetic conversion. We next evaluated the efficacy of anti-CD117 LNPs in mRNA delivery. To this end, we utilized a transgenic Ai14 mouse model containing a LoxP-flanked STOP cassette that blocks transcription of the fluorescent protein TdTomato. Upon Cre recombination (via Cre mRNA insertion), the STOP cassette is excised, and the cells constitutively express TdTomato (Figure 14a). We measured the level of gene editing in HSPCs after treatment with our Ab-LNPs using flow cytometry. Furthermore, we longitudinally tracked mature immune cells in the peripheral blood after LNP administration and examined TdTomato expression levels in edited offspring at 2, 4, 6, 8, and 14 weeks (Figure 14b). First, the dose response of anti-CD117 LNP (anti-mouse CD117, clone 2B8, ALC-0315) was evaluated at 0.1, 0.3, and 1 mg kg -1 The Cre mRNA dose was 0.3 mg kg -1 With a single dose of mRNA (6 μg), highly efficient delivery was observed with approximately 75% of TdTomato+ cells in both the HSPC and LT-HSC populations 48 hours after administration. Increasing the mRNA dose to 20 μg transfected almost all (~90%) of the HSPC and LT-HSC populations (Figure 14d). -1 Unconjugated LNPs at a Cre mRNA dose showed approximately 25% TdTomato expression, indicating that conventional non-targeting LNP formulations have a low ability to transfect HSPCs. Incorporation of HSPC-targeting ligands significantly improved transduction levels. The stemness of transfected HSPCs was maintained both short-term and long-term, as demonstrated by analysis of peripheral blood populations (erythroid, myeloid, B, and T cells). After 2 weeks, CD11b+ myeloid cells, consisting of granulocytes and monocytes, were already 90% TdTomato+. Because B and T cells are long-lived cells, TdTomato expression levels in these cell types naturally lagged behind myeloid cells at 2 weeks (-32% for B cells and -3.3% for T cells), but by 14 weeks had risen to -70% TdTomato+ B cells and -50% TdTomato+ T cells (Figure 14e). Analysis of T cell subsets (CD4 and CD8) was performed 4 weeks after LNP administration, when TdTomato expression levels in T cells became more apparent. At each time point, CD4 T cells had a higher population of TdTomato cells compared to CD8 T cells (Figure 14f). Erythrocytes also showed nearly 100% TdTomato expression at week 14 (Figure 14g). Overall, nearly all HSPCs were transfected, resulting in high levels of corrected progeny in all analyzed immune cell populations.

[0109] Example 13: PEG lipids Ab-LNP formulations containing Cre mRNA and different PEG lipids were administered at 0.3 mg kg -1 Ai14 mice were injected with a dose of 100 mg / kg / day. 48 hours after injection, the mice were sacrificed and the right hind limbs were harvested for flow cytometry analysis. TdTomato expression was evaluated in bone marrow LSK cells. The results are shown in Figure 15. Statistics were performed by one-way analysis of variance with Tukey's multiple comparison test (*P<0.05, **P<0.01).

[0110] Example 14: Dexamethasone pretreatment improved clinical signs after LNP administration. Mice were pretreated with PBS or 9 mg / kg dexamethasone (intraperitoneal injection) 1 hour before lipid nanoparticle (LNP) administration. Clinical examination was performed 6 hours after LNP administration. The results are shown in Table 13.

[0111] [Table 12]

[0112] [Table 13] < / mrna> < / sirna>

Claims

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

2. 2. The lipid composition of claim 1, wherein the lipid nanoparticles comprise a PEG lipid conjugated to a targeting molecule.

3. The lipid composition of claim 1, wherein the ionizable lipid has at least one ionizable amino group and at least one biodegradable group, and the biodegradable group is represented by -O(CO)O-, -O(CO)-, -(CO)O-, or S-S.

4. The lipid composition according to claim 1, wherein the ionizable lipid is a compound represented by the following formula (4): 【Chemistry 1】 During the ceremony, X is -NR １ - or -O-, R １ is a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R ２１ -L １ -R ２２ represents a group represented by -, and R ２１ represents a hydrocarbon group having 1 to 24 carbon atoms, and L １ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or 【Chemistry 2】 indicates R ２２ represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R ２ and R ３ are each independently a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R ３１ -L ２ -R ３２ represents a group represented by -, and R ３１ represents a hydrocarbon group having 1 to 24 carbon atoms, and L ２ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or 【Transformation 3】 indicates R ３２ represents a divalent linking group, 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 and R ５ , R １０ and R ５ , R ５ and R １２ , R ４ and R ６ , R ５ and R ６ , R ６ and R ７ , R ６ and R １０ , R １２ and R ７ , and R ７ and R ８ any one or more pairs of may be linked to each other to form a 4- to 7-membered ring optionally containing an O atom, The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, -NR ４５ R ４６ an amino group represented by the formula: ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ 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, 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, -NR ４５ R ４６ an amino group represented by —O(CO)O—R ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ 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, provided that a+b is 1 or more, and c+d is 1 or more.

5. The lipid composition of claim 1, wherein the ionizable lipid is a compound represented by formula (1). 【Chemistry 4】 During the ceremony, R １ and R ２ each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R ３ represents a hydrocarbon group having 2 to 8 carbon atoms, and R １ , R ２ and R ３ The hydrocarbon group represented by is —OH, COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , and O-R ５６ and optionally substituted with one or more substituents selected from R ４ represents a hydrocarbon group having 1 to 8 carbon atoms, R ５ and R ６ are each independently a hydrocarbon group having 1 to 8 carbon atoms, or R ８ -L １ -R ９ where R ５ and R ６ are both hydrocarbon groups having 1 to 8 carbon atoms, R ７ is -R １０ -L ２ -R １１ -L ３ -R １２ indicates, 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, R ５３ , R ５４ , R ５５ , and R ５６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ５８ may be substituted with The aryl group having 6 to 20 carbon atoms is —OH, —COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , -O-R ５６ or -(C1-C12 hydrocarbon group)-R ５７ may be substituted with R ５８ represents a hydrocarbon group having 1 to 12 carbon atoms, R ５７ is -OH, COOH, -NR ６１ R ６２ , -OC(O)OR ６３ , -C(O)O-R ６４ , —OC(O)—R ６５ , or -O-R ６６ Shows. 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, R ６３ , R ６４ , R ６５ , and R ６６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ６８ may be substituted with The aryl group having 6 to 20 carbon atoms is —OH, —COOH, —NR ６１ R ６２ , -OC(O)OR ６３ , -C(O)O-R ６４ , —OC(O)—R ６５ , -O-R ６６ or -(C1-C12 hydrocarbon group)-R ６７ may be substituted with R ６８ represents a hydrocarbon group having 1 to 12 carbon atoms, 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, R ９ , and R １２ The hydrocarbon group represented by is an aryl group, —OC(O)O—R ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , or S-R ５８ and R ５３ , R ５４ , R ５５ , and R ５８ is defined as above, R １１ The hydrocarbon group represented by is —OC(O)O—R ５３ , -C(O)O-R ５４ or —OC(O)—R ５５ and R ５３ , R ５４ , and R ５５ The definition of is as above.

6. The lipid composition of claim 1, wherein the ionizable lipid is a compound represented by formula (5). 【Transformation 5】 In the formula, 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 is a hydroxyl group, -G ２０ -CH(R ５５ ) (R ５６ ), -N(R ５８ ) (R ５９ ), or -G ２０ -R ６０ represents a 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, Substituent B is —N(R ６１ ) (R ６２ ) and 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 is -S-(CO)-NR ６４ - indicates R ６４ Ha,-L ３０ -G ２０ -CH(R ５５ ) (R ５６ ) represents a group represented by a represents 0 or 1; L ３０ represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G １０ is -O(CO)-, -(CO)O-, -O(CO)O- or -N(C(O)R ６３ ) - indicates 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 is —(CO)O —R ６５ , or -O(CO) -R ６５ represents a 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 represented by L ４０ represents a hydrocarbon group having 1 to 6 carbon atoms, R ６６ and R ６７ represents a hydrocarbon group or 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 compounds represented by the following formula: 【Transformation 6】 【change】 【change】 【change】 【change】 【change】

8. The lipid composition of claim 1 , wherein the lipid nanoparticles comprise a sterol.

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. 2. 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 for hematopoietic stem / progenitor cells is CD34, CD105, CD117, or CD184 (CXCR4).

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

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

18. A method for 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 a subject.

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

20. 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 side effects associated with administration of anti-CD117 antibody-modified LNPs, comprising administering a therapeutically effective amount of an inflammation-reducing agent to a subject prior to administering 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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