Ionizable lipids

Abstract

The present invention relates generally to the field of ionizable (also called cationic) lipids, and in particular provides a novel type of such lipid represented by formula (I). The present invention further provides a method for producing such lipids and their uses, particularly in the preparation of nanoparticle compositions, more particularly nanoparticle compositions containing nucleic acids. The present invention further provides vaccine formulations comprising the nanoparticle compositions based on the ionizable lipids disclosed herein.

Description

[Technical Field]

[0001] The present invention relates generally to the field of ionizable (also called cationic) lipids, and in particular to a novel type of such lipid represented by formula (I). The present invention further provides methods for producing such lipids and their uses, in particular in the preparation of nanoparticle compositions, more specifically in nanoparticle compositions containing nucleic acids. The present invention further provides vaccine formulations comprising nanoparticle compositions based on the ionizable lipids disclosed herein. [Background technology]

[0002] Nucleic acid-based drugs are being studied in an increasing number of therapeutic areas. Nevertheless, targeted delivery of nucleic acids such as plasmid DNA, messenger RNA, short interfering RNA, single guide RNA, and microRNA to tissues and cells remains a major challenge due to their negative charge, size, and instability. Numerous nanoparticle carrier systems have been studied to encapsulate and deliver nucleic acids. These nanoparticles need to combine efficient and stable encapsulation of nucleic acids during storage and in the extracellular environment with maximum cellular uptake and efficient release of the payload from endosomes to the cytosol.

[0003] Lipid-based nanoparticles are clinically used to deliver small interfering RNAs and mRNA vaccines, representing the most advanced class of RNA delivery vehicles. Lipid-based nanoparticles typically consist of cationic or ionizable lipids that can be protonated at acidic pH, helper phospholipids, PEGylated lipids, and sterols. Each component has a specific function related to the stability and activity of LNPs. Sterols and PEGylated lipids are essential for the structure and stability of LNPs, while phospholipids contribute to stability and endosomal evasion. Cationic ionizable lipids are thought to be the main drivers of activity and tolerability by controlling mRNA encapsulation, uptake into cells, and escape from endosomes. Although LNPs are effective nucleic acid delivery vehicles, they can induce dose-limiting toxicities such as complement activation-related pseudoallergies, inflammatory cytokine release, and cytotoxicity due to the accumulation of non-degradable ionizable lipids on the cell membrane. Therefore, further improvements to the chemistry of cationic or ionizable lipids are needed to enhance the efficacy and safety of LNP-delivered nucleic acid drugs. [Overview of the project] [Problems that the invention aims to solve]

[0004] In other words, the present invention relates to a new class of ionizable lipids, as defined by the claims, having improved properties compared to the currently available classes of ionizable lipids. [Means for solving the problem]

[0005] In a first embodiment, the present invention provides lipids, particularly ionizable lipids represented by formula (I): [ka] During the ceremony, R1 and R2 are independently -H and -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20Selected from alkynyl, wherein the -C 1-20 alkyl, -C 2-20 alkenyl, and -C 2-20 each of the alkynyl may optionally further contain one or more heteroatoms and / or may optionally be substituted with one to three -O-(C=O)-R7, -(C=O)-O-R7, C 1-20 alkyl, -C 2-20 alkenyl, and -C 2-20 alkynyl, and the total number of C atoms of R1 and R2 together is at least 8; R3 and R4 are each independently -C 1-6 alkyl; or R3 and R4 together with the N atom to which they are attached form a 5- to 10-membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may optionally further contain one or more additional N atoms and / or may optionally be substituted with one to three substituents selected from -C 1-6 alkyl; and, each of R5 and R6 is independently selected from -CH2- and -O-CH2-; each of R7 is selected independently from -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; the -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl may each optionally further contain one or more heteroatoms and / or may optionally be substituted with one to three -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; m and n are each independently integers selected from 1, 2, 3, and 4; X is selected from -O-, -S-, -S-S-, -O-(C=O)-, -O-(C=O)-O-, -(C=N-NH2)-, -O-CR8R9-O-, and -S-CR8R9-S-; R8 and R9 are -H and -C respectively. 1-6 Alkyl and -C 3-6 Selected independently from cycloalkyl groups; Y is selected from -NH- and -O-; Z is -C 1-6 It is alkylene.

[0006] In certain embodiments, the present invention provides lipids, in particular ionizable lipids as defined herein and represented by formula (II): [ka] During the ceremony, R3 and R4 are each independently set to -C 1-6 They are alkyl, or R3 and R4, together with the N atom to which they are bonded, form a 5-10 membered aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 It may be substituted with 1 to 3 substituents selected from alkyl groups; and R5 and R6 are selected independently from -CH2- and -O-CH2-, respectively; R7 is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Selected independently of alkynyl, and the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl may optionally further contain one or more heteroatoms and / or optionally 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 It may also be substituted with alkynyl; and The total number of carbon atoms in both R7 portions is at least 5; m and n are, independently, integers selected from 1, 2, 3, and 4; X is selected from -O-, -S-, -SS-, -O-(C=O)-, -O-(C=O)-O-, -(C=N-NH2)-, -O-CR8R9-O-, and -S-CR8R9-S-; R8 and R9 are -H and -C respectively. 1-6 Alkyl and -C 3-6 Selected independently from cycloalkyl groups; Y is selected from -NH- and -O-; Z is -C 1-6 It is alkylene.

[0007] In yet another embodiment, the present invention provides lipids, in particular ionizable lipids as defined herein and represented by any one of formulas (IIIa), (IIIb), or (IIIc): [ka] During the ceremony, R3 and R4 are each independently set to -C 1-6 They are alkyl, or R3 and R4, together with the N atom to which they are bonded, form a 5-10 member aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally C 1-6 It may be substituted with 1 to 3 substituents selected from alkyl groups; and R5 and R6 are selected independently from -CH2- and -O-CH2-, respectively; R7 is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Selected independently of alkynyl, and the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl may optionally further contain one or more heteroatoms and / or optionally 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl, -C2-20 It may also be substituted with alkynyl; and The total number of carbon atoms in both R7 portions is at least 5; m and n are, independently, integers selected from 1, 2, 3, and 4; R8 and R9 are -H and -C respectively. 1-6 Alkyl and -C 3-6 Selected independently from cycloalkyl groups; Y is selected from -NH- and -O-; Z is -C 1-6 It is alkylene.

[0008] The present invention further provides lipids, in particular ionizable lipids, selected from the list defined herein, including: [ka]

[0009] In further embodiments, the present invention provides lipids, in particular ionizable lipids as defined herein and represented by any of the formulas (IVa), (IVb), and (IVc): [ka] During the ceremony, R1 and R2 are independently -H and -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Selected from alkynyl, the -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Each alkynyl may optionally further contain one or more heteroatoms and / or optionally 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 It may be substituted with an alkynyl atom, and the total number of C atoms in R1 and R2 combined is at least 8; R3 and R4 are each independently set to -C 1-6 They are alkyl groups; or R3 and R4, together with the N atoms to which they are bonded, form a 5-10 member aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 It may be substituted with 1 to 3 substituents selected from alkyl groups; and Each of R5 and R6 is selected independently from -CH2- and -O-CH2-; Each of R7 is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Selected independently of Alkinnir; Said-C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl may optionally further contain one or more heteroatoms and / or optionally 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Often treated with alkinyl; m and n are, independently, integers selected from 1, 2, 3, and 4; R8 and R9 are -H and -C respectively. 1-6 Alkyl and -C 3-6 Selected independently from cycloalkyl groups; Y is selected from -NH- and -O-; Z is -C 1-6 It is alkylene.

[0010] In further embodiments, the present invention provides lipids, particularly ionizable lipids selected from the list defined herein, including: [ka]

[0011] In yet another embodiment, the present invention provides lipids, in particular ionizable lipids as defined herein, wherein the total number of carbon atoms in R1 and R2 combined is at least 14.

[0012] The present invention further provides lipids, in particular ionizable lipids as defined herein, wherein R5 and R6 are each -CH2-.

[0013] In further embodiments, the present invention provides lipids, particularly ionizable lipids as defined herein, where m and n are identical and are integers selected from 1, 2, 3, and 4, preferably 2.

[0014] In yet another embodiment, the present invention provides lipids, in particular ionizable lipids as defined herein, where Y is -NH-.

[0015] In a further embodiment, the present invention provides lipid nanoparticles or lipid nanoparticle compositions comprising lipids, particularly ionizable lipids as defined herein. The nanoparticle compositions may further comprise phospholipids, sterols, and PEG lipids.

[0016] In yet another embodiment of the present invention, the lipid nanoparticles or lipid nanoparticle composition as defined herein further comprises an active agent, in particular nucleic acids, preferably mRNA.

[0017] In a further embodiment, the present invention provides the use of lipids, in particular ionizable lipids as defined herein, in the production of lipid nanoparticles or lipid nanoparticle compositions.

[0018] In a final embodiment, the present invention provides a pharmaceutical composition comprising lipid nanoparticles or a lipid nanoparticle composition as defined herein and a pharmaceutically acceptable agent.

[0019] The present invention also provides pharmaceutical compositions as defined herein for use in human medicine and / or veterinary medicine.

[0020] In yet another embodiment, the present invention provides lipids, in particular ionizable lipids as defined herein and represented by formula (V): [ka] During the ceremony, R3 and R4 are each independently set to -C 1-6 They are alkyl, or R3 and R4, together with the N atom to which they are bonded, form a 5-10 membered aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 It may be substituted with 1 to 3 substituents selected from alkyl groups; and R5 and R6 are selected independently from -CH2- and -O-CH2-, respectively; R7 is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Selected independently of alkynyl, and the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl may optionally further contain one or more heteroatoms and / or optionally 1 to 3 -O-(C=O)-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 It may also be substituted with alkynyl; and The total number of carbon atoms in both R7 portions is at least 5; m and n are, independently, integers selected from 1, 2, 3, and 4; Y is selected from -NH- and -O-; Z is -C 1-6 It is alkylene.

[0021] The present invention further provides lipids, in particular ionizable lipids selected from the list defined herein, including: [Chemical formula]

[0022] In yet another embodiment, the present invention provides a lipid, particularly an ionizable lipid defined herein and represented by either formula (VIa) or (VIb): [Chemical formula] Wherein: R3 and R4 are each independently -C 1-6 alkyl, or R3 and R4 together with the N atom to which they are attached form a 5- to 10-membered aromatic or non-aromatic heterocyclic ring; said heterocyclic ring may optionally further contain one or more additional N atoms and / or may optionally be substituted with 1 to 3 substituents selected from -C 1-6 alkyl; and R5 and R6 are each independently selected from -CH2- and -O-CH2-; R7 is each independently selected from -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl, wherein each of said -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl may optionally further contain one or more heteroatoms and / or may optionally be substituted with 1 to 3 -O-(C=O)-R7, -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; and the total number of C atoms in both R7 moieties is at least 5; m and n are each independently an integer selected from 1, 2, 3, and 4; o and p are each independently an integer selected from 1 to 10; Y is selected from -NH- and -O-; Z is -C 1-6 It is alkylene.

[0023] In yet another embodiment, the present invention provides ionizable lipids selected from the following list: [ka] JPEG0007867217000011.jpg204101JPEG0007867217000012.jpg206108JPEG0007867217 000013.jpg206108JPEG0007867217000014.jpg207112JPEG0007867217000015.jpg20697 [Brief explanation of the drawing]

[0024] With particular reference to the drawings, it is emphasized that the details shown are for illustrative purposes only, illustrating different embodiments of the invention. They are presented to the effect that they provide what is considered to be the most useful and straightforward explanation of the principles and conceptual aspects of the invention. In this regard, no attempt has been made to show structural details of the invention in more detail than is necessary for a basic understanding of the invention. The description accompanied by the drawings will make it clear to those skilled in the art how some embodiments of the invention can actually be embodied.

[0025] [Figure 1] Relative mean fluorescence intensity of eGFP expression in HEK293T cells when incubated with LNPs at mRNA concentrations of 50 ng and 200 ng / well (measured as a multiple increase in eGFP MFI compared to untreated cells). [Figure 2-1] The relative MFI of eGFP expression upon transfection into various cell types, namely HEK293T cells (A), TS / A cells (B), CT26 cells (C), and B16F10 cells (D), is shown. [Figure 2-2] Same as above [Figure 2-3] Same as above [Figure 2-4] Same as above [Figure 3-1] The viability of various cell types after transfection with LNPs, namely HEK293T (A) and CT26 (B), is shown. [Figure 3-2] Same as above [Figure 4-1] The relative MFI of eGFP expression upon transfection of various cell types, namely HEK293T (A) and CT26 (B), by the LNPs shown. [Figure 4-2] Same as above [Figure 5-1] Fluc mRNA expression in CT26 tumors (A) or liver (B) after injection into the tumor, as measured by in vivo luminescence (photons / s / cm2 / sr), and body weight of mice before and after intratumoral administration of S-Ac-Dog and MC-3-based LNPs (C). [Figure 5-2] Same as above [Figure 5-3] Same as above [Figure 6-1] Fluc mRNA expression in B16F10 tumors (A) or liver (B) after injection into the tumor, as measured by in vivo luminescence (photons / s / cm2 / sr), and tumor / liver ratio of fluc expression after intratumoral injection of each LNP into B16F10 tumors (C). [Figure 6-2] Same as above [Figure 6-3] Same as above [Figure 7] The proportion of E7-specific CD8 T cells after intramuscular immunization was evaluated by flow cytometry using E7 mRNA encapsulated in LNPs containing each ionizable lipid. [Figure 8] The percentage of E7-specific CD8 T cells in the blood of C57BL / 6 mice measured by flow cytometry after intramuscular immunization with mRNA LNPs containing the indicated ionizable lipids. All LNPs were formulated with an ionizable lipid / DSPC / DMG-PEG2000 lipid molar ratio of 50 / 10 / 38.5 / 1.5. Mice underwent two immunizations with 5 μg mRNA on day 1 and day 7. [Figure 9] The thickness of the muscle at the injection site was measured before injection (d0), 1 day after injection (d1), and 4 days after injection (d4) for each LNP (E7 dose 5 μg). All LNPs were formulated with lipid / DSPC / DMG-PEG2000 with a lipid molar ratio of 50 / 10 / 38.5 / 1.5. [Figure 10] Anti-HA IgG1 and IgG2a antibody titers during intramuscular immunization with LNPs containing the indicated ionizable lipids. Mice underwent two intramuscular immunizations with mRNALNP (2 μg HA) on days 1 and 21. Blood samples were collected on days 21 and 35 to evaluate anti-HA antibody titers. The indicated LNPs containing ionizable lipids were formulated with ionizable lipids / DSPC / DMG-PEG2000 in a lipid molar ratio of 50 / 10 / 38.5 / 1.5. [Figure 11] Percentage of spleen IFNg-positive CD8 T cells during intramuscular immunization with LNPs containing the indicated ionizable lipids. Mice received two intramuscular immunizations with mRNA LNPs (2 μg HA) on days 1 and 21. Splenocytes were acquired on day 35 and either restimulated with a pool of overlapping HA peptides or left unstimulated. Subsequently, the percentage of IFNg+ CD8 T cells was measured by flow cytometry. [Figure 12] The magnitude of the E7-specific CD8 T cell response measured in blood after intramuscular vaccination with LNPs containing S-Ac7-Dog, S-Ac7-DHDa, or MC-3 as ionizable lipids. Mice were immunized twice with 5 μg of E7 mRNA on days 1 and 21. Blood samples were analyzed by flow cytometry on days 7 and 27. [Figure 13] Flow cytometry evaluation of the percentages of IFNg+, IFNg+ TNFα+, IFNg+ granzyme b (Grnz)+, and IFNg+ CD107+ CD8 T cells in the spleen after in vitro stimulation with the E7-derived peptide RAHYNIVT. Mice were immunized twice with 5 μg of E7 mRNA on day 1 and day 21. [Figure 14]Percentages of Cy5-positive macrophages, dendritic cells (cDC1 and cDC2 subsets), B cells, and T cells (CD4+ and CD8+ T cell subsets) in the spleen, measured by flow cytometry 24 hours after intravenous administration to C57BL / 6 mice. Mice were administered 10 μg of peptide and 10 μg of IMDQ or an equivalent dose. n=3. [Figure 15-1] Percentages of activated marker-positive dendritic cells (cDC1 and cDC2 subsets), B cells, and T cells (CD4+ and CD8+ T cell subsets) in the spleen, measured by flow cytometry 24 hours after intravenous administration to C57BL / 6 mice. Mice were administered 10 μg of peptide and 10 μg of IMDQ or an equivalent dose. n=3. [Figure 15-2] Same as above [Figure 16] Percentage of tetramer-positive CD8+ T cells in the blood, measured by flow cytometry one week after two intravenous doses (with a 2-week interval) in C57BL / 6 mice. Mice were administered 10 μg of peptide and 10 μg of IMDQ or an equivalent dose. n=5. [Figure 17] Anti-S1 spike protein IgG antibody titer in intramuscular immunization using LNPs containing S-Ac7-DOg as an ionizable lipid. C57BL / 6 mice were injected once with LNPs containing 25 μg of the TLR3 agonist polyI:C. 25 μg of S1 spike protein was either mixed in or bound to the LNP surface via His6-Ni2+ interaction. Blood samples were collected 7 days after immunization and analyzed by ELISA. [Figure 18] Anti-ovalbumin (OVA) IgG antibody titers in response to intramuscular immunization using LNP containing S-Ac7-DOg as an ionizable lipid. C57BL / 6 mice were injected once with LNP containing 10 μg of the TLR9 agonist CpG. 50 μg of OVA was mixed with the LNP, and blood samples were collected 7 days after immunization and analyzed by ELISA. [Modes for carrying out the invention]

[0026] Next, the present invention will be described further. Various aspects of the present invention will be defined in more detail in the following description. Each of the aspects defined in this way may be combined with any other aspect unless otherwise explicitly stated. In particular, any feature indicated as preferred or advantageous may be combined with another feature indicated as preferred or advantageous.

[0027] Unless otherwise indicated by context, the asterisk is used herein to indicate the point in which a depicted monovalent or divalent radical binds to a structure in which it is involved and in which it forms part.

[0028] As already mentioned above, in a first embodiment, the present invention provides lipids, in particular ionizable lipids represented by formula (I): [ka] During the ceremony, R1 and R2 are independently -H and -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Selected from alkynyl, the -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Each alkynyl may optionally further contain one or more heteroatoms and / or optionally 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 It may be substituted with an alkynyl atom, and the total number of C atoms in R1 and R2 combined is at least 8; R3 and R4 are each independently set to -C 1-6They are alkyl; or R3 and R4, together with the N atom to which they are bonded, form a 5-10 member aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally C 1-6 It may be substituted with 1 to 3 substituents selected from alkyl groups; and, Each of R5 and R6 is selected independently from -CH2- and -O-CH2-; Each of R7 is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Selected independently of Alkinnir; Said-C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl may optionally further contain one or more heteroatoms and / or optionally 1 to 3 -O-(C=O)-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Often treated with alkinyl; m and n are, independently, integers selected from 1, 2, 3, and 4; X is selected from -O-, -S-, -SS-, -O-(C=O)-, -O-(C=O)-O-, -(C=N-NH2)-, -O-CR8R9-O-, and -S-CR8R9-S-; R8 and R9 are -H and -C respectively. 1-6 Alkyl and -C 3-6 Selected independently from cycloalkyl groups; Y is selected from -NH- and -O-; Z is -C 1-6 It is alkylene.

[0029] In other words, the present invention also provides lipids, and in particular ionizable lipids represented by formula (I): [ka] During the ceremony, R1 and R2 are independently -H and -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Selected from alkynyl, the -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Each alkynyl can optionally contain 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 It may be substituted with an alkynyl atom, and the total number of C atoms in R1 and R2 combined is at least 8; R3 and R4 are each independently set to -C 1-6 They are alkyl groups; or R3 and R4, together with the N atoms to which they are bonded, form a 5-10 member aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 It may be substituted with 1 to 3 substituents selected from alkyl groups; and, Each of R5 and R6 is independently -CH2-; Each of R7 is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Selected independently of Alkinnir; Said-C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl has 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Often treated with alkinyl; m and n are, independently, integers selected from 1, 2, 3, and 4; X is selected from -O-, -S-, -SS-, -O-(C=O)-, -O-(C=O)-O-, -(C=N-NH2)-, -O-CR8R9-O-, and -S-CR8R9-S-; R8 and R9 are -H and -C respectively. 1-6 Alkyl and -C 3-6 Selected independently from cycloalkyl groups; Y is selected from -NH- and -O-; Z is -C 1-6 It is alkylene.

[0030] When describing the compounds / lipids of the present invention, unless otherwise indicated by the context, the terms used should be interpreted according to the following definitions.

[0031] The term "alkyl" can refer to the compound C, either by itself or as part of another substituent. x H 2x+1 This refers to a fully saturated hydrocarbon of the formula (wherein x is a number of 1 or more). Generally, the alkyl groups of the present invention contain 1 to 20 carbon atoms. The alkyl groups may be linear or branched and may be substituted as shown herein. Where a subscript is used after a carbon atom below, the subscript indicates the number of carbon atoms that the specified group may contain. For example, C 1-4 Alkyl refers to an alkyl group with 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, butyl and their isomers (e.g., n-butyl, i-butyl, t-butyl); pentyl and its isomers, hexyl and its isomers, heptyl and its isomers, octyl and its isomers, nonyl and its isomers; decyl and its isomers, undecyl and its isomers, dodecyl and its isomers, tridecyl and its isomers, tetradecyl and its isomers, pentadecyl and its isomers, hexadecyl and its isomers, heptadecyl and its isomers, octadecyl and its isomers, nonadecyl and its isomers, eicosanyl and its isomers.

[0032] The term "optionally substituted alkyl" refers to an alkyl group in which one or more substituents (e.g., 1 to 4 substituents, e.g., 1, 2, 3, or 4 substituents) are optionally substituted at any available bond site. Non-limiting examples of such substituents include esters, carboxylic acids, alkyl moieties, alkene moieties, and alkyne moieties.

[0033] In the context of the present invention, the alkyl, alkene, and alkyne moieties as defined herein may further comprise one or more heteroatoms, for example, a carbon atom in the alkyl, alkene, or alkyne chain may be substituted with a heteroatom selected from N, S, or O.

[0034] As used herein, the terms “alkenyl” or “alkene” mean a linear, cyclic, or branched hydrocarbon radical containing at least one carbon-carbon double bond, unless otherwise specified. Examples of alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, hexenyl, and hexadienyl, whether at terminal or internal positions. Generally, the alkenyl or alkene moieties of the present invention contain 2 to 20 carbon atoms. Optionally substituted alkenyls refer to alkenyls having one or more substituents (e.g., 1, 2, 3, or 4) selected from those defined above for substituted alkyl groups.

[0035] As used herein, the terms “alkynyl” or “alkyne” mean a linear or branched hydrocarbon radical containing at least one carbon-carbon triple bond, unless otherwise indicated. Examples of alkynyl groups include ethynyl, E- and Z-propynyl, isopropynyl, E- and Z-butynyl, E- and Z-isobutynyl, E- and Z-pentynyl, E,Z-hexynyl, and the like. Generally, the alkenyl or alkene moieties of the present invention contain 2 to 20 carbon atoms. Optionally substituted alkynyls refer to alkynyls having one or more substituents (e.g., 1, 2, 3, or 4) selected from those defined above for substituted alkyls.

[0036] The term "cycloalkyl" refers to a cyclic alkyl group, i.e., a monovalent saturated or unsaturated hydrocarbyl group having one, two, or three cyclic structures, either alone or as part of another substituent. Cycloalkyls include all saturated or partially saturated (including one or two double bonds) hydrocarbon groups containing one to three rings, including monocyclic, bicyclic, or polycyclic alkyl groups. Cycloalkyls may contain three or more carbon atoms in the ring, and according to the present invention, generally contain 3 to 15 carbon atoms. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, adamantanyl, and cyclodecyl, with cyclopropyl being particularly preferred. "Optionally substituted cycloalkyl" refers to a cycloalkyl having one or more substituents (e.g., one to three substituents, e.g., one, two, three, or four substituents) selected from those defined above for substituted alkyls.

[0037] If a defined alkyl group is divalent, that is, has two single bonds to bond to two other groups, it is called an "alkylene" group. Non-limiting examples of alkylene groups include methylene, ethylene, methylmethylene, trimethylene, propylene, tetramethylene, ethylethylene, 1,2-dimethylethylene, pentamethylene, and hexamethylene. Similarly, if the alkenyl group and alkynyl group defined above are divalent groups having single bonds to bond to two other groups, they are called "alkenylene" and "alkynylene," respectively.

[0038] As used herein, either alone or as part of another group, the term “heterocyclic” refers to a non-aromatic fully saturated or partially unsaturated cyclic group having at least one heteroatom, at least one carbon-carbon-containing ring (e.g., a 3- to 13-membered monocyclic system, a 7- to 17-membered dicyclic system, or a 10- to 20-membered tricyclic system, or a total of 3- to 10 ring atoms). Each ring of a heterocyclic group containing heteroatoms may have 1, 2, 3, or 4 heteroatoms selected from nitrogen, oxygen, and / or sulfur atoms, the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heterocyclic group may be bonded to any heteroatom or carbon atom of the ring or ring system, insofar as the valence allows. The rings of a polycyclic heterocyclic group may be condensed, bridged, and / or bonded via one or more spiroatoms. An optionally substituted heterocycle refers to a heterocycle having one or more substituents (e.g., 1 to 4 substituents, or e.g., 1, 2, 3, or 4) selected from those defined above with respect to the substituted alkyl. Non-restrictive examples of heterocycles include piperidinyl, azepanyl, and morpholinyl.

[0039] As used herein, the term “aryl” (also referred to herein as an aromatic heterocycle) refers to a polyunsaturated aromatic hydrocarbyl group having a single ring (i.e., phenyl) or multiple aromatic rings fused to each other (e.g., naphthalene or anthracene) or covalently bonded, typically containing 6 to 10 atoms, with at least one ring being aromatic. The aromatic ring may optionally contain 1 to 3 additional rings fused to it (either cycloalkyl, heterocyclyl, or heteroaryl). Aryls are also intended to include partially hydrogenated derivatives of the carbocyclic systems listed herein. Non-limiting examples of aryls include phenyl, etc.

[0040] The aryl rings or heterocycles defined herein may be substituted with one or more substituents (e.g., 1 to 5 substituents, e.g., 1, 2, 3, or 4) at any available bond point. Non-limiting examples of such substituents include halogens, hydroxyls, oxo, nitros, aminos, hydrazines, aminocarbonyls, azides, cyanos, alkyls, cycloalkyls, alkenyls, alkynyls, cycloalkylalkyls, alkylaminos, alkoxys, -SO2-NH2, aryls, heteroaryls, aralkyls, haloalkyls, haloalkoxys, alkoxycarbonyls, alkylaminocarbonyls, heteroarylalkyls, alkylsulfonamides, heterocyclyls, alkylcarbonylaminoalkyls, aryloxys, alkylcarbonyls, acyls, arylcarbonyls, aminocarbonyls, alkylsulfoxides, -SO2R a (In the formula, R a The elements are selected from alkyl or cycloalkyl, alkylthio, carboxyl, etc.

[0041] When the carbon atoms of the aryl group are substituted with heteroatoms, the resulting ring is referred to herein as a heteroaryl ring.

[0042] As used herein, either alone or as part of another group, the term “heteroaryl” refers to a ring system comprising 5 to 12 carbon atoms in an aromatic ring, or 1 to 3 rings that are fused or covalently bonded, typically containing 5 to 8 atoms; at least one of which is aromatic, and one or more carbon atoms in these rings are substituted with oxygen, nitrogen, or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. Such rings may be fused to aryl, cycloalkyl, heteroaryl, or heterocyclyl rings. Non-limiting examples of such heteroaryls include pyridinyl, azepinyl, and the like.

[0043] An "optionally substituted heteroaryl" refers to a heteroaryl having one or more substituents (e.g., 1 to 4 substituents, e.g., 1, 2, 3, or 4) selected from those defined above for a substituted aryl.

[0044] As used herein, the term "oxo" refers to the =O group.

[0045] As used herein, the terms “alkoxy” or “alkyloxy” refer to the formula -OR b (In the formula, R b This refers to a radical having (where is alkyl). Preferably, the alkoxy is C1-C 10 These are alkoxy, C1-C6 alkoxy, or C1-C4 alkoxy. Non-limiting examples of suitable alkoxys include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, and hexyloxy. When the oxygen atom of the alkoxy group is substituted with sulfur, the resulting radical is called a thioalkoxy. A "haloalkoxy" is an alkoxy group in which one or more hydrogen atoms in the alkyl group are substituted with halogens. Non-limiting examples of suitable haloalkoxys include fluoromethoxy, difluoromethoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy, 1,1,2,2-tetrafluoroethoxy, 2-fluoroethoxy, 2-chloroethoxy, 2,2-difluoroethoxy, 2,2,2-trichloroethoxy; trichloromethoxy, 2-bromoethoxy, pentafluoroethyl, 3,3,3-trichloropropoxy, and 4,4,4-trichlorobutoxy.

[0046] The terms "carboxy," "carboxyl," or "hydroxycarbonyl" refer to a -CO2H group, either by itself or as part of another substituent. Thus, a carboxyalkyl is an alkyl group as defined above, having at least one substituent that is -CO2H.

[0047] The term "alkoxycarbonyl" refers to a compound that is attached to an alkyl radical, either by itself or as part of another substituent, i.e., -C(=O)OR e (In the formula, R e This refers to the carboxyl group that forms an alkyl group (as defined above).

[0048] The term "alkylcarbonyloxy" can be used by itself or as part of another substituent, such as -OC(=O)R e (In the formula, R e This refers to alkyl (as defined above).

[0049] Whenever the term “substituted” is used in this invention, it means that one or more hydrogen atoms on the atom indicated by the expression using “substituted” are replaced by a selection from the indicated group, provided that the valence does not exceed the normal valence of the indicated atom, and that the substitution results in a chemically stable compound, i.e., a compound robust enough to withstand isolation from the reaction mixture to a useful purity and formulation into a therapeutic agent.

[0050] If a group is optionally substituted, such a group may be substituted one or more times, preferably once, twice, or three times. The substituents can be selected from the group including, for example, halogens, hydroxyls, oxos, nitros, amides, carboxyls, aminos, cyanohaloalkoxys, and haloalkyls.

[0051] As used herein, terms such as "optionally substituted alkyl, aryl, or cycloalkyl" or "optionally substituted alkyl, aryl, or cycloalkyl" refer to optionally substituted alkyl, optionally substituted aryl, and optionally substituted cycloalkyl.

[0052] Furthermore, if a group is divalent, that is, if it has two single bonds to bond to two other groups, each of its occurrences can be in either direction within the molecule, even if not specifically indicated in the structural formula or definition of the R group. For example, -CH2- and -O-CH2- as part of R5 means that R5 may be represented not only by -O-CH2-O-CH2-, -CH2-O-CH2O-, but also by -O-CH2-CH2-O-, etc. In particular, any combination of chemically feasible -CH2-, -O-CH2-, and -CH2-O- moieties is assumed for R5 and R6 in the context of the present invention.

[0053] In the context of this invention, the term lipid means a chemically defined substance that is insoluble in water but soluble in alcohols, ethers, and chloroform, among other things. Ionizable lipids or cationic lipids are lipids that typically consist of three sections: an amine head group, a linker moiety, and a hydrophobic tail. In the context of compounds or lipids, the term "ionizable" (or cationic) means that ions (usually H) are present. + This means the presence of uncharged groups in the compound or lipid that can dissociate by generating ions and thus become positively charged themselves. Alternatively, any uncharged groups in the compound or lipid may generate electrons and thus become negatively charged.

[0054] In the context of the present invention, the linker portion can be selected from a variety of different linkers, but disulfide, ketal, and ether linkers are particularly preferred. That is, in order to obtain lipid characteristics, the compound of the present invention contains lipid tails represented by R1 and R2, and the total number of carbon atoms of both groups combined is at least 8, for example, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20. That is, in the context of the present invention, for example, R1 may contain 3 carbon atoms, while R2 may contain 5 carbon atoms, so the total number of carbon atoms of both groups combined is at least 8. This also means that R1 and R2 do not have to be the same, but in certain embodiments they may be the same as each other.

[0055] The present invention provides two distinct categories of lipids: lipids in which the lipid tail is directly bonded to the amide moiety (represented by formulas IVa, IVb, and IVc) and lipids in which the lipid tail is bonded to the amide moiety via a carboxylic acid-containing linker moiety (represented by formulas II, IIIa, IIIb, and IIIc).

[0056] In other words, in certain embodiments, the present invention provides lipids, in particular ionizable lipids as defined herein and represented by formula (II): [ka] During the ceremony, R3 and R4 are each independently set to -C 1-6 They are alkyl, or R3 and R4, together with the N atom to which they are bonded, form a 5-10 membered aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 It may be substituted with 1 to 3 substituents selected from alkyl groups; and R5 and R6 are -CH2- respectively; R7 is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 It is selected independently of alkinyl, Said-C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl can optionally contain 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 It may also be substituted with alkynyl; and The total number of carbon atoms in both R7 portions is at least 5; m and n are, independently, integers selected from 1, 2, 3, and 4; X is selected from -O-, -S-, -SS-, -O-(C=O)-, -O-(C=O)-O-, -(C=N-NH2)-, -O-CR8R9-O-, and -S-CR8R9-S-; R8 and R9 are -H and -C respectively. 1-6 Alkyl and -C 3-6 Selected independently from cycloalkyl groups; Y is selected from -NH- and -O-; Z is -C 1-6 It is alkylene.

[0057] In yet another embodiment, the present invention provides lipids, in particular ionizable lipids as defined herein and represented by any one of formulas (IIIa), (IIIb), or (IIIc): [ka] During the ceremony, R3 and R4 are each independently set to -C 1-6They are alkyl, or R3 and R4, together with the N atom to which they are bonded, form a 5-10 membered aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 It may be substituted with 1 to 3 substituents selected from alkyl groups; and R5 and R6 are -CH2- respectively; R7 is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Selected independently of alkynyl, and the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl can optionally contain 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 It may also be substituted with alkynyl; and The total number of carbon atoms in both R7 portions is at least 5; m and n are, independently, integers selected from 1, 2, 3, and 4; R8 and R9 are -H and -C respectively. 1-6 Alkyl and -C 3-6 Selected independently from cycloalkyl groups; Y is selected from -NH- and -O-; Z is -C 1-6 It is alkylene.

[0058] The present invention further provides lipids, in particular ionizable lipids, selected from the list defined herein, including: [ka]

[0059] In yet another embodiment, the present invention provides lipids, in particular ionizable lipids as defined herein and represented by any of the formulas (IVa), (IVb), and (IVc): [ka] During the ceremony, R1 and R2 are independently -H and -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Selected from alkynyl, the -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Each alkynyl can optionally contain 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 It may be substituted with an alkynyl atom, and the total number of C atoms in R1 and R2 combined is at least 8; R3 and R4 are each independently set to -C 1-6 They are alkyl groups; or R3 and R4, together with the N atoms to which they are bonded, form a 5-10 member aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 It may be substituted with 1 to 3 substituents selected from alkyl groups; and R5 and R6 are each -CH2-; m and n are, independently, integers selected from 1, 2, 3, and 4; R8 and R9 are -H and -C respectively. 1-6 Alkyl and -C 3-6 Selected independently from cycloalkyl groups; Y is selected from -NH- and -O-; Z is -C 1-6 It is alkylene.

[0060] In further embodiments, the present invention provides lipids, particularly ionizable lipids selected from the list defined herein, including: [ka]

[0061] In yet another embodiment, the present invention provides lipids, in particular ionizable lipids represented by formula (V) as defined herein: [ka] During the ceremony, R3 and R4 are each independently set to -C 1-6 They are alkyl, or R3 and R4, together with the N atom to which they are bonded, form a 5-10 membered aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 It may be substituted with 1 to 3 substituents selected from alkyl groups; and R5 and R6 are -CH2- respectively; R7 is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Selected independently of alkynyl, and the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl can optionally contain 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 It may also be substituted with alkynyl; and The total number of carbon atoms in both R7 portions is at least 5; m and n are, independently, integers selected from 1, 2, 3, and 4; Y is selected from -NH- and -O-; Z is -C 1-6 It is alkylene.

[0062] The present invention further provides lipids, in particular ionizable lipids selected from the list defined herein, including: [ka]

[0063] In yet another embodiment, the present invention provides lipids, in particular ionizable lipids represented by either formula (VIa) or (VIb) as defined herein: [ka] During the ceremony, R3 and R4 are each independently set to -C 1-6 They are alkyl, or R3 and R4, together with the N atom to which they are bonded, form a 5-10 membered aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 It may be substituted with 1 to 3 substituents selected from alkyl groups; and R5 and R6 are -CH2- respectively; R7 is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Selected independently of alkynyl, and the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl can optionally contain 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 It may also be substituted with alkynyl; and The total number of carbon atoms in both R7 portions is at least 5; m and n are, independently, integers selected from 1, 2, 3, and 4; o and p are each independently an integer selected from 1 to 10; Y is selected from -NH- and -O-; Z is -C 1-6 an alkylene.

[0064] In a very specific embodiment of the present invention, one or more of the following apply: R1 and R2 are each independently -H, -C 1-20 alkyl, -C 2-20 alkenyl, and -C 2-20 alkynyl, wherein each of said -C 1-20 alkyl, -C 2-20 alkenyl, and -C 2-20 alkynyl may optionally further contain one or more heteroatoms and / or may optionally be substituted with one or three -O-(C=O)-R7, C 1-20 alkyl, -C 2-20 alkenyl, and -C 2-20 alkynyl, and the total number of C atoms of R1 and R2 combined is at least 8; R1 and R2 are each independently -H, -C 1-20 alkyl, -C 2-20 alkenyl, and -C 2-20 alkynyl, wherein each of said -C 1-20 alkyl, -C 2-20 alkenyl, and -C 2-20 alkynyl may optionally be substituted with one to three -O-(C=O)-R7, -(C=O)-O-R7, C 1-20 alkyl, -C 2-20 alkenyl, and -C 2-20 alkynyl, and the total number of C atoms of R1 and R2 combined is at least 8; R3 and R4 are each independently -C 1-6is alkyl; or R3 and R4 together with the N atom to which they are attached form a 5- to 10-membered aromatic or non-aromatic heterocyclic ring; said heterocyclic ring may optionally further contain one or more additional N atoms, and / or, optionally, -C 1-6 may be substituted with 1 to 3 substituents selected from 1-6 alkyl; and, each of R5 and R6 is independently selected from -CH2- and -O-CH2-; each of R5 and R6 is -CH2-; each of R7 is -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; said -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl may each optionally further contain one or more heteroatoms, and / or, optionally, 1 to 3 -O-(C=O)-R7, -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; each of R7 is -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; said -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl may each optionally be substituted with 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; m and n are each independently an integer selected from 1, 2, 3 and 4; X is selected from -O-, -S-, -S-S-, -O-(C=O)-, -O-(C=O)-O-, -(C=N-NH2)-, -O-CR8R9-O-, and -S-CR8R9-S-; each of R8 and R9 is -H, -C 1-6Alkyl and -C 3-6 Selected independently from cycloalkyl groups; Y is selected from -NH- and -O-; Z is -C 1-6 It is alkylene.

[0065] In another specific embodiment, the present invention provides lipids, particularly ionizable lipids selected from the list defined herein, including: [ka] JPEG0007867217000027.jpg201105JPEG0007867217000028.jpg189105JPEG00078672170 00029.jpg206105JPEG0007867217000030.jpg208108JPEG0007867217000031.jpg208108

[0066] All lipids as defined herein may exist as different isomers / stereoisomers. In particular, lipids as defined herein may exist in trans or cis configurations, such as when they contain double bonds. In one preferred embodiment, lipids as defined herein exist in cis configuration. In the context of the present invention, the term "cis" indicates that the functional groups are on the same side of the plane, and the term "trans" means that the functional groups are on opposite sides.

[0067] In yet another embodiment, the present invention provides lipids, in particular ionizable lipids as defined herein, wherein the total number of carbon atoms in R1 and R2 combined is at least 14, for example, at least 15, at least 17, at least 18, at least 19 or at least 20.

[0068] The present invention further provides lipids, in particular ionizable lipids as defined herein, wherein each R5 and R6 is independently -CH2-, i.e., both groups are -CH2-.

[0069] In a further embodiment, the present invention provides lipids, particularly ionizable lipids as defined herein, where m and n are the same and an integer selected from 1, 2, 3, 4, for example, 1, 2, 3, 4, and preferably 2.

[0070] In yet another embodiment, the present invention provides lipids, in particular ionizable lipids as defined herein, where Y is -NH-.

[0071] In a further embodiment, the present invention provides lipid nanoparticles or lipid nanoparticle compositions comprising lipids, in particular ionizable lipids as defined herein.

[0072] In the context of this invention, the term lipid nanoparticles (LNPs), also known as solid lipid nanoparticles, refers to nanoparticles containing lipids. These are commonly used as drug delivery systems or pharmaceutical formulations. LNPs as drug delivery media were first approved in 2018 and are currently used in several RNA-based vaccine candidates. Lipid nanoparticles are typically spherical with an average diameter of 10 to 1000 nanometers and have a lipid core matrix capable of solubilizing lipophilic molecules. The term lipid is used herein in a broad sense and includes triglycerides, diglycerides, monoglycerides, fatty acids, steroids (e.g., cholesterol), and waxes. Biological membrane lipids such as phospholipids, sphingomyelin, bile acids, and sterols are commonly used as stabilizers for LNPs.

[0073] In this specification, the term “nanoparticles” refers to any particles having a diameter suitable for systemic administration, particularly intravenous administration, of nucleic acids, typically having a diameter of less than 1,000 nanometers (nm), preferably less than 500 nm, more preferably less than 200 nm, for example, between 50 and 200 nm, and preferably between 80 and 160 nm.

[0074] In other words, in the context of the present invention, the nanoparticles disclosed herein further comprise one or more additional lipids, such as phospholipids, sterols, and / or PEG lipids, which may or may not act as stabilizers.

[0075] In the context of the present invention, the terms "PEG lipid" or "PEGylated lipid" mean any suitable lipid modified with a PEG (polyethylene glycol) group. Particularly suitable PEG lipids in the context of the present invention are characterized by being C18-PEG lipids, C14-PEG lipids (e.g., DMG-PEG or DMG-PEG2000), or C16-PEG lipids.

[0076] C18-PEG lipids contain a polyethylene glycol moiety that determines the molecular weight of the lipid and a fatty acid tail containing 18 carbon atoms. In certain embodiments, the C18-PEG2000 lipid is selected from a list including (distearoyl-based)-PEG2000 lipids such as DSG-PEG2000 lipid (2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000) or DSPE-PEG2000 lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]); or (dioleoil-based)-PEG2000 lipids such as DOG-PEG2000 lipid (1,2-dioleoil-rac-glycerol) or DOPE-PEG2000 lipid (1,2-dioleoil-sn-glycero-3phosphoethanolamine-N-[amino(polyethylene glycol)-2000]). [ka]

[0077] C14-PEG lipids contain a polyethylene glycol moiety that determines the molecular weight of the lipid and a fatty acid tail containing 14 carbon atoms. In certain embodiments, the C14-PEG2000 lipid is dimyristoyl-based and has two C14 tail portions, selected from a list of (dimiristoyl-based)-PEG2000 lipids such as DMG-PEG2000 lipid (1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol 2000) or 2-dimiristoyl-sn-glycero-3-phosphoethanolamine glycol-2000 (DMPE-PEG2000). [ka]

[0078] In the context of the present invention, the term "phospholipid" means a lipid molecule consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. The two components are most often linked by a glycerol molecule, and therefore, the phospholipids of the present invention are preferably glycerol-phospholipids. Furthermore, the phosphate group is often modified with a simple organic molecule such as choline (i.e., giving phosphocholine) or ethanolamine (i.e., giving phosphoethanolamine).

[0079] Suitable phospholipids within the scope of the present invention can be selected from the list including the following: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-didodecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinate-sn-glycero-3-phosphocholine (OChem PC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof.

[0080] In a more specific embodiment, the phospholipid is selected from the list including the following: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), and mixtures thereof.

[0081] In the context of the present invention, the term "sterol," also known as steroid alcohol, is a subgroup of steroids that are naturally present in plants, animals, and fungi, or that can be produced by certain bacteria. In the context of the present invention, any suitable sterol, preferably cholesterol, may be used, selected from the list including cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol, sitosterol, and stigmasterol.

[0082] In certain embodiments of the present invention, one or more of the following apply: - The LNP comprises approximately 35 mol% to 65 mol% of the ionizable lipid; - The LNP contains approximately 5 mol% to 25 mol% of the phospholipid; - The LNP contains approximately 0.5 mol% to 3.0 mol% of the PEG lipid; The balance is maintained by the amount of the aforementioned sterols.

[0083] In yet another embodiment of the present invention, the lipid nanoparticles or lipid nanoparticle composition as defined herein further comprises cargo molecules such as pharmaceutically active substances (e.g., small molecules), or biomolecules such as peptides, proteins, or nucleic acids. In a particular embodiment, the cargo may be nucleic acids such as DNA or RNA, preferably mRNA. In another particular embodiment, the cargo may be a TLR agonist such as the TLR3 agonist polyI:C, or the TLR9 agonist CpG.

[0084] Before being loaded onto lipid nanoparticles, the cargo molecules may be further modified to induce polyanionicity throughout the molecule. This can be done, for example, by attaching them to a Glu10 moiety, as illustrated in the Examples section. The Glu10 moiety is a 10-unit glutamic acid moiety that enhances the polyanionicity of the molecule it attaches to.

[0085] In other words, the lipid nanoparticles and lipid nanoparticle compositions of the present invention are particularly suitable for intracellular delivery of cargo molecules. Accordingly, the present invention provides for the use of lipid nanoparticles and lipid nanoparticle compositions as defined herein for intracellular delivery of cargo molecules.

[0086] In certain embodiments, the lipid nanoparticles or lipid nanoparticle compositions defined herein further comprise nucleic acids, preferably mRNA.

[0087] In the context of the present invention, “nucleic acid” refers to deoxyribonucleic acid (DNA), or preferably ribonucleic acid (RNA), more preferably mRNA. According to the present invention, nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. According to the present invention, nucleic acids may be single-stranded or double-stranded, in the form of linear or covalently bound molecules forming a ring. Nucleic acids can be used for introduction into cells, i.e., cell transfection, in the form of RNA, which can be prepared, for example, by in vitro transcription from a DNA template. Furthermore, RNA can be modified before application by sequence stabilization, capping, and / or polyadenylation.

[0088] In the context of this invention, the term "RNA" refers to a molecule containing ribonucleotide residues, preferably a molecule composed entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. This term includes isolated RNA such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide substances to or within the RNA, for example, to one or more nucleotides of the RNA. Nucleotides in an RNA molecule may also include non-natural nucleotides or non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. These modified RNAs are called analogs. Nucleic acids may be contained in a vector. As used herein, the term “vector” includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phages, viral vectors such as adenoviruses or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes, or analogues of natural RNA.

[0089] According to the present invention, the term "RNA" includes, and preferably relates to, "mRNA," meaning "messenger RNA," and relates to a "transcript" that is produced using DNA as a template and codes for a peptide or protein. mRNA typically includes a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR). mRNA has a limited half-life both intracellularly and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the present invention, RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to those skilled in the art. For example, various in vitro transcription kits are commercially available.

[0090] In further embodiments, the present invention provides a pharmaceutical composition comprising one or more LNPs as defined herein and a pharmaceutically acceptable agent such as a carrier or excipient. Such a pharmaceutical composition is particularly suitable as a vaccine. Accordingly, the present invention also provides a vaccine comprising one or more LNPs according to the present invention.

[0091] In the context of the present invention, the term “vaccine” as used herein means any formulation intended to provide adaptive immunity (antibody and / or T-cell response) to a disease. To this end, a vaccine as used herein comprises at least one nucleic acid molecule, e.g., an mRNA molecule encoding an antigen on which an adaptive immune response is initiated. This antigen may exist as a microorganism, a protein or peptide, or in a weakened or dead form of the nucleic acid-encoding antigen. In the context of the present invention, an antigen means a protein or peptide that is recognized as foreign by the host’s immune system and thereby stimulates the production of antibodies against it for the purpose of combating such antigen. Vaccines may be prophylactic (e.g., to prevent or mitigate the effects of a future infection by a natural or “wild” pathogen) or therapeutic (e.g., to actively treat or alleviate the symptoms of an ongoing disease). The act of administering a vaccine is referred to as vaccination.

[0092] The vaccine of the present invention can be used to induce an immune response, particularly an immune response against disease-related antigens or cells expressing disease-related antigens, such as an immune response against cancer. That is, the vaccine can be used for prophylactic and / or therapeutic treatment of diseases involving disease-related antigens or cells expressing disease-related antigens, such as cancer. Preferably, the immune response is a T-cell response. In one embodiment, the disease-related antigen is a tumor antigen. The antigen encoded by RNA contained in the nanoparticles described herein is preferably a disease-related antigen or induces an immune response against disease-related antigens or cells expressing disease-related antigens.

[0093] The present invention also provides LNPs, pharmaceutical compositions, and vaccines according to the present invention for use in human medicine or veterinary medicine. The use of LNPs, pharmaceutical compositions, and vaccines according to the present invention in humans or veterinary medicine is also intended. Finally, the present invention provides methods for the prevention and treatment of human and veterinary diseases by administering LNPs, pharmaceutical compositions, and vaccines according to the present invention to subjects in need.

[0094] The present invention further provides the use of LNPs, pharmaceutical compositions, or vaccines according to the present invention for immunogenic delivery of one or more nucleic acid molecules. Therefore, the LNPs, pharmaceutical compositions, and vaccines of the present invention are highly useful in the treatment of several human and veterinary diseases. Accordingly, the present invention provides LNPs, pharmaceutical compositions, and vaccines for use in the treatment of cancer or infectious diseases.

[0095] The lipid nanoparticles of the present invention can be prepared according to the protocols specified in the Examples section. More generally, LNPs can be prepared using methods including: - A step of preparing a first alcohol composition comprising the ionizable lipid, the phospholipid, the sterol, the PEG lipid, and a suitable alcohol solvent. - A step of preparing a second aqueous composition comprising one or more nucleic acids and an aqueous solvent, - A step of mixing the first composition and the second composition in a microfluidic mixing device.

[0096] More specifically, the lipid components are mixed at appropriate concentrations in an alcohol medium such as ethanol. An aqueous composition containing nucleic acids is then added to this mixture, and it is subsequently loaded into a microfluidic mixing device.

[0097] The objective of microfluidic mixing is to completely and rapidly mix multiple samples (i.e., lipid phase and nucleic acid phase) within a microscale apparatus. Such mixing of samples is typically achieved by enhancing the diffusion effect between different types of flows. For this purpose, several microfluidic mixing apparatuses can be used, such as those outlined in Lee et al., 2011. A particularly suitable microfluidic mixing apparatus according to the present invention is the Nano Assemblr from Precision Nanosystems.

[0098] Other techniques suitable for preparing the LNPs of the present invention include dispersing the components in a suitable dispersion medium, such as aqueous and alcoholic solvents, and applying one or more of the following methods: ethanol dilution, simple hydration, sonication, heating, vortexing, ether injection, French press, cholic acid, Ca 2+ Methods include fusion, freeze-thaw, reverse-phase evaporation, T-junction mixing, microfluidic hydrodynamic focusing, and staggered herringbone mixing.

[0099] The ionizable lipids of the present invention can be prepared according to the reaction schemes provided in the following examples, but those skilled in the art will understand that these are merely illustrative examples of the present invention and that they can be prepared by any of several standard synthetic processes commonly used by those skilled in organic chemistry. [Examples]

[0100] Example 1: Preparation of lipids

[0101] 1. General information

[0102] Unless otherwise specified, all glassware was oven-dried before use, and all reactions were carried out under an argon atmosphere using standard Schlenk technique. Dry solvents were purchased from Acros Organics or Sigma-Aldrich and used without further purification. All reagents were purchased from commercial suppliers and used without further purification unless otherwise specified. The progress of the reactions was monitored by thin-layer chromatography (TLC) performed on aluminum plates coated with 0.2 mm thick Kieselgel F254. Visualization was achieved by ultraviolet light (254 nm) or staining with potassium permanganate. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck and co.). Mass spectra were obtained using FinniganMAT 8200 (70 eV) or Agilent 5973 (70 eV) by electrospray ionization (ESI) or electron impulse ionization (EI). All 1H NMR and 13C NMR spectra were recorded with a Bruker AV-400 in chloroform-d1 or DMSO-d6. Chemical shifts are expressed in parts per million (ppm) relative to tetramethylsilane, using the solvent peak as the internal standard (CDCl3: 1 H = 7.26 ppm, 13 C = 77.16 ppm; CD3SOCD3: 1 H = 2.50 ppm, 13 C = 39.52 ppm). The coupling constant is expressed in Hz. 1 ¹H NMR resolution patterns were specified as singlets (s), broads (brd), doublets (d), triplets (t), quartets (q), pentets (p), sextets (se), septets (sep), octets (o), or combinations thereof. Uninterpretable resolution patterns were specified as multilines (m).

[0103] 2. Lipid synthesis

[0104] 2.1 The general synthesis pathway of lipids represented by the structure of Equation I, or more specifically, the structure of Equation IVa, is shown below.

[0105] [ka]

[0106] Synthesis of Compound 3 Amine 2 (1.0 equivalent) and Et3N (1.5 equivalents) were first dissolved in CHCl3 / hexane / THF (1 / 1 / 1). Next, the prepared solution was added dropwise at 0°C to a stirred solution of compound 1 (Amano et al., 2017) (3.0 equivalents) in CH2Cl2. The resulting mixture was vigorously stirred and warmed to room temperature over 2 hours. The solvent was then removed under vacuum, the remaining residue was dissolved in CH2Cl2, washed with saturated citric acid (aqueous solution) and brine, dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 15:1 to 10:1) to obtain compound 3. [ka] Yield: 62%. Colorless oil. 1 H NMR (400 MHz, chloroform-d) δ8.29 (d, J = 9.2Hz, 2H, H17 & H19), 7.40 (d, J = 9.3 Hz, 2H, H16 & H20), 4.54 (t, J =6.6 Hz, 2H, OCH2), 4.34 (t, J = 6.4 Hz,2H, OCH2), 3.18(brd, 4H, H22& H34), 3.03 (t, J = 6.6 Hz,2H, SCH2), 2.97 (t, J =6.4 Hz, 2H, SCH2), 1.54-1.48 (m, 4H, H23 & H35), 1.25 (brd,36H), 0.88 (t, J = 6.7 Hz, 6H, H33 & H45) ppm. 13C NMR (100 MHz, クロロホルム-d) δ 156.0 (C=O), 155.6 (Ar-C, quaternary), 152.5 (C=O), 125.5 (ArC-H), 121.9 (ArC-H), 67.0 (C2 or C7), 62.8 (C2 or C7), 47.8 (C22 orC34),47.2 (C22 or C34), 38.2 (C3 or C6), 36.9 (C3 or C6), 32.1, 29.81, 29.79,29.76,29.6, 29.5, 28.8 (C23 or C35), 28.3 (C23 or C35), 27.0, 22.8, 14.3 (C33& C45) ppm. LRMS(ESI) (m / z): [M+H] + (C 36 H 63 The calculated value of O7N2S2) is 699.4, and the measured value is: 699.4.

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[0107] Synthesis of Compound 5 A amine 4 (1.2 equivalents) was added to a stirred solution of compound 3 (1.0 equivalent) in CH2Cl2 at room temperature, followed by Et3N (1.5 equivalents). The reaction mixture was vigorously stirred at room temperature for 2 hours. The organic phase was first washed with saturated Na2CO3 (aqueous solution) until it turned off-white, then washed with brine, dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 3:1 to 1:1, then changed to CH2Cl2 / CH3OH = 15:1) to obtain compound 5. The name of compound 5 is shown immediately below its structure. [ka] Yield: 75%. Colorless oil. 1H NMR (400 MHz, クロロホルム-d) δ5.48 (brd, 1H,H12), 4.34-4.29 (m, 4H, OCH2), 3.30 (q, J = 5.8 Hz, 2H, H13), 3.21-3.14 (m, 4H, H20 & H32), 2.95-2.90 (m, 4H, H5 & H8), 2.48 (t, J =6.2 Hz, 2H, H14), 2.28 (s, 6H, H18 &H19), 1.54-1.47 (m, 4H, H21 &H33), 1.26 (s, 36H), 0.88 (t, J = 6.7 Hz,6H, H31 & H43) ppm. 13 C NMR (100 MHz, クロロホルム-d) δ 156.0 (C2 & C11), 63.0 (OCH2), 62.6 (OCH2), 58.3 (C14), 47.8 (C20 or C32), 47.2 (C20 orC32), 45.2(C18 & C19), 38.4(C13), 38.2 (C5 or C8), 38.0 (C5 or C8),32.1, 29.82,29.79, 29.76, 29.6, 29.5, 28.8 (C21 or C33), 28.3 (C21 orC33), 27.0, 22.8, 14.3(C31 &C43) ppm. LRMS (ESI) (m / z): [M+H) + (C 34 H 70 The calculated value of O4N3S2) is 648.5, and the measured value is: 648.4.

[0108]

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[0109]

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[0110]

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[0111]

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[0112]

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[0113] 2.2 The general synthesis pathway of lipids represented by the structure of formula I, or more specifically, the structure of formula IVb, is shown below. [ka]

[0114] Synthesis of Compound 7 Compound 6 (Shenoi et al., 2012) (3.58 g, 21.8 mmol, 1.0 equivalent) and Et3N (9.1 mL, 65.4 mmol, 3.0 equivalents) were dissolved in CH2Cl2 (20 mL) at 0°C in a stirred solution of 4-nitrophenyl chloroformate (11.0 g, 54.5 mmol, 2.5 equivalents) in CH2Cl2 (80 mL). The reaction mixture was vigorously stirred and warmed to room temperature over 12 hours. The reaction was then quenched with saturated Na2CO3 (aqueous solution). The organic phase was separated, washed with saturated Na2CO3 (aqueous solution) until the color was off-white, then washed with brine, dried over Na2SO4, filtered, and concentrated to obtain compound 7 (6.00 g, yield 56%) as a yellowish solid, which was used without further purification.

[0115] Synthesis of compound 8 (1) Amine 4 (1.0 equivalent) was added dropwise at 0°C to a stirred solution of compound 7 (3.0 equivalents) and Et3N (3.0 equivalents) in CH2Cl2. The reaction mixture was vigorously stirred at 0°C for 5 hours. The solvent was then removed under reduced pressure. The residue was dissolved in the smallest possible amount of Et2O and cooled to 0°C. After 6 hours, excess compound 7 began to precipitate from the solution. The solution was then decanted and concentrated under reduced pressure. The resulting crude product was subjected to the above procedure two more times to precipitate the same amount of compound 7 from the mixture. By this method, >80% of compound 7 can be recycled and used. (2) After precipitation three times, the above mixture was dissolved in CH2Cl2. To this solution, Et3N (8.0 equivalents) and amine 2 (1.5 to 3.0 equivalents depending on the purity of the resulting mixture), which had been previously dissolved in CHCl3 / hexane / THF, were added. The reaction mixture was vigorously stirred at room temperature for 5 hours. (3) Next, Ac2O (5.0 equivalents) was added to the mixture and the solution was stirred at room temperature for a further 5 hours. Note: This step is to convert the excess amount of amine 2 into acetylamide, otherwise it would be very difficult to remove amine 2 from the final product. (4) Next, the solvent was removed under reduced pressure and the mixture was redissolved in ethyl acetate. The organic phase was first washed with saturated Na2CO3 (aqueous solution) until it turned off-white, then washed with brine, dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (CH2Cl2 / CH3OH (+0.1% Et3N) = 30:1 to 15:1) to obtain compound 8. The name of compound 8 is shown immediately below its structure. [ka] Yield: 28%. Colorless oil. 1HNMR (400 MHz, クロロホルム-d) δ 5.60 (s, 1H, H12), 4.204.15 (m, 4H, H2 & H8), 3.64-3.60 (m, 4H, H3 & H7), 3.28-3.23 (m, 2H, H16), 3.19-3.14 (m, 4H, H23 & H24), 2.42-2.39 (m, 2H,H17), 2.22 (s, 6H, H19& H20), 1.53-1.47 (m, 4H, H25 & H36), 1.36 (s, 6H, H14 &H15), 1.26(brd, 36H), 0.89-0.86 (t, (6H, H35 & H46) ppm. 13 C NMR (100 MHz, クロロホルム-d) δ156.5 (C9 & C11), 100.1 (C5), 64.5 (C2 or C8), 64.3 (C2 orC8), 59.6 (C3 orC7), 59.5 (C3 or C7), 58.5 (C17), 47.7 (C23 or C24), 47.1 (C23or C24), 45.5(C19 & C20), 38.7 (C16), 32.1, 29.80, 29.76, 29.61, 29.49,28.8, 28.3,27.0, 25.0 (C14 & C15), 22.8 (C34 & C45), 14.3 (C35 &C46) ppm. LRMS(ESI) (m / z): [M+H] + (C 37 H 76 O6N3) calculated as 658.5, measured value: 658.5

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[0116]

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[0117]

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[0118] The general synthesis pathway of lipids represented by the structure of formula I, or more specifically, the structure of formula IIIa, is shown below. [ka]

[0119] Synthesis of compound 12 A solution of 3-(Boc-amino)-1,2-propanediol (2.43 g, 12.7 mmol, 1.0 equivalent) and oleoyl chloride (8.6 mL, 26.0 mmol, 2.05 equivalents) in CH2Cl2 (50 mL) was stirred at 0°C, and Et3N (5.3 mL, 38.1 mmol, 3.0 equivalents) was added dropwise. The reaction mixture was then covered with aluminum foil and vigorously stirred at 0°C for a further 4 hours. The reaction was then quenched with saturated Na2CO3 (aqueous solution). The organic phase was separated, washed with brine, dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 15:1 to 10:1) to obtain compound 12 as a colorless solid (4.06 g, 44%). The spectral data for compound 12 was in good agreement with the data reported in the literature (Luo et al., 2016).

[0120] Synthesis of compound 13 Next, compound 12 was dissolved in a mixed solvent of CH2Cl2 / CF3COOH (10 mL / 10 mL). After stirring at room temperature for 30 minutes, the solvent was removed under reduced pressure, and the crude product 13 was further dried under vacuum and used without further purification.

[0121] Synthesis of Compound 15 Compound 13 (1.0 equivalent), Compound 1 (2.0 equivalents), DMAP (0.2 equivalents), and Et3N (5.0 equivalents) were dissolved in DM at room temperature. The resulting mixture was then covered with aluminum foil and stirred at room temperature for 24 hours, after which amine 14 (2.5 equivalents) was added. After 5 hours, the solvent DMF was removed under reduced pressure, and the crude residue was redissolved in ethyl acetate. The organic phase was first washed with saturated Na2CO3 (aqueous solution) until the color became off-white, then washed with brine, dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (CH2Cl2 / CH3OH = 30:1 to 15:1) to obtain compound 15. The name of compound 15 is shown immediately below its structure. [ka] Yield: 30%. Colorless のオイル. 1 HNMR (400 MHz, クロロホルム-d) δ 5.50(brd, 1H, NH),5.37-5.32 (m, 5H,H19, H20, H35, H36 &NH), 5.13-5.08 (m, 1H, H2), 4.33-4.26(m, 5H, H47, H53 & H1), 4.13 (dd, J = 12.0, 5.7 Hz, 1H, H1), 3.48-3.34 (m,2H, H3), 3.32-3.28 (m, 2H, H57), 2.92 (t, J = 6.4 Hz, 4H, H48 & H52), 2.50(t, J = 5.9 Hz, 2H, H58), 2.33-2.27 (m, 10H, H27, H28, H60 & H62), 2.03-1.98(m, 8H, H18, H21, H34 & H37), 1.631.58 (m, 4H, H12 & H43), 1.30-1.25(m, 40H), 0.88 (t, J = 7.0 Hz,6H, H11 & H44) ppm. 22.8, 14.3 (C11 &C44) ppm. LRMS (ESI)(m / z): [M+H] + (C 49 H 92 O8N3S2), the calculated value is 914.6, and the measured value is: 914.5.

[0122]

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[0123] 2.4 The general synthesis pathway of lipids represented by the structure of formula I, or more specifically, the structure of formula IIIb, is shown below. [ka]

[0124] Compound 13 (1.0 equivalent), Compound 7 (2.0 equivalents), DMAP (0.2 equivalents), and Et3N (10.0 equivalents) were dissolved in DMF at room temperature. The resulting mixture was then covered with aluminum foil and stirred at room temperature for a further 24 hours, after which amine 14 (2.5 equivalents) was added. After 5 hours, Ac2O (3.0 equivalents) was added, and the mixture was stirred at room temperature for a further 12 hours. Note: Ac2O was added to facilitate the removal of unidentified impurities from the final product. The solvent DMF was then removed under reduced pressure, and the crude residue was redissolved in ethyl acetate. The organic phase was first washed with saturated Na2CO3 (aqueous solution) until the color was off-white, then washed with brine, dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (CH2Cl2 / CH3OH (+0.1% Et3N) = 30:1 to 15:1) to obtain compound 16. The name of compound 16 is shown immediately below its structure.

[0125] [ka] Yield: 20%. Colorless oil. 1HNMR (400 MHz、クロロホルム-d) δ 5.72(t, J = 5.4Hz, 1H, NH), 5.47 (s, 1H, NH), 5.40-5.30 (m, 4H,H19, H20, H35, H36), 5.11-5.07(m, 1H, H2), 4.32-4.08 (m, 6H, Including H1, H47, H53),4.37-3.61 (m, 4H, H48, H52), 3.48-3.22 (m, 4H, H3& H61), 2.41 (t, J = 5.9 Hz, 2H, H62), 2.33-2.23 (m, 10H, H27, H28, H64,H65), 2.04-1.96 (m, 8H, H18, H21, H34, H37), 1.62-1.58 (m, 4H, H26 & H29),1.36 (s, 6H, H57 & H58) 1.34-1.25 (m, 40H), 0.88 (t, J = 7.0 Hz, 6H, H11& H44) ppm. 13¹¹C NMR (100 MHz, chloroform-d) δ 173.5 (ester C=O), 173.2 (ester C=O), 156.89 (carbamate C=O), 156.86 (carbamate C=O), 130.2 (alkene carbon), 129.86 (alkene carbon), 129.84 (alkene carbon), 100.0 (C50), 70.6 (C2), 67.4, 67.3, 64.5 (C47 or C53), 64.2 (C47 or C53), 62.8 (C1), 59.5 (C48 or C52), 59.3 (C48 or C52), 58.3 (C62), 45.2 (C64, C65), 41.3 (C3), 38.4 (C61), 34.4 (C27 or C28), 34.2 (C27 or C28), 32.0, 29.92, 29.88, 29.87, 29.81, 29.7, 29.47, 29.46, 29.37, 29.35, 29.29, 29.26, 29.25, 27.37 (allyl carbon), 27.33 (allyl carbon), 25.00, 24.94 (C58, C59), 22.8, 14.3 (C11, C44) ppm. LRMS(ESI) (m / z): [M+H] + (C 52 H 98 O 10 The calculated value for N3 is 924.7, while the measured value is 924.6.

[0126] [ka] Yield: 18%. Colorless oil. 1HNMR (400 MHz, クロロホルム-d) δ5.79-5.77 (m, 1H,NH), 5.38-5.31 (m, 4H, H19, H20, H35, H36), 5.13-5.09 (m, 1H, H2), 4.32-4.10 (m,6H, Including H1, H47, H53), 3.69-3.60(m, 4H,H48 & H52), 3.52-3.43 (m, 1H, H3), 3.39-3.27 (m, 3H, H3 & H61), 2.77-2.70 (m,6H, H62, H64, H69), 2.33-2.28 (m, 4H, H27 & H28), 2.04-1.96(m, 8H, H18, H21, H34, H37), 1.70-1.58 (m, 12H, H65-68 & H26, H29), 1.36(s, 6H, H58 & H59), 1.34-1.24(m, 40H), 0.89-0.86 (m, 6H, H11 & H44)ppm. 13 C NMR (100 MHz, クロロホルム-d) δ 173.5 (エステルC=O), 173.2 (エステルC=O), 156.9 (カルバメートC=O), 130.2 (Arocarbon), 129.85 (Arocarbon), 129.84 (Arocarbon), 100.0 (C50), 70.6 (C2), 67.6, 67.5, 67.4,64.5, 64.2, 62.9 (C1), 59.5, 59.3, 56.5 (C62), 55.38, 55.35,41.3 (C3), 39.6,36.6, 34.4 (C27 or C28), 34.2 (C27 or C28), 32.0, 29.91, 29.88,29.87, 29.85, 29.7, 29.47,29.46, 29.37, 29.35, 29.33, LRMS (ESI)(m / z): [M+H] + (C 56 H 104 O 10The calculated value of N3) is 978.8, and the measured value is: 978.7.

[0127]

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[0128] The general synthesis pathway of lipids represented by the structure of formula I, or more specifically, the structure of IIIc, is shown below. [ka] Compound 13 (1.0 equivalent), Compound 17 (Yaeger et al., 2004) (2.0 equivalents), DMAP (0.2 equivalents), and Et3N (5.0 equivalents) were dissolved in DMF at room temperature. The resulting mixture was then covered with aluminum foil and stirred at room temperature for 24 hours, after which amine 14 (2.5 equivalents) was added. The solvent DMF was then removed under reduced pressure, and the crude residue was redissolved in ethyl acetate. The organic phase was first washed with saturated Na2CO3 (aqueous solution) until the color became off-white, then washed with brine, dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (CH2Cl2 / CH3OH = 30:1 to 15:1) to obtain Compound 18. The name of Compound 18 is shown immediately below its structure. [ka] Yield: 24%. Colorless oil. 1 HNMR (400 MHz, Chloroform-d) δ5.41-5.32 (m, 5H,H19, H20, H35, H36 & NH),5.17 (t, J = 6.2 Hz, 1H, NH), 5.12-5.07 (m, 1H,H2), 4.27 (dd, J = 12.0, 4.4 Hz, 1H, H1), 4.22 (t, J = 4.7 Hz, 4H, H47&H52), 4.12 (dd, J = 12.0, 5.7 Hz,1H, H1), 3.67 (t, J = 4.7 Hz,4H, H48& H51), 3.48-3.34 (m,2H, H3), 3.30-3.25 (m, 2H, H56), 2.44 (t, J = 6.1 Hz,2H, H57),2.31 (td, J = 7.6, 2.3 Hz, 4H, H27 & H28), 2.25 (s, 6H, H59 &H61), 2.00 (q, J= 6.5 Hz, 8H, H18, H21, H34 & H37),1.63-1.58 (m, 4H, H12& H43), 1.32-1.25(m, 40H), 0.88 (t, J = 7.0 Hz, 6H, H11 & H44) ppm. 13CNMR (100 MHz, クロロホルム-d) δ 173.6, 173.2, 156.6, 156.5,130.2, 129.85, 129.84, 70.4, 69.7, 69.6, 64.3, 63.9, 62.7, 58.3,45.2, 41.4,38.4, 34.4, 34.2, 32.0, 29.91, 29.86, 29.7, 29.5, 29.35, 29.33, 29.27,29.25,29.23, 27.36, 27.32, 25.01, 24.99, 22.8, 14.3. LRMS (ESI) (m / z): [M+H] + (C 49 H 92 The calculated value of O9N3) is 866.7, and the measured value is: 866.6.

[0129]

change

[0130] [ka] Yield: 16%. Colorless oil. 1HNMR (400 MHz、クロロホルム-d) δ 5.94(s, 1H, NH),5.395.29 (m, 4H, H19, H20, H35, H36), 5.23 (s, 1H, NH), 5.10 (p, J = 5.2 Hz,1H, H2), 4.294.19 (m, 5H, including H1, H47, H52), 4.12(dd, J = 12.0, 5.7 Hz,1H, H1), 3.68-3.65(m, 4H, H48, H51), 3.48-3.34 (m, 2H, H3),3.30-3.23 (m, 2H,H56), 2.95 (brd, 2H, H58 or H65 or H61), 2.58-2.47 (m, 3H, H58 or H65 orH61),2.31 (td, J = 7.6, 2.7 Hz, 4H, H27, H28), 2.03-1.98 (m, 8H, H18, H21, H34,H37),1.81-1.54 (m, 14H, H26, H29, H57, H62, H63, H64, H66), 1.29-1.25 (m,40H),0.93-0.86 (m, 9H, H11, H44, H67) ppm. 13¹¹C NMR (100 MHz, chloroform-d) δ 173.6 (ester C=O), 173.3 (ester C=O), 156.8 (carbamate C=O), 156.2 (carbamate C=O), 70.5 (C2), 69.66 (C48 or C51), 69.60 (C48 or C51), 64.3 (C47 or C52), 63.9 (C47 or C52), 62.7 (C1), 51.0 (C58, C61, C65. Signals are canceled out and peaks are weak), 41.4 (C3), 34.4 (C27 or C28), 34.2 (C27 or C28), 32.0, 29.90, 29.86, 29.7, 29.46, 29.45, 29.35, 29.33, 29.27, 29.24, 29.22, 27.36 (allyl carbon), 27.31 (allyl carbon), 25.01, 24.98, 22.8, 14.3 (C11 & C44), 10.3 (C67) ppm. LRMS (ESI) (m / z): [M+H] + (C 55 H 102 The calculated value for O9N3 was 948.8, while the measured value was 948.7.

[0131] 2.5 The general synthesis pathway of lipids represented by the structure of formula I, or more specifically, the structure of formula V, is shown below. [ka]

[0132] Synthesis of compound 19 N-BOC-diethanolamine (5.43 g, 0.03 mol) and 2-hexyldecanoic acid were mixed in a 30 mL CH2Cl2 solution at 0°C with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (13.1 g, 0.06 mol), TEA (6.42 g, 0.06 mol), and DMAP (0.66 mg, 0.005 mol). The reaction mixture was then covered with aluminum foil and vigorously stirred at 0°C for 1 hour. The reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was then filtered and concentrated. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 15:1) to obtain compound 19 as a brown oil (2.84 g, 17.00%).

[0133] Synthesis of compound 20 Next, compound 19 was dissolved in a mixed solvent of CH2Cl2 / CF3COOH (10 mL / 10 mL). After stirring at room temperature for 30 minutes, the solvent was removed under reduced pressure, and the crude product 20 was further dried under vacuum and used without further purification.

[0134] Synthesis of Compound 21 Compound 20 (1.0 equivalent), Compound 1 (2.0 equivalents), DMAP (0.2 equivalents), and Et3N (5.0 equivalents) were dissolved in DMF at room temperature. The resulting mixture was then covered with aluminum foil and stirred at room temperature for 24 hours, after which amine 14 (2.5 equivalents) was added. After 5 hours, the solvent DMF was removed under reduced pressure, and the crude residue was redissolved in ethyl acetate. The organic phase was first washed with saturated Na2CO3 (aqueous solution) until the color became off-white, then washed with brine, dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography ((CH2Cl2 / CH3OH = 30:1 to 10:1)) to obtain Compound 21. The name of Compound 21 is shown immediately below its structure. [ka] Yield: 16%. Colorless oil. 1HNMR (400 MHz, クロロホルム-d)4.39-4.28 (m, 4H, H56& H58), 4.23-4.16 (m, 4H, H23 & H28), 3.58-3.51(m, 4H, H57 & H59),3.38-3.29 (m, 2H, H33), 2.99-2.88 (m, 4H, H24 &H27),2.61-2.51 (m, 2H, H34), 2.41-2.27 (m, 8H, H4, H36, H37 & H39),1.64-1.38 (m, 8H,H6, H7, H40 & H41), 1.32-1.20 (brd, 40H), 0.87 (t, J = 6.8Hz, 12H, H17, H19, H51 & H53) ppm. LRMS (ESI) (m / z): [M+H] + (C 48 H 89 The calculated value of O8N3S2) is 876.6, and the measured value is: 876.5.

[0135]

change

[0136] [ka] Yield: 20%. Colorless oil. 1 HNMR (400 MHz, Chloroform-d)4.40-4.27 (m, 4H, H54& H56), 4.24-4.16 (m, 4H, H23 & H28), 3.55 (m,4H, H55 & H57),3.38-3.28 (m, 2H, H33), 3.03-2.89 (m, 6H, H24, H27 &H63),3.03-2.28 (m, 2H, H4 & H37), 2.18-1.68 (m, 10H, H34, H60, H61, H62& H64) 1.65-1.38(m, 8H, H6, H7, H40 & H41), 1.25 (brd, 40H), 1.01 (t, J= 7.4 Hz, 3H, H65) 0.88 (t, J = 6.8 Hz, 12H, H17, H19, H51 & H53) ppm. LRMS(ESI) (m / z): [M+H] + (C 52 H 99 The calculated value for O8N3S2 is 958.7, and the measured value is 958.7.

[0137] [ka] Yield: 68%. Colorless oil. 1H NMR (400 MHz, Chloroform-d) 4.39-4.25 (m, 4H,H54,H56), 4.25-4.15 (m, 4H, H23,H28),3.58-3.51 (m, 6H, H33,H55 & H57),2.99-2.88 (m, 8H, H24, H27, H58 & H63), 2.40-2.26(m, 2H, H4 & H37),2.00-1.64 (m, 10H, H34, H59, H60, H61 & H62), 1.63-1.38 (m, 8H,H6, H7, H38& H39), 1.32-1.20 (brd, 40H), 0.87 (t, J = 6.8 Hz, 12H, H17,H19, H49 &H51) ppm. LRMS (ESI) (m / z): [M+H] + (C 50 H 95 The calculated value for O8N3S2 is 930.7, while the measured value is 930.6.

[0138] The general synthesis pathway of lipids represented by the structure of formula I, or more specifically, the structure of formula VIa, is shown below.

[0139] [ka]

[0140] 2.7 The general synthesis pathway of lipids represented by the structure of formula I, or more specifically, the structure of formula VIb, is shown below. [ka]

[0141] Example 2: In vitro experiment

[0142] Materials and methods:

[0143] mRNA synthesis:

[0144] mRNA encoding eGFP and firefly luciferase was prepared in vitro by T7-mediated transcription from a linear DNA template (peTheRNAvs3 vector) incorporating 5' and 3' UTRs and a polyA tail. The final mRNA utilized Cap1 and 100% uridine was replaced with N1-methyl-pseudo-uridine.

[0145] LNP synthesis:

[0146] Lipid-based nanoparticles were generated by microfluidic mixing of mRNA solution and lipid solution in sodium acetate buffer (100 mM, pH 4) at a volume ratio of 2:1 at a rate of 9 mL / min using a NanoAssemblr Benchtop (PrecisionNanosystems). The lipid solution contained a mixture of the target ionizable lipids: DSPC, DOPC, or DOPE (Avanti), cholesterol (Sigma), and DMG-PEG2000 (Sunbright GM-020, NOF Corporation). The four lipids were mixed in six different molar ratios. LNPs were dialyzed against TBS (TBS volume 10,000 times the LNP volume) using a Slide-a-lyzer dialysis cassette (20K MWCO, 3 mL, ThermoFisher). Size, polydispersity, and zeta potential were measured using a Zetasizer Nano (Malvern). mRNA encapsulation was measured using a standard Ribogreen RNA assay (Invitrogen).

[0147] Cell line: HEK-TS / A-CT26-B16

[0148] The following summarizes the optimal culture conditions for each cell type, including growth media, subculturing ratios, and recommendations for medium renewal. To harvest adherent cells, the used growth medium was discarded, the cells were washed twice with phosphate-buffered saline (PBS) (Sigma), and then the cells were loosened with trypsin-EDTA (0.05%) (Gibco, Thermo Fisher Scientific). The medium should be renewed every 2-3 days, whenever the cells reach approximately 70% confluence. Cell viability was measured using a Vi-Cell XR CellViability Analyzer (Beckman Coulter).

[0149] Table 1: Culture conditions according to cell type [Table 1]

[0150] Transfection

[0151] Cells were plated into 96-well plates at a density of 20–30 × 10⁴ cells / 100 μl of complete growth medium (specific to each cell type). Transfection was performed when the cells reached 70–90% confluence. Positive control lipofectamine (MessengerMAX, Invitrogen) was diluted with OptiMEM (sera-reduced, Gibco) and incubated for 10 minutes. Meanwhile, eGFP mRNA and LNPs encapsulating eGFP mRNA were diluted with OptiMEM to mRNA content concentrations of 200 and 50 ng / well, respectively. mRNA:lipid complexes were incubated in a 1:1 ratio for 5 minutes, and four were added to each condition. Cells were incubated at 37°C and 5% CO₂ for 24 hours. Subsequently, cells were harvested using 1×TrypLE selective enzyme (Gibco) and stained with the viability / death marker SYTOX blue (Life Technologies) in FACS buffer (PBS supplemented with 1% bovine serum albumin (BSA) and 0.09% azide (all from Sigma)). After the addition of the viability / death marker, cells were immediately obtained using an Attune Nxt flow cytometer (Thermo Fisher Scientific). Lipofectamine alone (lipofectamine treatment without mRNA addition) and Blanco cells or untreated (UT) cells were included as negative controls.

[0152] Flow cytometry:

[0153] E7-specific CD8 T cell response: Blood was collected in heparin-coated tubes on days 14 and 25 after tumor inoculation. Red blood cells were lysed, and the remaining white blood cells were stained with a survival dye. After incubation and washing, APC-labeled E7 blood was collected. (RAHYNIVTF) -Dextramer (Immudex) was added and incubated at room temperature for 30 minutes. Excess dextramer was washed off, and an antibody mixture against surface molecules CD3 and CD8 was added to the cells and incubated at 4°C for 30 minutes. [Table A]

[0154] eGFP expression To evaluate eGFP expression, cells were stained with SYTOX blue. eGFP expression levels were determined within a gate of SYTOX blue-negative cells. Relative mean fluorescence intensity (rel MFI) was calculated by dividing the MFI value of the expression marker by the MFI value of untransfected cells.

[0155] Data was acquired using an Attune Nxt cytometer and analyzed with FlowJo software. Flow cytometry data was analyzed using FlowJo version 10 software.

[0156] Tumor inoculation and intratumor injection: Balb / c mice were used for CT26 tumor inoculation. C57 / BL6 mice were used for B16F10 tumor inoculation. For subcutaneous tumor inoculation preparation, mice were anesthetized with 2.5% isoflurane and the injection site was shaved. The injection site is typically the posterior / lateral aspect of the lower left flank. For inoculation purposes, cells should be cultured for approximately one week, and after thawing, passaged between 3 and 5 times. The chilled tumor cell solution was subcutaneously injected at a dose of 0.5 × 10⁶ cells / 50 μl PBS. Tumor growth was measured every 2 to 3 days using a Caliper device. Tumor size was calculated using the following formula: (tumor width * tumor width * tumor length) / 2. The tumor had an average volume of 50–100 mm². 3 Once the target was reached, LNP containing firefly luciferase mRNA (10 μg mRNA in 20 μl TBS buffer) or control buffer (TBS) was injected into the tumor. After injection, the mice were continuously monitored for 5–10 minutes until they were fully awake without any signs of pain or complications.

[0157] In vivo bioluminescence: Fluc mRNA expression in tumors and the liver was evaluated at 6 and 24 hours post-injection. Bioluminescence could be measured via in vivo imaging (IVISSpectrum System) by intraperitoneal injection of D-luciferin (Promega) into mice. Bioluminescence is generated by an oxidation reaction between the enzyme luciferase, derived from firefly mRNA encapsulated in LNPs, and its substrate, D-luciferin. Regions of interest (ROIs) in tumors and the liver were specified using Living Image Software (PerkinElmer), and then the mean radiance (p / s / cm²) within these ROIs was measured. 2 The ratio ( / sr) was calculated. To monitor the tolerability of the various LNP types used, body weight 6 and 24 hours post-injection was compared to baseline body weight on the randomization day.

[0158] Data analysis: All raw data was analyzed using Graph Pad Prism version 7 software.

[0159] Example 2.1: Expression level of reporter eGFP mRNA after in vitro transfection of HEK293T cells with the indicated LNP composition.

[0160] LNPs were generated with a standard molar ratio of approximately 50 / 10 / 38.5 / 1.5 for ionizable lipid / DOPE / cholesterol / DMG-PEG2000. MC-3 is an ionizable lipid used in Onpattro and is considered state-of-the-art technology. eGFP mRNA was encapsulated in all LNPs as a reporter mRNA with a molar ratio of mRNA / ionizable lipid of 1 / 10.

[0161] Table 2: [Table 2]

[0162] As detailed in Table 2, all LNPs showed high encapsulation efficiency when measured by the RiboGreen assay. Size and polydispersity index were assessed by dynamic light scattering (DLS) using MalvernZetasizer. MC-3 is an ionizable lipid used in Onpattro and is considered a state-of-the-art technology.

[0163] Figure 1 shows the relative mean fluorescence intensity of eGFP expression in HEK293T cells (measured as a multiple of increase in eGFPMFI compared to untreated cells) when incubated with the indicated LNPs at mRNA concentrations of 50 ng and 200 ng / well, or with MC3 as a positive control. As is clear from this figure, overall, the LNPs of the present invention perform at an equal or better level than the positive control sample.

[0164] Example 2.2: Expression levels of reporter eGFP mRNA after in vitro transfection of HEK293T and cancer cell line (CT26-B16F10-TS / A) with the indicated LNP composition.

[0165] LNPs were produced with a standard molar ratio of approximately 50 / 10 / 38.5 / 1.5 for ionizable lipids / phospholipids / cholesterol / DMG-PEG2000. MC-3 is an ionizable lipid used in Onpattro and is considered state-of-the-art technology. All LNPs were formulated using DOPE, except for MC-3-based LNPs formulated with DSPC as the phospholipid. eGFP mRNA was encapsulated in all LNPs as a reporter mRNA with an mRNA / ionizable lipid molar ratio of 1 / 10. Table 3: [Table 3]

[0166] Figure 2 again demonstrates that LNPs perform comparably to or better than the positive control MC3 in multiple cell lines. Specifically, LNPs containing ionizable lipids combining an SS linker motif and a DOg acyl chain were the most efficient in cell transfection. Regarding the amine group, Ac7 performed better than Ac6e and Adm.

[0167] Example 2.3: Evaluation of the relationship between mol% PEG lipids and the nitrogen / phosphate (N:P) ratio To evaluate the effects of %DMG-PEG2000 and the molar ratio of ionizable lipids to mRNA on the physicochemical properties and cell transfectability of LNPs, nine mRNALNPs were generated using 3 mol% DMG-PEG2000 with three different N:P ratios. All LNPs were based on S-Ac7-Dog as the ionizable lipid. eGFP mRNA was encapsulated to allow evaluation of transfection efficiency in vitro.

[0168] Table 4: Size and PDI of the indicated LNP composition as measured by DLS. [Table 4]

[0169] Figures 3A and 3B show that none of the LNPs significantly affected the viability of the transfected HEK293T cells and CT26 cells, respectively.

[0170] The LNP size increased with decreasing % DMG-PEG2000. LNPs containing 1.5% DMG-PEG2000 showed improved transfection efficiency across all N:P ratios tested. For transfecting HEK293T cells, N:P 10 was the most efficient (Figure 4A). For transfecting CT26 tumor cells, N:P 20 was superior to N:P 10 and N:P 5 ratios (Figure 4B).

[0171] Example 2.4: In vivo mRNA expression upon injection of mRNALNP into subcutaneously proliferating CT26 tumors.

[0172] CT26 tumor cells were subcutaneously inoculated into Balb / c mice. The tumors had an average volume of 50-100 mm². 3 Once the target was reached, the tumor was injected with either mRNA LNP (10 μg mRNA; 20 μl volume; TBS buffer) or a control buffer. Fluc mRNA expression in the tumor (Figure 5A) and liver (Figure 5B) was evaluated by in vivo bioluminescence measurements at 6 and 24 hours post-injection. mRNA delivery with S-Ac7-Dog-based LNP resulted in similar Fluc expression levels in the tumor compared to MC-3-based benchmark LNP, but off-target expression was significantly reduced in the liver. No weight loss was observed with mRNA delivery using S-Ac-Dog (Figure 5C), while significant weight loss occurred with delivery using MC-3.

[0173] Example 2.5: In vivo mRNA expression in tumor-derived mRNA LNP injection of subcutaneously proliferating B16F10 tumors.

[0174] CT26 tumor cells were subcutaneously inoculated into Balb / c mice. The tumors had an average volume of 100 mm³. 3 Once the target was reached, the tumor was injected with either the respective mRNA LNP (10 μg mRNA; 20 μl volume; TBS buffer) or the control buffer. Fluc mRNA expression in the tumor (Figure 6A) and liver (Figure 6B) was evaluated by in vivo bioluminescence measurements at 6 and 24 hours post-injection. mRNA delivery with S-Ac7-Dog-based LNP resulted in increased Fluc expression levels in the tumor compared to MC-3-based benchmark LNP, but showed a significant decrease in off-target expression in the liver (Figure 6C). No weight loss was observed with S-Ac-Dog mRNA delivery, but significant weight loss occurred with MC-3 delivery.

[0175] Example 2.6: Induction of T cell response during intramuscular vaccination

[0176] C57BL / 6 mice were intramuscularly vaccinated on days 1 and 8 with 10 μg of E7 mRNA encapsulated in LNPs containing either S-Ac7-Dog or MC-3 as an ionizable lipid. The LNPs were formulated with a molar ratio of S-Ac7-Dog / mRNA of 10:1. E7-specific CD8 T cell responses were measured by flow cytometry 6 days after each vaccination (Figure 7).

[0177] Example 3: Induction of antigen-specific CD8 T cells upon intramuscular vaccination with mRNA LNP

[0178] Materials and methods: Intramuscular injection and assessment of muscle thickness: All mice were housed under specific pathogen-free conditions, and animal experiments were conducted according to protocols and guidelines approved by the Ghent University Animal Care and Use Committee (ECD20 / 100). mRNA LNP (50 μl volume, 5 μg mRNA) in TBS was injected into the biceps femoris muscle of the mice. Muscle thickness at the injection site was measured using an electronic external measurement gauge (K220T, Kroeplin) on days 1 and 4 post-injection.

[0179] Flow cytometry (evaluation of E7-specific CD8 T cell response): Anna 100 μl of whole blood for flow cytometry staining was collected on day 7 after the initial immunization and on day 14 after the booster immunization. After erythrocyte lysis (RBC lysis buffer, 420302 Biolegend), whole blood cells were subjected to mouse BD Fc block at 0.5 μg / test. 商標 Incubated with (BD, 553142) and Zombie Aqua viability dye (Biolegend, 423102). After incubation and washing, 5 μl / test of APC-labeled E7 was used. (RAHYNIVTF)-Dextramer (Immudex, JA2195) was added and incubated at room temperature (RT) for 30 minutes. After the washing step, cell surface staining was performed during a 30-minute incubation with the antibody mixture in the following amounts of FACS buffer (PBS, 2 mMEDTA, 1% BSA) for each test: CD3 PerCP-eFluor710 (Invitrogen, 46-0032-82), CD8-V450 (BD Biosciences, 560469), KLRG1 (Biolegend, 138408), and CD127-BV605 (Biolegend, 135041). After subsequent washing, stem samples were acquired using a 3-laser AtuneNxt flow cytometer (ThermoFisher, A29003), and the data were analyzed using FlowJo v10.7.1 software (BD Biosciences, FlowJo portal account).

[0180] LNP production LNP formulations were prepared using a modified procedure of the previously described method for siRNA LNP synthesis. All formulations were prepared aseptically with an N / P ratio of 10. All lipid components were dissolved in ethanol in a molar ratio of 50:10:38.5:1.5 mol% (ionizable lipids / DSPC / cholesterol / DMG-PEG2000). DSPC, cholesterol, and DMG-PEG2000 were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA), and the ionizable lipids were synthesized in-house. The final lipid concentration in ethanol was fixed at 10 mg / mL. E7 mRNA was dissolved in 100 mM acetate buffer (Sigma Aldrich, Saint-Louis, Missouri, USA) at pH 4. The ethanol and aqueous phases were mixed using a microfluidic mixer (NanoAssemblr® BenchTop (Precision Nanosystems, Vancouver, BC)) in a 2:1 (water:ethanol) ratio. The formulation was then dialyzed for 18 hours against 1X sterile TBS pH 7.4 (Sigma Aldrich, Saint-Louis, Missouri, USA) using a MWCO 20,000 Da Slide-A-Lyzer® dialysis cassette (Thermo Scientific, Massachusetts, USA). The purified formulation was then concentrated using an Amicon ultracentrifuge filter (EMD Millipore, Massachusetts, USA). Finally, the exact concentration and EE % were determined by a ribogreen assay before final dilution with TBS (100 μg / mL E7 mRNA per formulation). The size and PDI of all formulations were characterized using a dynamic light scattering zetasizing nano ZS (Malvern Pananlytical Ltd., Malvern, UK), and then stored at 4°C. The physicochemical properties of each formulation are shown in Table 5.

[0181] Table 5: List of relevant physicochemical properties of various LNP formulations. These include the type of ionizable lipid, composition, mole fraction of each component, size and PDI determined by dynamic light scattering, and encapsulation efficiency. Encapsulation efficiency was measured by the Ribogreen assay. [Table 5]

[0182] result: Each mRNA LNP vaccine induced a potent E7-specific CD8 T cell response during intramuscular immunization, which was clearly influenced by the chemical properties of the ionizable lipid (Figure 8). In contrast to LNPs formulated with MC-3, the ionizable lipid used for Onpattro delivery, LNPs formulated with the target ionizable lipid did not cause significant edema at the injection site (measured by a relative increase in muscle thickness) (Figure 9).

[0183] Example 4: Induction of anti-HA (hemagglutinin) immune response upon intramuscular mRNA vaccination.

[0184] Materials and methods:

[0185] Flow cytometry (ICS): Collected 2x10e6 splenocytes were stimulated with a peptide library containing 139 peptides derived from HA / Puerto Rico / 8 / 1934 H1N1 (PepMix® Influenza A, JPT, PM-INFA-HAPR) at a concentration of 1 μg / peptide / ml. Splenocytes treated with 20 ng / ml PMA (79346-1MG, Sigma) and 1 μg / ml ionomycin (I0634-1MG, Sigma) were used as positive controls. 0.065 μg / sample of CD107a-BV711 (564348, BD) antibody was added along with the activating stimulus. Cells were stimulated for 5 hours. One hour after the start of stimulation, 1X GolgiPlug (555028, BD) was added to stop cytokine secretion. Next, cells were incubated with a cocktail of surface-staining antibodies: 0.125 ug / test CD4-FITC (Biolegend, 100509), 0.02 ug / test Thy1.2-Alexa700 (Biolegend, 140323), and 0.05 ug / test CD8-eFluor450 (eBioscience, 48-0081-82). Cells were fixed and permeabilized using the BDCytofix / Cytoperm Plus Fixation / Permeabilization Solution Kit (555028, BD) according to the manufacturer's guidelines. Cells were stained with permeabilization buffer containing the following concentrations of mAbs: 0.03 ug / test IFNg-PE (BD, 554412), 0.065 ug / test CD154-PerCP-eFluor710 (ebioscience, 46-1541-80), 0.62 ug / test Granz-AF647 (Biolegend, 515406), 0.125 ug / test IL2-BV605 (BD, 563911), and 0.065 ug / ml TNFα-BV785 (Biolegend, 506341). Cells were analyzed using an Attune Nxt flow cytometer (Thermo Fisher, A29003).Mononuclear cells were gated using forward and side scattering, then duplets were identified using FCS-A and FCS-H scattering, and finally dead cells were gated out based on fixable dead cell staining fluorescence. All additional gates are shown and, where applicable, are based on fluorescence minus one control. Data acquisition and analysis were performed using FlowJo v10.7.1 software (BDBiosciences).

[0186] Evaluation of mouse endpoint immunoglobulin titers 100 μl of whole mouse blood was collected in serum gel tubes (SarsTedt) on days 21 and 35. Serum was separated from the blood clot by centrifugation at 10000 g at 4°C for 10 minutes. A black flat-bottomed Maxisoape 96-well plate (437111, Life Technologies) was coated overnight at 4°C with 100 μl of 1 μg / ml recombinant H1N1 (A / Puerto Rico / 8 / 1934) HA protein (Sino Biological, 11684-V08H) in carbonate / bicarbonate buffer (0.1 M, pH 9.6). Subsequently, the plate was blocked for 2 hours with 100 μl of 3% BSA (05479-250 g, Sigma) in PBS (w / v). Subsequently, the plate was washed three times with PBS / 0.1% Tween (10113103, Fisher Scientific). Serial dilutions of serum samples were added to the plates (first 100-fold serum dilution for day 21, and first 1000-fold serum dilution for day 35; 5-fold dilution step). After incubation at room temperature for 2 hours, the plates were washed 5 times and a solution of HRP-conjugated rabbit anti-mouse IgG1 (1:15,000, Biorad, OBT1508P) or HRP-conjugated goat anti-mouse IgG2a (1:8000, STAR133P Biorad) was added for a further 1 hour. After the final wash, the plates were treated with fluorescent Amplex UltraRed reagent (A36006, Invitrogen) according to the manufacturer's instructions. The plates were λ-scaled on a Tecan Infinite 200 Pro. ex =540nm, λ emReads were taken at 590 nm. The dilution of serum from TBS-treated mice was the mean value of fluorescence measured in the TBS sample plus a standard deviation of 3, which helped determine the cutoff. All points above the cutoff were considered below the limit of quantification. The 5PL curve was fitted to the dilution data, and the endpoint titer was then calculated at the intersection of the modeled curve and the cutoff.

[0187] LNP production: LNP formulations were prepared as described above. All formulations were prepared aseptically with an N / P ratio of 10. The size and PDI of all formulations were characterized using a dynamic light scattering zetasizing nano ZS (Malvern Pananlytical Ltd., Malvern, UK), and then stored at 4°C. The physicochemical properties of each formulation are shown in Table 6.

[0188] Table 6: List of relevant physicochemical properties of various LNP formulations. These include the type and composition of ionizable lipids, the mole fraction of each component, their size (determined by dynamic light scattering), PDI, and encapsulation efficiency. Encapsulation efficiency was measured by the Ribogreen assay. [Table 6]

[0189] result: Each mRNA LNP vaccine induced an anti-HA antibody titer. A clear increase in titer was observed after booster immunization (Figure 10). Furthermore, the mRNA LNP vaccines also induced an IFNg+ CD8 T cell response to HA, which was influenced by the chemical properties of the ionizable lipids used (Figure 11).

[0190] Example 5: Induction of E7-specific CD8 T cell response during intramuscular vaccination

[0191] Materials and methods: Flow cytometry (evaluation of E7-specific CD8 T cell response): 100 μl of whole blood for flow cytometry staining was collected 7 days after primary immunization and 14 days after booster immunization. After erythrocyte lysis (RBC lysis buffer, 420302 Biolegend), whole blood cells were incubated with 0.5 μg / sample of mouse BD Fc Block® (BD, 553142) and Zombie Aqua viability dye (Biolegend, 423102). After incubation and washing, 5 μl / sample of APC-labeled E7 was incubated. (RAHYNIVTF) -Dextramer (Immudex, JA2195) was added and incubated at room temperature (RT) for 30 minutes. After the washing step, cell surface staining was performed during a 30-minute incubation with the antibody mixture in the following amounts of FACS buffer (PBS, 2 mMEDTA, 1% BSA) for each test: The samples used were CD3 PerCP-eFluor710 (Invitrogen, 46-0032-82), CD8-V450 (BD Biosciences, 560469), KLRG1 (Biolegend, 138408), and CD127-BV605 (Biolegend, 135041). Samples were acquired using a 3-laser AtuneNxt flow cytometer (ThermoFisher, A29003), and data were analyzed using FlowJo software (FlowJo_v10.7.1, FlowJo portal account).

[0192] LNP production: LNP formulations were prepared as described above. All formulations were prepared aseptically with an N / P ratio of 10. The size and PDI of all formulations were characterized using a dynamic light scattering zetasizing nano ZS (Malvern Pananlytical Ltd., Malvern, UK), and then stored at 4°C. The physicochemical properties of each formulation are shown in Table 7.

[0193] Table 7: List of relevant physicochemical properties of various LNP formulations. These include the type and composition of ionizable lipids, the mole fraction of each component, their size (determined by dynamic light scattering), PDI, and encapsulation efficiency. Encapsulation efficiency was measured by the Ribogreen assay. [Table 7]

[0194] result: Each mRNA LNP vaccine induced a potent E7-specific CD8 T cell response during intramuscular immunization, which was clearly influenced by the chemical properties of ionizable lipids (Figure 12). S-Ac7-DHDa-based LNPs induced a superior T cell response compared to MC-3 (Figure 13).

[0195] Example 6: Intravenous immunization with LNPs co-encapsulated with peptide antigen and imidazoquinoline TLR7 / 8 agonist

[0196] Materials and methods Lipid nanoparticle (LNP) formulations The minimal epitope amino acid sequence of E7 (RAHYNIVTF) was extended with 10 glutamate residues and adjacent amino acid sequences (QAEPD) and 2 serine residues (SS) derived from the amino acid sequence of the native E7 protein. The resulting peptide (EEEEEEEEEEESSQAEPDRAHYNIVTF) is further called GLU10-E7. SSQAEPDRAHYNIVTF was used as an unformulated control and is also called E7. The TLR7 / 8 agonist 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline-4-amine (IMDQ) was conjugated with a peptide containing 10 glutamate residues. This conjugate is further called GLU10-IMDQ. For fluorescence-based tracking, a Cy5-labeled peptide containing 10 glutamate residues (also known as GLU10-Cy5) was used. For LNP formulations, aqueous phases containing GLU10-E7 (or GLU10-Cy5 for fluorescence-based tracking experiments) and GLU10-IMDQ were prepared in 25 mM acetate buffer (pH 5.2) with either GLU10-E7 or GLU10-IMDQ. The organic phase was prepared by dissolving S-Ac7-DOG, DOPE, cholesterol, and DMG-PEG in a molar ratio of 50:10:38.5:1.5. The ratio of peptides to ionizable lipids was fixed at an N:C ratio of 5:1 (N: ionized amine from ionizable lipid, C: carboxylic acid from glutamic acid residue). The organic solution and aqueous solution were mixed in a 1:3 ratio by dropping the organic phase into a vigorously agitated aqueous solution. Subsequently, the formed LNPs were dialyzed against PBS for 12 hours using a 3.5 kDa cutoff dialysis membrane to remove ethanol.

[0197] Results and Methods Compared to the unformulated soluble peptide (GLU10-Cy5), the LNP-formulated peptide (LNP (GLU10)) significantly increases peptide uptake by macrophages and dendritic cells (cDC1 and cDC2 subsets) in the spleen after intravenous administration (Figure 14). Additionally, B cells and, to a small extent, T cells begin to bind to the peptide. Co-formulation of GLU10-IMDQ with LNP further increases peptide uptake in the spleen by macrophages, cDC1 dendritic cells, B cells, and T cells (CD4+ and CD8+ T cell subsets).

[0198] LNPs containing the TLR7 / 8 agonist GLU10-IMDQ can activate dendritic cells (cDC1 and cDC2 subsets), B cells, and T cells (CD4+ and CD8+ T cell subsets) in the spleen after intravenous administration (Figure 15).

[0199] Compared to unformulated soluble peptide E7 antigen and soluble peptide E7 antigen adjuvanted with the TLR7 / 8 agonist IMDQ, LNPs containing GLU10-E7 peptide antigen and GLU10-IMDQ induce a potent increase in tetramer-positive CD8 T cells in the blood of immunized mice after two inoculations two weeks apart. Administration of the antigen and TLR7 / 8 agonist within the same LNP induces a higher response than separate LNP populations containing either the antigen or the TLR7 / 8 agonist (Figure 16).

[0200] Example 7: Intramuscular immunization with poly(I:C) LNP and protein antigens

[0201] Materials and methods Lipid nanoparticle (LNP) formulations The LNP preparation was prepared using N / Pratio 10. All lipid components were dissolved in ethanol in a molar ratio of 50:10:38.5:1.5 mol% (S-Ac7-DOg / DOPE / cholesterol / DSG-PEG2000). Low molecular weight poly(I:C) was dissolved in 100 mM acetate buffer (pH 4). The ethanol phase and aqueous phase were mixed in a 1:3 ratio by adding the organic phase dropwise to the vigorously stirred aqueous solution. Subsequently, the formed LNP was dialyzed against PBS for 12 hours using a 3.5 kDa cutoff dialysis membrane to remove the ethanol.

[0202] Results and Methods Compared to naive poly I:C, formulations of poly I:C in LNPs increase the titer of anti-S1 spike protein IgG antibodies. These titers are further increased when the S1 Spike protein binds to the LNP surface (Figure 17).

[0203] Example 8: Intramuscular immunization with CpG LNP and protein antigens

[0204] Materials and methods Lipid nanoparticle (LNP) formulations The LNP preparation was prepared with an N / P ratio of 10. All lipid components were dissolved in ethanol in a molar ratio of 50:10:38.5:1.5 mol% (S-Ac7-DOg / DOPE / cholesterol / DSG-PEG2000). CpG was dissolved in 100 mM acetate buffer (pH 4). The ethanol phase and aqueous phase were mixed in a 1:3 ratio by adding the organic phase dropwise to a vigorously stirred aqueous solution. Subsequently, the formed LNP was dialyzed against PBS for 12 hours using a 3.5 kDa cutoff dialysis membrane to remove the ethanol.

[0205] Results and Methods Compared to natural CpG, formulation of CpG into LNP increases the titer of anti-ovalbumin (OVA) IgG antibodies (Figure 18).

[0206] References 1. Amano, Y.; Umezawa, N.; Sato, S.; Watanabe, H.; Umehara, T.; Higuchi, T. Activation of lysine-specific demethylase 1 inhibitorpeptide by redox-controlled cleavage of a traceless linker. Bioorg. Med.Chem.2017, 25, 1227-1234. 2. Shenoi, RA;Lai, BFL; Kizhakkedathu, JNSynthesis, characterization, and biocompatibility of biodegradable hyperbranched polyglycerols from acid-cleavable ketal group functionalized initiators.Biomacromolecules 2012, 13, 3018-3030. 3. Luo, C.; Miao, L.; Zhao, Y.; Musetti, S.; Wang, Y.; Shi, K.; Huang, L. A novel cationic lipid with intrinsic antitumor activity tofacilitate gene therapy of TRAIL DNA. Biomaterials 2016, 102, 239-248. 4. Yeager, AR;Finney, NS The first direct evaluation of the two-active site mechanism forchitin synthase. J. Org. Chem.2004, 69, 613-618.

[0207] Drawing terminology well viability survival rate Debris is coming out within the gate. blanc Lipo only Relative MFI eGFP Relative MFI eGFP withingate alive positive positive Average radiance Tumor 6 hours after IT injection 24hrs after IT-injection Liver Body weight Tumor expression Liver expression Tumor / liver ratio E7-specific CD8 Tcells E7-specific CD8 T cells Immunization Vaccination muscle thickness injected injection noninjected endpoint titers endpoint potency titer stimulated unstimulated Cytokine production spleen macrophages cells cells % tetramer + cells of CD8 + cells anti-S1 IgG titer Dilution factor endpoint titer Spike protein conjugated anti-OVA IgG

Claims

【Request Item 1】 【Chemistry 1】 During the ceremony, R 1 and R 2 are each independently, -H, -C 1-20 alkyl, -C 2-20 alkenyl, and -C 2-20 alkynyl selected from, wherein said -C 1-20 alkyl, -C 2-20 alkenyl, and -C 2-20 alkynyl each may optionally be substituted with 1 to 3 -O-(C=O)-R 7 , -(C=O)-O-R 7 , C 1-20 alkyl, -C 2-20 alkenyl, and -C 2-20 alkynyl, and R 1 and R 2 the total number of C atoms combined is at least 8; R 3 and R 4 Each of these is independently -C 1-6 Alkyl; or R 3 and R 4 These, together with the N atoms to which they are bonded, form a 5- to 10-membered aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 It may be substituted with 1 to 3 substituents selected from alkyl groups; and, R 5 and R 6 Each of these independently, -CH 2 -and; R 7 Each of these is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Selected independently of Alkinnil; Said-C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl can optionally contain 1 to 3 -O-(C=O)-R 7 , -(C=O)-OR 7 , -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Often substituted with alkynyl; m and n are both 2; X is -O-, -SS-, and -O-CR 8 R 9 Selected from -O-; R 8 and R 9 Each of them is -H, -C 1-6 Alkyl and -C 3-6 Selected independently from cycloalkyl; Y is -NH-; and Z is -C 1-6 It is alkylene. An ionizable lipid represented by formula (I). 【Request Item 2】 【Chemistry 2】 During the ceremony, R 3 and R 4 Each of these can be used independently with -C 1-6 Alkyl, or R 3 and R 4 These, together with the N atoms to which they are bonded, form a 5- to 10-membered aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 It may also be substituted with 1 to 3 substituents selected from alkyl groups; R 5 and R 6 Each is independent of -CH 2 -and; R 7 These are -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 It is selected independently of alkinyl, Said-C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl can optionally contain 1 to 3 -O-(C=O)-R 7 , -(C=O)-OR 7 , -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 It may also be substituted with alkynyl; and Both R 7 The total number of carbon atoms in the part is at least 5; m and n are both 2; X is -O-, -SS-, and -O-CR 8 R 9 Selected from -O-; R 8 and R 9 These are -H and -C respectively. 1-6 Alkyl and -C 3-6 Selected independently from cycloalkyl; Y is -NH-; and Z is -C 1-6 It is alkylene. An ionizable lipid according to claim 1, represented by formula (II). 【Request Item 3】 【Chemistry 3】 During the ceremony, R 3 and R 4 Each of these can be used independently with -C 1-6 Alkyl, or R 3 and R 4 These, together with the N atoms to which they are bonded, form a 5- to 10-membered aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 They may be substituted with 1 to 3 substituents selected from alkyl groups; and R 5 and R 6 These are -CH 2 - and; R 7 These are -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 It is selected independently of alkinyl, The foregoing -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 each of alkynyl may optionally be substituted with 1 to 3 -O-(C=O)-R 7 , -(C=O)-O-R 7 , -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; and Both R 7 The total number of carbon atoms in the part is at least 5; m and n are both 2; R 8 and R 9 are each independently selected from -H, -C 1-6 alkyl and -C 3-6 cycloalkyl; Y is -NH-; and Z is -C 1-6 It is alkylene. An ionizable lipid according to claim 1 or 2, represented by any one of formulas (IIIa), (IIIb), or (IIIc). 【Request Item 4】 【Chemistry 4】 During the ceremony, R 1 and R 2 These are -H and -C, respectively, independently. 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Selected from alkynyl, the -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Each alkynyl can optionally contain 1 to 3 -O-(C=O)-R 7 , -(C=O)-OR 7 , C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 It may be substituted with an alkynyl, and R 1 and R 2 The total number of C atoms combined is at least 8; R 3 and R 4 Each of these is independently -C 1-6 Alkyl; or R 3 and R 4 These, together with the N atoms to which they are bonded, form a 5- to 10-membered aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 It may be substituted with 1 to 3 substituents selected from alkyl groups; R 7 Each of these is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Selected independently of Alkinnil; Said-C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl can optionally contain 1 to 3 -O-(C=O)-R 7 , -(C=O)-OR 7 , -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Often substituted with alkynyl; R 5 and R 6 Each of these is -CH 2 -and-O-CH 2 -Selected independently of; m and n are both 2; R 8 and R 9 Each of them is -H, -C 1-6 Alkyl and -C 3-6 Selected independently from cycloalkyl; Y is -NH-; and Z is -C 1-6 It is alkylene. An ionizable lipid according to claim 1, represented by any one of formulas (IVa), (IVb), or (IVc). 【Request Item 5】 【Chemistry 5】 During the ceremony, R 3 and R 4 Each of these can be used independently with -C 1-6 Alkyl, or R 3 and R 4 These, together with the N atoms to which they are bonded, form a 5- to 10-membered aromatic heterocycle or non-aromatic heterocycle; the heterocycle may optionally further contain one or more additional N atoms, and / or optionally, -C 1-6 They may be substituted with 1 to 3 substituents selected from alkyl groups; and R 5 and R 6 These are -CH 2 - and -O-CH 2 - Selected independently of; R 7 These are -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 It is selected independently of alkinyl, Said-C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each alkynyl can optionally contain 1 to 3 -O-(C=O)-R 7 , -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 It may also be substituted with alkynyl; and Both R 7 The total number of carbon atoms in the part is at least 5; m and n are both 2; Y is -NH-; and Z is -C 1-6 It is alkylene. An ionizable lipid according to claim 1, represented by formula (V). 【Request Item 6】 【Transformation 6】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 An ionizable lipid according to any one of claims 1 to 5, selected from the list including the above chemical formula.

7. R 1 and R 2 The ionizable lipid according to any one of claims 1 to 3 or 5, wherein the total number of carbon atoms is at least 14.

8. Lipid nanoparticles or lipid nanoparticle compositions comprising an ionizable lipid according to any one of claims 1 to 7.

9. Lipid nanoparticles or lipid nanoparticle composition according to claim 8, further comprising phospholipids, sterols and / or PEG lipids.

10. Lipid nanoparticles or lipid nanoparticle composition according to claim 8 or 9, further comprising an active agent.

11. The lipid nanoparticle or lipid nanoparticle composition according to claim 8 or 9, further comprising nucleic acids.

12. The lipid nanoparticle or lipid nanoparticle composition according to claim 8 or 9, further comprising mRNA.

13. Use of an ionizable lipid according to any one of claims 1 to 7 in the production of lipid nanoparticles or a lipid nanoparticle composition.

14. A pharmaceutical composition comprising lipid nanoparticles or a lipid nanoparticle composition according to any one of claims 8 to 12 and a pharmaceutically acceptable agent.

15. A pharmaceutical product comprising the pharmaceutical composition described in claim 14.

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