Ionizable lipids

Abstract

The present invention relates generally to the field of ionizable (also referred to as cationic) lipids, and particularly provides novel such lipids as represented by any chemical formula disclosed herein. The invention further provides methods for preparing such lipids and uses thereof, in particular in the preparation of nanoparticle compositions, more particularly nanoparticle compositions comprising nucleic acids. It further provides vaccine formulations and pharmaceutical formulations comprising nanoparticle compositions based on ionizable lipids disclosed herein.

Description

Field of the Invention

[0001] The present invention relates generally to the field of ionizable (also called cationic) lipids, and in particular provides novel such lipids as represented by any of the formulas disclosed herein. The present invention further provides methods for preparing such lipids and uses thereof, in particular in the preparation of nanoparticle compositions, more particularly nanoparticle compositions comprising nucleic acids. It further provides vaccine formulations and pharmaceutical formulations comprising nanoparticle compositions based on the ionizable lipids disclosed herein. Background of the Invention

[0003] Increasingly, therapeutic areas are exploring drugs based on nucleic acids. However, due to their negative charge, size and instability, nucleic acids such as plasmid DNA, messenger RNA, short interfering RNA, single guide RNA and microRNA pose major challenges to the targeted delivery of tissues and cells. Many nanoparticle carrier systems have been explored to encapsulate and deliver nucleic acids. These nanoparticles need to be combined with the maximum cellular uptake and effective release of their payload from endosomes to the cytosol when nucleic acids are stored and in the extracellular environment.

[0004] Nanoparticles based on lipids are clinically used to deliver small interfering RNA and mRNA vaccines, and represent the most advanced types of RNA delivery vectors. Nanoparticles based on lipids are usually composed of cations or ionizable lipids, auxiliary phospholipids, PEGylated lipids and sterols that can be protonated at acidic pH. Each component has a special function in terms of LNP stability and activity. Sterols and PEGylated lipids are crucial for LNP structure and stability, and phospholipids can contribute to stability and endosomal escape. Cation or ionizable lipids are conversely considered to be the main driving factors of activity and tolerance by controlling mRNA encapsulation, cellular uptake and endosomal escape. Although it is an effective nucleic acid delivery vector, LNP can induce dose-limiting toxicity, such as pseudoallergy, inflammatory cytokine release and cytotoxicity associated with complement activation by accumulating non-degradable ionizable lipids into cell membranes. Therefore, further improvement of cation or ionizable lipid chemistry is needed to improve the efficacy and safety of the nucleic acid drug delivered by LNP.

[0005] The present invention therefore relates to a new type of ionizable lipids as defined by one set of claims of the present invention, which have improved properties over currently available types of ionizable lipids. SUMMARY OF THE INVENTION

[0007] In a first aspect, the present invention provides a lipid, in particular an ionizable lipid represented by formula (I)

[0008]

[0009] in

[0010] R1 and R2 are each independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Each of the alkynyl groups may be optionally substituted with 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl substitution;

[0011] wherein the total number of C atoms in R1 and R2 together is at least 8;

[0012] wherein R1 and R2 are different from each other;

[0013] R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH;

[0014] Each occurrence of R7 is independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution;

[0015] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0016] -

[0017] Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl;

[0018] R5 is selected from -NH- and -O-; and

[0019] R6 is -C 1-6 Alkylene-.

[0020] In a specific embodiment, the present invention provides a lipid, in particular an ionizable lipid as defined herein and represented by any one of formula (Ia), (Ib) and (Ic)

[0021]

[0022] in

[0023] R1 and R2 are each independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Each of the alkynyl groups may be optionally substituted with 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl substitution;

[0024] wherein the total number of C atoms in R1 and R2 together is at least 8;

[0025] wherein R1 and R2 are different from each other;

[0026] R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH;

[0027] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution;

[0028] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0029] Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl;

[0030] R5 is selected from -NH- and -O-; and

[0031] R6 is -C 1-6 Alkylene-.

[0032] In yet further embodiments, the present invention provides lipids, in particular ionizable lipids as defined herein and represented by any one of formula (II) or (III)

[0033]

[0034] in

[0035] R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH;

[0036] R7 and R7" are independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution; and the total number of C atoms in R7 and R7" together is at least 6;

[0037] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0038] o and p are each independently an integer selected from 1 to 10;

[0039] -

[0040] Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl;

[0041] R5 is selected from -NH- and -O-;

[0042] R6 is -C 1-6 Alkylene-; and

[0043] wherein o and p are different from each other; or wherein R7 and R7″ are different from each other.

[0044] The present invention further provides lipids, in particular ionizable lipids as defined herein and represented by formula (IIa)

[0045]

[0046] in

[0047] R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH;

[0048] R7 and R7" are independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution; and the total number of C atoms in R7 and R7" together is at least 6;

[0049] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0050] o and p are each independently an integer selected from 1 to 10;

[0051] R5 is selected from -NH- and -O-;

[0052] R6 is -C 1-6 Alkylene-; and

[0053] wherein o and p are different from each other; and / or wherein R7 and R7″ are different from each other.

[0054] The invention further provides lipids, in particular ionizable lipids as defined herein and selected from the list comprising:

[0055]

[0056]

[0057] In yet further embodiments, the invention provides lipids, in particular ionizable lipids as defined herein; wherein the total number of C atoms in R1 and R2 together is at least 14.

[0058] The present invention further provides lipids, in particular ionizable lipids as defined herein; wherein the total number of C atoms in R7 and R7" together is at least 12.

[0059] In a further embodiment, the invention provides a lipid, in particular an ionizable lipid as defined herein; wherein m and n are the same and are an integer selected from 1, 2, 3 and 4; preferably 2.

[0060] The invention further provides lipids, in particular ionizable lipids as defined herein; wherein R5 is -NH-.

[0061] In a further aspect, the invention provides lipid nanoparticles or lipid nanoparticle compositions comprising lipids, in particular ionizable lipids as defined herein. The nanoparticle compositions may also comprise phospholipids, sterols and PEG lipids.

[0062] In yet further embodiments of the present invention, the lipid nanoparticle or lipid nanoparticle composition as defined herein further comprises an active agent, in particular a nucleic acid, preferably mRNA.

[0063] In a further aspect, the present invention provides the use of a lipid, in particular an ionizable lipid as defined herein, in the preparation of a lipid nanoparticle or a lipid nanoparticle composition.

[0064] In a final aspect, the present invention provides a pharmaceutical composition comprising a lipid nanoparticle or a lipid nanoparticle composition as defined herein and a pharmaceutically acceptable agent.

[0065] The invention also provides a pharmaceutical composition as defined herein for use in human and / or veterinary medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Now with specific reference to the accompanying drawings, it should be emphasized that the details shown are by way of example and are only for the purpose of illustrative discussion of different embodiments of the present invention. They are provided in order to provide what are considered to be the most useful and easiest descriptions of the principles and concepts of the present invention. In this regard, no attempt is made to illustrate the structural details of the present invention in more detail than is necessary for a basic understanding of the present invention. The description in conjunction with the accompanying drawings makes it apparent to those skilled in the art how several forms of the present invention can be implemented in practice.

[0068] Figure 1 : In vivo bioluminescence measurements at 4 and 24 h after injection with corresponding mRNA LNPs (5 μg mRNA, 100 μL volume; TBS buffer) or control buffer.

[0069] Figure 2 : IgG1 antibody titers against HA at day 21 (prime) and day 35 (boost) after injection with corresponding mRNA LNPs (2 μg mRNA, TBS buffer) or control buffer.

[0070] Figure 3 : hEPO expression at different time points after injection with corresponding mRNA LNPs (10 μg mRNA, TBS buffer) or control buffer. A) Area under the curve analysis. B) hEPO serum concentration. DETAILED DESCRIPTION OF THE INVENTION

[0072] The present invention will now be further described. In the following paragraphs, different aspects of the present invention are defined in more detail. Unless clearly indicated to the contrary, each aspect so defined can be combined with any other one or more aspects. In particular, any feature indicated as preferred or advantageous can be combined with any other one or more features indicated as preferred or advantageous.

[0073] Unless the context indicates otherwise, asterisks are used herein to indicate the point at which a depicted monovalent or divalent group is attached to a structure to which it refers, and that the group forms a part of that structure.

[0074] As mentioned above, in a first aspect, the present invention provides a lipid, in particular an ionizable lipid represented by formula (I):

[0075]

[0076] in

[0077] R1 and R2 are each independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl; wherein the -C1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Each of the alkynyl groups may be optionally substituted with 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl substitution;

[0078] wherein the total number of C atoms in R1 and R2 together is at least 8;

[0079] wherein R1 and R2 are different from each other;

[0080] R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH;

[0081] Each occurrence of R7 is independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution;

[0082] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0083] -

[0084] Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl;

[0085] R5 is selected from -NH- and -O-; and

[0086] R6 is -C 1-6 Alkylene-.

[0087] Therefore, the present invention also provides lipids, in particular ionizable lipids represented by any one of formula (Ia), (Ib) and (Ic)

[0088]

[0089] in

[0090] R1 and R2 are each independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Each of the alkynyl groups may be optionally substituted with 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl substitution;

[0091] wherein the total number of C atoms in R1 and R2 together is at least 8;

[0092] wherein R1 and R2 are different from each other;

[0093] R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH;

[0094] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution;

[0095] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0096] Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl;

[0097] R5 is selected from -NH- and -O-; and

[0098] R6 is -C 1-6 Alkylene-.

[0099] When describing the compounds / lipids of the present invention, unless the context indicates otherwise, the terms used should be interpreted according to the following definitions:

[0100] The term "alkyl" by itself or as part of another substituent refers to a radical of the formula C x H 2x+1 A fully saturated hydrocarbon of , wherein x is a number greater than or equal to 1. Typically, the alkyl groups of the present invention contain 1 to 20 carbon atoms. Alkyl groups may be straight or branched and may be substituted as indicated herein. When a subscript is used after a carbon atom herein, the subscript refers to the number of carbon atoms that the designated group may contain. Thus, for example, C 1-4 Alkyl refers to an alkyl group of one to four carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, butyl and isomers thereof (e.g., n-butyl, isobutyl and tert-butyl); pentyl and isomers thereof, hexyl and isomers thereof, heptyl and isomers thereof, octyl and isomers thereof, nonyl and isomers thereof; decyl and isomers thereof, undecyl and isomers thereof, dodecyl and isomers thereof, tridecyl and isomers thereof, tetradecyl and isomers thereof, pentadecyl and isomers thereof, hexadecyl and isomers thereof, heptadecyl and isomers thereof, octadecyl and isomers thereof, nonadecyl and isomers thereof, eicosyl and isomers thereof.

[0101] The term "optionally substituted alkyl" refers to an alkyl group optionally substituted with one or more substituents (e.g., 1 to 4 substituents, e.g., 1, 2, 3, or 4 substituents) at any available point of attachment. Non-limiting examples of such substituents include esters, carboxylic acids, alkyl moieties, alkene moieties, alkyne moieties, ... and the like.

[0102] In the context of the present invention, alkyl, alkene and alkyne moieties as defined herein may also contain one or more heteroatoms, wherein for example a C atom in the alkyl, alkene or alkyne chain is replaced by a heteroatom such as selected from N, S or O.

[0103] Unless otherwise indicated, the term "alkenyl" or "alkene" as used herein refers to a straight, cyclic, or branched hydrocarbon group containing at least one carbon-carbon double bond. Examples of alkenyl groups include vinyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, hexenyl, hexadienyl, which are in terminal or internal positions, etc. Typically, the alkenyl or alkene moiety of the present invention contains 2 to 20 C atoms. Optionally substituted alkenyl refers to an alkenyl group with optionally one or more substituents (e.g., 1, 2, 3, or 4) selected from those substituents defined above for substituted alkyl.

[0104] Unless otherwise indicated, the term "alkynyl" or "alkyne" as used herein refers to a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond. 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, etc. Typically, the alkenyl or alkene moiety of the present invention contains 2 to 20 C atoms. Optionally substituted alkynyl refers to an alkynyl having optionally one or more substituents (e.g., 1, 2, 3 or 4) selected from those substituents defined above for substituted alkyl.

[0105] The term "cycloalkyl" itself or as a part of another substituent is a cyclic alkyl group, i.e. a monovalent, saturated or unsaturated hydrocarbon group with 1, 2 or 3 ring structures.Cycloalkyl includes all saturated or partially saturated hydrocarbon groups (containing 1 or 2 double bonds) containing 1 to 3 rings, including monocyclic, bicyclic or polycyclic alkyl groups.Cycloalkyl groups can contain 3 or more carbon atoms in the ring, and usually contain 3 to 15 atoms according to the present invention.The example of cycloalkyl groups includes but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, adamantyl and cyclodecyl, wherein cyclopropyl is particularly preferred. "Optionally substituted cycloalkyl" refers to a cycloalkyl with optionally one or more substituents (e.g. 1 to 3 substituents, e.g. 1, 2, 3 or 4 substituents), and the substituents are selected from those substituents defined above for substituted alkyl.

[0106] In the case where the alkyl group as defined is divalent, that is, there is two single bonds for being connected to two other groups, they are referred to as "alkylene" groups. Non-limiting examples of alkylene groups include methylene, ethylene, methylmethylene, trimethylene, propylene, tetramethylene, ethylethylene, 1,2-dimethylethylene, pentamethylene and hexamethylene. Similarly, in the case where the alkenyl group as defined above and the alkynyl group as defined above are respectively divalent groups with single bonds for being connected to two other groups, they are referred to as "alkenylene" and "alkynylene" respectively.

[0107] As used herein, the term "heterocycle" itself or as part of another group refers to a non-aromatic, fully saturated or partially unsaturated cyclic group (e.g., 3 to 13-membered monocyclic, 7 to 17-membered bicyclic or 10 to 20-membered tricyclic ring system, or containing a total of 3 to 10 ring atoms) having at least one heteroatom in at least one carbon-containing ring. Each ring of the heterocyclic group containing heteroatoms can have 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and / or sulfur atoms, wherein nitrogen and sulfur heteroatoms can be optionally oxidized, and nitrogen heteroatoms can be optionally quaternized. Where valence permits, the heterocyclic group can be attached to any heteroatom or carbon atom of the ring or ring system. The ring of the polycyclic heterocycle can be fused, bridged and / or connected by one or more spiral atoms. Optionally substituted heterocycle refers to a heterocycle with optionally one or more substituents (e.g., 1 to 4 substituents, or, for example, 1, 2, 3 or 4), the substituents being selected from those substituents defined above for substituted alkyl. Non-limiting examples of heterocycles include piperidinyl, pyrrolidinyl, azepanyl, morpholinyl, and the like.

[0108] The term "aryl" as used herein (also referred to herein as aromatic heterocycle) refers to a polyunsaturated aromatic hydrocarbon group having a single ring (i.e., phenyl) or multiple aromatic rings fused together (e.g., naphthalene or anthracene) or covalently linked, typically containing 6 to 10 atoms; wherein at least one ring is aromatic. The aromatic ring may optionally include one to three additional rings (cycloalkyl, heterocyclyl, or heteroaryl) fused thereto. Aryl is also intended to include partially hydrogenated derivatives of the carbocyclic ring systems listed herein. Non-limiting examples of aryl include phenyl, etc.

[0109] The aryl ring or heterocyclic ring as defined herein may be optionally substituted with one or more substituents (e.g., 1 to 5 substituents, e.g., 1, 2, 3, or 4) at any available point of attachment. Non-limiting examples of such substituents are selected from halogen, hydroxy, oxo, nitro, amino, hydrazine, aminocarbonyl, azido, cyano, alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkylalkyl, alkylamino, alkoxy, -SO2-NH2, aryl, heteroaryl, aralkyl, haloalkyl, haloalkoxy, alkoxycarbonyl, alkylaminocarbonyl, heteroarylalkyl, alkylsulfonamide, heterocyclyl, alkylcarbonylaminoalkyl, aryloxy, alkylcarbonyl, acyl, arylcarbonyl, aminocarbonyl, alkylsulfoxide, -SO2R a , alkylthio, carboxyl, etc., where R a is an alkyl group or a cycloalkyl group.

[0110] When a carbon atom in an aryl group is replaced with a heteroatom, the resulting ring is referred to herein as a heteroaryl ring.

[0111] The term "heteroaryl" as used herein, by itself or as part of another group, refers to, but is not limited to, an aromatic ring of 5 to 12 carbon atoms or a ring system containing 1 to 3 rings fused together or covalently linked, typically containing 5 to 8 atoms; at least one of which is aromatic, wherein one or more carbon atoms in one or more of these rings may be replaced by oxygen, nitrogen or sulfur atoms, wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. Such rings may be fused to an aryl, cycloalkyl, heteroaryl or heterocyclyl ring. Non-limiting examples of such heteroaryls include: pyridinyl, aza Ji et al.

[0112] "Optionally substituted heteroaryl" refers to a heteroaryl group having optionally one or more substituents (eg, 1 to 4 substituents, such as 1, 2, 3 or 4) selected from those defined above for substituted aryl.

[0113] The term "oxo" as used herein refers to the group =0.

[0114] As used herein, the term "alkoxy" or "alkyloxy" refers to a group having the formula -OR b A group in which R b Preferably, the alkoxy group is C1-C 10 Alkoxy, C1-C6 alkoxy or C1-C4 alkoxy. The non-limiting examples of suitable alkoxy include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy and hexyloxy. In the case where the oxygen atom in the alkoxy group is replaced by sulfur, the resulting group is referred to as thioalkoxy. "Haloalkoxy" is an alkoxy group in which one or more hydrogen atoms in the alkyl group are replaced by halogen. The non-limiting examples of suitable haloalkoxy 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, 4,4,4-trichlorobutoxy.

[0115] The term "carboxy" or "carboxyl" or "hydroxycarbonyl" by itself or as part of another substituent refers to the group -CO2H. Thus, a carboxyalkyl group is an alkyl group as defined above having at least one substituent which is -CO2H.

[0116] The term "alkoxycarbonyl" by itself or as part of another substituent refers to a carboxyl group attached to an alkyl group, i.e., forming a -C(=O)ORe , where R e As defined above for alkyl.

[0117] The term "alkylcarbonyloxy" by itself or as part of another substituent refers to -OC(=O)R e , where R e As defined above for alkyl.

[0118] Whenever the term "substituted" is used in the present invention, it is intended to indicate that one or more hydrogens on the indicated atom in the expression using "substituted" are replaced with a group selected from the indicated group, provided that the normal valency of the indicated atom is not exceeded and that the substitution results in a chemically stable compound, i.e., a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture and formulation into a therapeutic agent.

[0119] Where a group may be optionally substituted, such a group may be substituted one or more times, and preferably one, two or three times. The substituent may be selected from, for example, the group comprising halogen, hydroxy, oxo, nitro, amido, carboxyl, amino, cyano, haloalkoxy and haloalkyl.

[0120] As used herein, terms such as "alkyl, aryl, or cycloalkyl, each of which is optionally substituted" or "optionally substituted alkyl, aryl, or cycloalkyl" refer to optionally substituted alkyl, optionally substituted aryl, and optionally substituted cycloalkyl.

[0121] Furthermore, where a group is divalent, i.e. has two single bonds for attachment to two other groups, it may exist in either orientation in the molecule at each occurrence, even if not specifically indicated in the formula or definition of the R group. For example -(C=N-NH2)- as part of X means that X may be represented, for example, by -(C=N-NH2)- or alternatively by the reverse orientation as -(NH2-C=N)-.

[0122] In the context of the present invention, the term lipid refers to a chemically defined substance that is insoluble in water but particularly soluble in alcohol, ether and chloroform. Ionizable lipids or cationic lipids are lipids that are generally composed of three parts: an amine head group, a linker moiety and a hydrophobic tail. In the context of a compound or lipid, the term "ionizable" (or alternatively cationic) refers to the presence of any uncharged group in the compound or lipid that is capable of reacting with water by generating an ion (usually H + ions) and thus become positively charged themselves. Alternatively, any uncharged group in the compound or lipid can generate electrons and thus become negatively charged.

[0123] In the context of the present invention, the linker moiety can be selected from a variety of different linkers, however, disulfide, ketal and ether linkers are particularly preferred. Therefore, and in order to obtain their lipid characteristics, the compounds of the present invention comprise a lipid tail represented by R1 and R2, wherein the total number of C atoms of the two groups combined is at least 8, such as 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.

[0124] Thus, in the context of the present invention, R1 may, for example, contain 3 C atoms and R2 may contain 5 C atoms, so that the total number of C atoms of the two groups combined is at least 8. In the context of the present invention, R1 and R2 are in any case different from one another.

[0125] Thus, in a particular embodiment, the present invention provides a lipid, in particular an ionizable lipid as defined herein and in particular an ionizable lipid as defined herein and represented by any one of formula (II) or (III):

[0126]

[0127] in

[0128] R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH;

[0129] R7 and R7" are independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution; and the total number of C atoms in R7 and R7" together is at least 6;

[0130] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0131] o and p are each independently an integer selected from 1 to 10;

[0132] -

[0133] Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl;

[0134] R5 is selected from -NH- and -O-;

[0135] R6 is -C 1-6 Alkylene-; and

[0136] wherein o and p are different from each other; or wherein R7 and R7″ are different from each other.

[0137] In yet further embodiments, the invention provides lipids, in particular ionizable lipids as defined herein and represented by any one of formula (IIa), (IIb) or (IIc);

[0138]

[0139]

[0140] in

[0141] R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH;

[0142] R7 and R7" are independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution; and the total number of C atoms in R7 and R7" together is at least 6;

[0143] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0144] o and p are each independently an integer selected from 1 to 10;

[0145] R5 is selected from -NH- and -O-;

[0146] R6 is -C 1-6 Alkylene-;

[0147] Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl;

[0148] and wherein o and p are different from each other; and / or wherein R7 and R7″ are different from each other.

[0149] In yet further embodiments, the invention provides lipids, in particular ionizable lipids as defined herein and represented by any one of formula (IIIa), (IIIb) or (IIIc);

[0150]

[0151] in

[0152] R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH;

[0153] R7 and R7" are independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution; and the total number of C atoms in R7 and R7" together is at least 6;

[0154] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0155] o and p are each independently an integer selected from 1 to 10;

[0156] R5 is selected from -NH- and -O-;

[0157] R6 is -C 1-6 Alkylene-;

[0158] Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl;

[0159] and wherein o and p are different from each other; and / or wherein R7 and R7″ are different from each other.

[0160] In further specific embodiments, the present invention provides lipids, particularly ionizable lipids as defined herein and particularly ionizable lipids as defined herein and represented by any of formula (IV) or (V)

[0161]

[0162] in

[0163] R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH;

[0164] R7 and R7" are independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution; and the total number of C atoms in R7 and R7" together is at least 6;

[0165] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0166] o and p are each independently an integer selected from 1 to 10;

[0167] -

[0168] Each R8 and R9 is independently selected from -H, -C 1-6Alkyl and -C 3-6 Cycloalkyl;

[0169] and wherein o and p are different from each other; or wherein R7 and R7″ are different from each other.

[0170] In yet further embodiments, the invention provides lipids, in particular ionizable lipids as defined herein and represented by any one of formula (IVa), (IVb) or (IVc);

[0171]

[0172] in

[0173] R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH;

[0174] R7 and R7" are independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution; and the total number of C atoms in R7 and R7" together is at least 6;

[0175] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0176] o and p are each independently an integer selected from 1 to 10;

[0177] Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl;

[0178] and wherein o and p are different from each other; and / or wherein R7 and R7″ are different from each other.

[0179] In yet further embodiments, the invention provides lipids, in particular ionizable lipids as defined herein and represented by any one of formula (Va), (Vb) or (Vc);

[0180]

[0181] in

[0182] R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH;

[0183] R7 and R7" are independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution; and the total number of C atoms in R7 and R7" together is at least 6;

[0184] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0185] o and p are each independently an integer selected from 1 to 10;

[0186] Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl;

[0187] and wherein o and p are different from each other; and / or wherein R7 and R7″ are different from each other.

[0188] In another embodiment, the moiety -NR3R4 can be selected from the list comprising: -N-(C 1-6 In a very specific embodiment, the moiety -NR3R4 can be selected from the list comprising -N-(CH3)2, -pyrrolidinyl, -piperidinyl, or -azepanyl; in particular -N-(CH3)2, or -azepanyl.

[0189] In very specific embodiments, the present invention provides compounds according to any one of formula (I), (II), (III), (IV), (V) and any variants (a), (b), or (c) thereof, wherein one or more of the following apply:

[0190] R1 and R2 are each independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Each of the alkynyl groups may be optionally substituted with 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl substitution;

[0191] wherein the total number of C atoms in R1 and R2 together is at least 8;

[0192] wherein R1 and R2 are different from each other;

[0193] R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH;

[0194] Each occurrence of R7 is independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution;

[0195] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0196] -

[0197] Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl;

[0198] R5 is selected from -NH- and -O-; and

[0199] R6 is -C 1-6 Alkylene-.

[0200] In another very specific embodiment, the present invention provides compounds according to any one of formula (I), (II), (III), (IV), (V) and any variants (a), (b), or (c) thereof, wherein one or more of the following apply:

[0201] R1 and R2 are each independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl;

[0202] wherein the total number of C atoms in R1 and R2 together is at least 8;

[0203] wherein R1 and R2 are different from each other;

[0204] 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 5-10 membered non-aromatic heterocyclic ring;

[0205] Each occurrence of R7 is independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl;

[0206] m and n are each independently an integer selected from 1, 2, 3 and 4;

[0207] X is selected from -O-, -SS-, -O-CR8R9-O-;

[0208] Each R8 and R9 is independently selected from -H, -C 1-6 alkyl;

[0209] R5 is -NH-; and

[0210] R6 is -C 1-6 Alkylene-.

[0211] The invention further provides lipids, in particular ionizable lipids as defined herein and selected from the list comprising:

[0212]

[0213]

[0214] All lipids as defined herein can exist as different isomers / stereoisomers. In particular, lipids as defined herein can exist in trans or cis configurations, such as when they contain double bonds. In a preferred embodiment, lipids as defined herein exist in cis configurations. In the context of the present invention, the term "cis" indicates that the functional groups are on the same side of the plane, while "trans" refers to that they are on the opposite side.

[0215] In yet further embodiments, the invention provides lipids, in particular ionizable lipids as defined herein; wherein the total number of C atoms in R1 and R2 together is at least 14, such as at least 15, at least 17, at least 18, at least 19 or at least 20.

[0216] The present invention further provides lipids, in particular ionizable lipids as defined herein; wherein the total number of C atoms in R7 and R7" together is at least 12.

[0217] In a further embodiment, the invention provides a lipid, in particular an ionizable lipid as defined herein; wherein m and n are the same and are an integer selected from 1, 2, 3 and 4; such as 1 or 2 or 3 or 4; preferably 2.

[0218] The invention further provides lipids, in particular ionizable lipids as defined herein; wherein R5 is -NH-.

[0219] In a further aspect, the present invention provides lipid nanoparticles or lipid nanoparticle compositions comprising a lipid, in particular an ionizable lipid as defined herein.

[0220] In the context of the present invention, the term lipid nanoparticle (LNP), also referred to as solid lipid nanoparticle, refers to a nanoparticle comprising lipid. They are generally used as a drug delivery system or pharmaceutical preparation. LNP was first approved in 2018 as a drug delivery carrier, and is currently used in several vaccines based on candidate RNA. Lipid nanoparticles are generally spheres with an average diameter of 10 to 1000 nanometers, and have a lipid core matrix that can dissolve lipophilic molecules. The term lipid is used in this article with a broader meaning, and includes triglycerides, diglycerides, monoglycerides, fatty acids, steroids (such as cholesterol) and waxes. Biomembrane lipids such as phospholipids, sphingomyelin, bile acids and sterols are generally used as stabilizers in LNP.

[0221] As used herein, the term "nanoparticle" refers to any particle having a diameter that makes the particle suitable for systemic, in particular intravenous administration, in particular of nucleic acids, which typically has a diameter of less than 1000 nanometers (nm), preferably less than 500 nm, even more preferably less than 200 nm, such as, for example, 50 to 200 nm; preferably 80 to 160 nm.

[0222] Therefore, in the context of the present invention, the nanoparticles as disclosed herein further comprise one or more additional lipids, which may or may not act as stabilizers, such as phospholipids, sterols and / or PEG lipids.

[0223] In the context of the present invention, the term "PEG lipid" or alternatively "PEGylated lipid" refers to any suitable lipid modified with a PEG (polyethylene glycol) group. In the context of the present invention, particularly suitable PEG lipids are characterized by being C18-PEG lipids, C14-PEG lipids (e.g., DMG-PEG or DMG-PEG2000) or C16-PEG lipids.

[0224] C18-PEG lipids contain a polyethylene glycol moiety that defines the molecular weight of the lipid, and a fatty acid tail containing 18 C-atoms. In a specific embodiment, the C18-PEG2000 lipid is selected from the list comprising: (distearoyl-based)-PEG2000 lipids, such as DSG-PEG2000 lipids (2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol-2000) or DSPE-PEG2000 lipids (1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]); or (dioleoyl-based)-PEG2000 lipids, such as DOG-PEG2000 lipids (1,2-dioleoyl-rac-glycerol) or DOPE-PEG2000 lipids (1,2-dioleoyl-sn-glyceryl-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000])

[0225]

[0226] C14-PEG lipids contain a polyethylene glycol moiety that defines the molecular weight of the lipid, and a fatty acid tail comprising 14 C-atoms. In a specific embodiment, the C14-PEG2000 lipid is based on dimyristoyl, i.e., has 2 C14 tails, such as selected from a list comprising: (dimyristoyl)-PEG2000 lipids, such as DMG-PEG2000 lipids (1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol-2000) or 2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine alcohol-2000 (DMPE-PEG2000).

[0227]

[0228]

[0229] In the context of the present invention, the term "phospholipid" refers to a lipid molecule that consists of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. The two components are most commonly linked together by a glycerol molecule, and therefore, the phospholipids of the present invention are preferably glycero-phospholipids. In addition, the phosphate group is usually modified with a simple organic molecule such as choline (i.e., providing phosphorylcholine) or ethanolamine (i.e., providing phosphoethanolamine).

[0230] In the context of the present invention, suitable phospholipids may be selected from the list comprising: 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 (DLPC). PPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-undecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16Lyso PC), 1,2-dilinolenoyl-sn-glyceryl-3-phosphocholine, 1,2-arachidonoyl-sn-glyceryl-3-phosphocholine, 1,2-di-docosahexaenoyl-sn-glyceryl-3-phosphocholine, 1,2-diphytanoyl-sn-glyceryl-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dilinoleoyl Acyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-di-docosahexaenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dioleoyl-sn-glyceryl-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0231] In a more specific embodiment, said phospholipid is selected from the list comprising 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and mixtures thereof.

[0232] In the context of the present invention, the term "sterol", also referred to as sterol (steroid alcohol), is a subgroup of steroids naturally occurring in plants, animals and fungi or that can be produced by some bacteria. In the context of the present invention, any suitable sterol can be used, such as selected from the list comprising: cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol (nicasterol), sitosterol and stigmasterol; preferably cholesterol.

[0233] In specific embodiments of the present invention, one or more of the following apply:

[0234] - said LNP comprises between about 35 mol % and 65 mol % and between 35 mol % and 65 mol % of said ionizable lipid;

[0235] - said LNP comprises between about 5 mol% and 25 mol% and between 5 mol% and 25 mol% of said phospholipid;

[0236] - said LNP comprises about 0.5 mol% and 3.0 mol% and between 0.5 mol% and 3.0 mol% of said PEG lipid;

[0237] The balance is the amount of the sterol.

[0238] In yet another embodiment of the present invention, the lipid nanoparticle or lipid nanoparticle composition as defined herein further comprises a carrier (cargo) molecule, such as a pharmaceutically active agent (e.g., a small molecule) or a biomolecule, such as a peptide, a protein, or a nucleic acid. In a specific embodiment, the carrier can be a nucleic acid, such as DNA or RNA; preferably mRNA. In another specific embodiment, the carrier can be a TLR agonist, such as, for example, a TLR3 agonist poly I: C, or a TLR9 agonist CpG.

[0239] Before loading in lipid nanoparticles, the cargo molecules can be additionally modified to induce the overall polyanionic properties of the molecules. This can be carried out, for example, by bonding them to the Glu10 moiety as illustrated in the Examples section. The Glu10 moiety is a 10 glutamic acid moiety that increases the polyanionic properties of the molecule to which it is connected.

[0240] Thus, the lipid nanoparticles and lipid nanoparticle compositions of the invention are particularly suitable for intracellular delivery of their cargo molecules. Thus, the invention provides the use of lipid nanoparticles and lipid nanoparticle compositions as defined herein for intracellular delivery of cargo molecules.

[0241] In a particular embodiment, the lipid nanoparticle or lipid nanoparticle composition as defined herein further comprises a nucleic acid, preferably mRNA.

[0242] In the context of the present invention, "nucleic acid" is deoxyribonucleic acid (DNA) or preferably ribonucleic acid (RNA), more preferably mRNA. According to the present invention, nucleic acid includes genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. According to the present invention, nucleic acid can be in the form of a single-stranded or double-stranded and linear or covalently closed molecule to form a ring. Nucleic acid (for example, in the form of RNA, which can be prepared by DNA template by in vitro transcription) can be used to introduce into cells, i.e. transfected cells. In addition, RNA can be modified by stabilizing sequences, capping and / or polyadenylation before application.

[0243] In the context of the present invention, the term "RNA" refers to a molecule comprising ribonucleotide residues and preferably completely or substantially composed of ribonucleotide residues." Ribonucleotide " refers to a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA (such as partially purified RNA), substantially pure RNA, synthetic RNA, recombinant RNA, and RNA modified by adding, deleting, replacing and / or changing one or more nucleotides that are different from naturally occurring RNA. Such changes can include adding non-nucleotide materials, such as adding to one or more ends or inside of RNA, for example, at one or more nucleotides of RNA. The nucleotides in the RNA molecule can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. The RNA of these changes can be referred to as analogs. Nucleic acid can be contained in a vector. The term "vector" as used herein includes any vector known to the skilled person, including a plasmid vector, a cosmid vector, a phage vector such as lambda phage, a viral vector such as an adenovirus vector or a baculovirus vector, or an artificial chromosome vector such as a bacterial artificial chromosome (BAC), a yeast artificial chromosome, or an analog of naturally occurring RNA.

[0244] According to the present invention, the term "RNA" includes and preferably relates to "mRNA", which refers to "messenger RNA" and refers to a "transcript" that can be produced using DNA as a template and encodes a peptide or protein. mRNA generally comprises 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 in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to technicians. For example, there are a variety of commercially available in vitro transcription kits.

[0245] In a further aspect, the present invention provides a pharmaceutical composition comprising one or more LNPs as defined herein and a pharmaceutically acceptable agent, such as a carrier, an excipient, ... Such a pharmaceutical composition is particularly suitable as a vaccine. Therefore, the present invention also provides a vaccine comprising one or more LNPs according to the present invention.

[0246] In the context of the present invention, the term "vaccine" as used herein refers to any preparation intended to provide adaptive immunity (antibodies and / or T cell responses) against a disease. To this end, a vaccine as referred to herein contains at least one nucleic acid molecule, such as an mRNA molecule encoding an antigen against which an adaptive immune response is elicited. The antigen may be present in the form of a weakened or inactivated form of a microorganism, a protein or peptide, or an antigen encoding a nucleic acid. In the context of the present invention, an antigen refers to a protein or peptide that is recognized as foreign by the host's immune system, thereby stimulating the production of antibodies against such an antigen, with the purpose of counteracting such an antigen. A vaccine may be preventive (e.g., preventing or improving the effects of future infection by any natural or "wild" pathogen), or therapeutic (e.g., actively treating or alleviating the symptoms of an ongoing disease). The administration of a vaccine is referred to as vaccination.

[0247] The vaccine of the present invention can be used to induce an immune response, particularly an immune response to a disease-related antigen or a cell expressing an antigen associated with a disease, such as an immune response to cancer. Therefore, the vaccine can be used for preventive and / or therapeutic treatment of diseases involving a disease-related antigen or a cell expressing an antigen associated with a disease, 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 nanoparticle described herein is preferably an antigen associated with a disease, or induces an immune response to a disease-related antigen or a cell expressing an antigen associated with a disease.

[0248] The present invention also provides LNPs, pharmaceutical compositions and vaccines according to the present invention for use in human medicine or veterinary medicine. It is also intended to provide the use of LNPs, pharmaceutical compositions and vaccines according to the present invention for use in human medicine or veterinary medicine. Finally, the present invention provides methods for preventing and treating human and veterinary disorders by administering LNPs, pharmaceutical compositions and vaccines according to the present invention to subjects in need thereof. Such pharmaceutical compositions are particularly suitable for use in various fields, such as preventive vaccines, therapeutic vaccines, protein replacement therapies, gene editing, gene silencing, small molecule delivery, etc.

[0249] The present invention further provides the use of LNP, pharmaceutical composition or vaccine according to the present invention for immunogenic delivery of one or more nucleic acid molecules. Thus, the LNP, pharmaceutical composition and vaccine of the present invention are very useful in treating several human and veterinary disorders. Thus, the present invention provides LNP, pharmaceutical composition and vaccine of the present invention for use in treating cancer or infectious diseases.

[0250] The lipid nanoparticles of the present invention can be prepared according to the protocol specified in the Examples section. More generally, LNPs can be prepared using a method comprising:

[0251] - preparing a first alcohol composition comprising the ionizable lipid, the phospholipid, the sterol, the PEG lipid and a suitable alcohol solvent;

[0252] - preparing a second aqueous composition comprising the one or more nucleic acids and an aqueous solvent;

[0253] - mixing said first and second compositions in a microfluidic mixing device.

[0254] In more detail, the lipid components are combined in an alcohol vehicle such as ethanol at appropriate concentrations. An aqueous composition containing nucleic acids is added thereto, and then loaded into a microfluidic mixing device.

[0255] The purpose of microfluid mixing is to achieve thorough and rapid mixing of multiple samples (i.e. lipid phase and nucleic acid phase) in a microdevice. Such sample mixing is usually achieved by enhancing the diffusion effect between different material flows. In addition, several microfluid mixing devices can be used, such as, for example, those reviewed in Lee et al., 2011. A particularly suitable microfluid mixing device according to the present invention is the NanoAssemblr from Precision Nanosystems.

[0256] Other techniques suitable for preparing the LNPs of the present invention include dispersing the components in a suitable dispersion medium such as an aqueous solvent and an alcohol solvent, and applying one or more of the following methods: ethanol dilution method, simple hydration method, ultrasonic treatment, heating, vortexing, ether injection method, French press method, bile acid method, Ca 2+ Fusion method, freeze-thaw method, reverse phase evaporation method, T-junction mixing, microfluidic hydrodynamic focusing, staggered herringbone mixing, etc.

[0257] The ionizable lipids of the present invention can be prepared according to the reaction schemes provided in the examples below, but those skilled in the art will understand that these are only for illustration of the present invention and that the compounds of the present invention can be prepared by any of several standard synthetic methods commonly used by those skilled in the art of organic chemistry. Example

[0258] Preparation of lipids

[0259] 1. General Information

[0260] Unless otherwise noted, all glassware was oven dried before use, and all reactions were performed using standard Schlenk techniques under an argon atmosphere. Dry solvents were purchased from Acros Organics or Sigma-Aldrich and used without further purification. Unless otherwise noted, all reagents were purchased from commercial sources and used without further purification. The reaction process was monitored by thin layer chromatography (TLC) performed on an aluminum plate coated with 0.2 mm thickness of Kieselgel F254. Visualization was achieved by ultraviolet light (254 nm) or by staining with ninhydrin, cerium molybdate or potassium permanganate. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck and co.). Mass spectra were obtained using Finnigan MAT 8200 (70 eV), Agilent 5973 (70 eV). Electrospray ionization (ESI) or electron impact ionization (EI) were used to obtain mass spectra. All 1H NMR, 13C NMR NMR were recorded on a Bruker AV-400 in chloroform-d1 or DMSO-d6. The solvent peak was used as an internal standard (CDCl3: 1 H=7.26ppm, 13 C=77.16ppm;CD3SOCD3: 1 H=2.50ppm, 13 Chemical shifts are given in parts per million (ppm) with reference to tetramethylsilane (C = 39.52 ppm). Coupling constants are given in Hz. 1H NMR splitting patterns were assigned as singlet (s), broad (brd), doublet (d), triplet (t), quartet (q), quintet (qu), quintet (p), sextet (se), septet (sep), octet (o), or combinations thereof. Unexplained splitting patterns were assigned as multiplets (m).

[0261] 2. Lipid Synthesis

[0262] 2.1 A general route for synthesizing the lipids represented by the structure of Formula I is shown below.

[0263]

[0264] Synthesis of compound 3

[0265] To a stirred solution of compound 1 (1.0 eq.) and alcohol 2 (1.2 eq.) in CH2Cl2 at 0°C was added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.2 eq.), N,N-diisopropylethylamine (4.0 eq.) and DMAP (0.2 eq.) (Sabnis et al., 2018) under nitrogen. The reaction was stirred at room temperature for 18 hours. The reaction was then diluted with dichloromethane and washed with saturated Na2CO3 (aq.), brine and dried over Na2SO4. After filtration and vacuum concentration, the resulting residue was purified by silica gel column chromatography (ethyl acetate in hexanes: 1% to 4%) to give compound 3.

[0266] Synthesis of compound 6

[0267] Under nitrogen, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.3 eq.), Et3N (2 eq.) and DMAP (0.2 eq.) (Rajappan et al., 2020) were added to a stirred solution of compound 4 (1.0 eq.) and alcohol 5 (1.0 eq.) in CH2Cl2 at 0°C. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. The organic phase was washed with saturated Na2CO3 (aqueous solution), water and brine in sequence. The organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The crude product 6 was used directly without further purification.

[0268] Synthesis of compound 7

[0269] Compound 6 is dissolved in a mixed solvent of CH2Cl2 / CF3COOH (10mL / 10mL). After stirring at room temperature for 1 hour, the solvent is removed under reduced pressure and redissolved in CH2Cl2. First, the organic phase is washed with saturated Na2CO3 (aqueous solution), then washed with salt water, and then dried over Na2SO4. After filtering and vacuum concentration, the gained residue is further purified to obtain compound 7 by silica gel column chromatography (CH2Cl2 / MeOH / NH4OH (0.5%): 9 / 1).

[0270] Synthesis of compound 8

[0271] To a stirred solution of compound 7 in anhydrous DMF, anhydrous KCO (3 equivalents) and a solution of compound 3 in DMF were added. The reaction was vigorously stirred for 5 h at 80 ° C under nitrogen. After cooling to room temperature, the solid was removed by filtration, and DMF was removed under reduced pressure. Finally, the crude residue was purified by silica gel column chromatography (hexane / ethyl acetate=100:0 to 85:15) to obtain compound 8.

[0272] Synthesis of compound 12

[0273] Compound 10 (1.0 eq.) (Amano et al., 2017), compound 8 (2.1 eq.), DMAP (0.2 eq.) and Et3N (5.0 eq.) were dissolved in DMF at room temperature. The resulting mixture was stirred at room temperature for 24 h, and then amine 11 (3.5 eq.) was added. After 5 h, 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 its 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 12.

[0274] The name of compound 12 is given just below its structure.

[0275]

[0276] Yield: 18% colorless oil. 1H-NMR (400MHz, chloroform-d) δ5.70-5.61(m,1H,H56),5.57-5.48(m,2H,H57 and NH),4.87(qu,J=6.3Hz,1H,H27),4.63(d,J=6.9Hz,2H,H54),4.38-4.28(m,4H,H2&H7),3.73(q,J =7.0Hz,2H,H14),3.26-3.12(m,4H,H21&H22),2.99-2.88(m,4H,H3&H6),2.53-2.42(m,2H,H15),2 .40-2.16(m,10H,H48,H51,H64&H65);2.10(q,J=7.0Hz,2H,H58),1.67-1.59(m,4H,H24&H47),1. 59-1.47(m,8H,H16,H18,H29&H30),1.36-1.21(m,44H);0.88(t,J=7.00Hz,9H,H37,H49,H63)ppm. 13 C-NMR (100 MHz, chloroform-d) δ 173.6 (ester C=O), 156.8 (carbamate C=O), 155.8 (carbamate C=O), 135.4 (olefin carbon), 123.4 (olefin carbon), 74.1 (C27); 62.82 (C54); 62.5, 60.2, 50.7, 45.1, 38.1, 37.9, 34.7, 34.3, 34.1, 31.8, 29.6, 29.5, 29.2, 29.1, 27.3, 26.6, 25.3, 25.1, 22.7, 14.1 (C37, C49, C63) ppm. LRMS (ESI) (m / z): [M+H] + (C 52 H 99 N3O8S2) calculated value: 958.7, found value: 958.6.

[0277]

[0278] Yield: 16% colorless oil. 1H-NMR (400 MHz, chloroform-d) δ 5.69-5.61 (m, 1H, H56), 5.58-5.48 (m, 1H, H57), 4.87 (qu, J = 6.3 Hz, 1H, H27), 4.63 (d, J = 6.9 Hz, 2H, H54), 4.37-4.29 (m, 4H, H2&H7), 3.52-3.36 (m, 2H, H14), 3.27-3.11 (m, 4H, H64&H69), 3.04-2.83 (m, 10H, H15, H3, H 6,H21&H22),2.36-2.21(m,4H,H48,H51),2.1(q,J=7.1Hz,2H,H58),1.88-1.76(m,4H,H65&H68),1.72-1.57(m,8H,H6 6, H67, H24&H47), 1.56-1.45 (m, 8H, H16, H18, H29 & H30), 1.42-1.21 (m, 44H); 0.88 (t, J=6.75Hz, 9H, H37, H49, H63) ppm. 13 C-NMR (100 MHz, chloroform-d) δ 173.6 (ester C=O), 153.5 (carbamate C=O), 151.7 (carbamate C=O), 135.4 (olefin carbon), 123.4 (olefin carbon), 74.1 (C27); 62.7 (C54), 60.2, 57.6, 55.4, 37.8, 34.7, 34.3 34.1, 31.9, 29.5, 29.5, 29.4, 29.2, 29.1 27.5, 26.9, 26.7, 25.3, 25.1, 22.7, 14.1 (C37, C49, C63) ppm. LRMS (ESI) (m / z): [M+H] + (C 55 H 105 The calculated value of N3O8S2 is 1012.8, the measured value is 1012.6.

[0279]

[0280] Yield: 14% colorless oil. 1H-NMR (400MHz, chloroform-d) δ4.87 (qu, J = 6.2 Hz, 1H, H27), 4.37-4.27 (m, 4H, H2&H7), 4.08 (t, J = 6.8 Hz, 2H, H54), 3.35-3.24 (m, 2H, H14), 3.23-3.12 (m, 4H, H21&H22), 3.02-2.88 (m, 4H, H3&H6), 2.50- 2.40(m,2H,H15),2.35-2.24(m,10H,H48,H51,H64&H65),1.71-1.58(m,6H,H56,H24&H47),1.57 -1.44(m,8H,H16,H18,H29&H30),1.42-1.21(m,48H),0.89(t,J=6.75Hz,9H,H37,H49,H63)ppm. 13 C-NMR (100 MHz, chloroform-d) δ 173.6 (ester C=O), 156.4 (carbamate C=O), 155.8 (carbamate C=O), 74.1 (C27), 64.4, 62.8, 62.5, 51.8, 45.11, 38.0, 37.9, 34.7, 34.3, 34.1, 31.9, 29.5, 29.2, 29.1, 28.6, 26.7, 25.9, 25.3, 25.1, 24.9, 22.7, 14.1 (C37, C49, C63) ppm. LRMS (ESI) (m / z): [M+H] + (C 52 H 101 N3O8S2) calculated value: 960.7, measured value: 960.6

[0281]

[0282] Yield: 19% colorless oil. 1H-NMR (400 MHz, chloroform-d) δ4.87 (qu, J = 6.3 Hz, 1H, H27), 4.37-4.29 (m, 4H, H2&H7), 4.11-4.1 (t, J = 6.9 Hz, 2H, H54), 3.47-3.32 (m, 2H, H14), 3.26-3.10 (m, 4H, H64&H69), 2.99-2.90 (m, 4H, H21&H22), 3.04-2.83 (m, 4H, H3&H6), 2.8 8-2.76(m,2H,H15),2.35-2.24(m,4H,H48,H51),1.85-1.71(m,4H,H65&H68),1.69-1.59(m,10H,H56,H66,H67, H24&H47),1.55-1.44(m,8H,H16,H18,H29&H30),1.42-1.21(m,48H),0.89(t,J=6.75Hz,9H,H37,H49,H63)ppm. 13 C-NMR (100 MHz, chloroform-d) δ 173.6 (ester C=O), 155.7 (carbamate C=O), 149.1 (carbamate C=O), 74.1 (C27), 64.4, 64.0, 62.8, 62.7, 59.4, 55.3, 52.9, 52.8, 50.3, 50.0; 37.9, 34.7, 34.3, 34.1, 31.9, 29.5, 29.2, 29.1, 28.6, 28.1 26.9, 26.7, 25.9, 25.3, 25.1 24.9, 22.7 14.1 (C37, C49, C63) ppm. LRMS (ESI) (m / z): [M+H] + (C 56 H 107 The calculated value of N3O8S2 is 1014.8, the measured value is 1014.6.

[0283]

[0284] Yield: 15% colorless oil. 1H-NMR (400MHz, chloroform-d) δ4.91-4.77 (m, 2H, H27&H54), 4.36-4.28 (m, 4H, H2&H7), 3.40-3.33 (m, 2H, H14), 3.24-3.13 (m, 4H, H21&H22), 2.99-2.90 (m, 4H, H3&H6), 2.68-2.57 (brd, 2H, H15), 2.44-2. 36(brd,4H,H48,H51),2.33-2.22(m,6H,H66,H67),1.68-1.56(m,4H,H24&H47),1.57-1.44(m,1 2H, H16, H18, H29, H30, H56, H64), 1.42-1.21 (m, 48H), 0.89 (t, J = 6.75Hz, 9H, H37, H49, H63) ppm. 13 C-NMR (100 MHz, chloroform-d) δ 173.7 (ester C=O), 162.8 (carbamate C=O), 155.8 (carbamate C=O), 75.4 (C27), 74.1, 62.8, 62.6, 58.0, 44.8, 37.9, 34.7, 34.1, 33.6, 31.8, 29.5, 29.5, 29.2; 29.1, 26.9, 26.7, 25.3, 25.1, 22.7, 14.1 (C37, C49, C63) 9.60 (C65) ppm. LRMS (ESI) (m / z): [M+H] + (C 54 H 105 N3O8S2) calculated value: 988.7, measured value: 988.6

[0285]

[0286] Yield: 17% colorless oil. 1H-NMR (400 MHz, chloroform-d) δ4.91-4.77 (m, 2H, H27&H54), 4.36-4.28 (m, 4H, H2&H7), 3.40-3.33 (brd, 2H, H14), 3.24-3.13 (m, 4H, H66&H71), 2.99-2.90 (m, 4H, H21&H22), 2.88-2.76 (brd, 6H, H15, H3&H6), 2.36-2.2 2(m,4H,H48,H51),1.87-1.70(brd,4H,H67,H70),1.69-1.57(m,8H,H24,H47,H68,H69),1.57-1.44(m, 12H, H16, H18, H29, H30, H56, H64), 1.41-1.20 (m, 48H), 0.89 (t, J = 6.75Hz, 12H, H37, H49, H63&H65) ppm. 13 C-NMR (100 MHz, chloroform-d) δ 173.6 (ester C=O), 164.3 (carbamate C=O), 155.8 (carbamate C=O), 75.2 (C27), 74.1, 62.8, 62.7, 55.3, 52.9, 50.7, 37.9, 34.7, 34.1, 33.61, 31.9, 29.6, 29.5, 29.2, 29.1, 26.9, 26.7, 25.3, 25.1, 22.7, 14.1 (C37, C49, C63) 9.60 (C65) ppm. LRMS (ESI) (m / z): [M+H] + (C 58 H 111 N3O8S2) calculated value 1042.8, measured value: 1042.7

[0287]

[0288] Yield: 13% colorless oil. 1H-NMR (400MHz, chloroform-d) δ4.87 (qu, J=6.3, 2H, H26&H46), 4.37-4.29 (m, 4H, H2&H7), 3.33-3.25 (m, 2H, H14), 3.24-3.14 (m, 4H, H21&H22), 3.00-2.88 (m, 4H, H3&H6), 2.52-2.42 m, 2H, H15)2 .36-2.22(m,10H,H64,H67,H70&H75),1.68-1.57(m,4H,H63,H66),1.57-1.47(m,12H,H16, H18, H28, H29, H48, H49), 1.41-1.20 (m, 56H), 0.89 (t, J=6.8Hz, 12H, H36, H43, H56&H68)ppm. 13 C-NMR (100 MHz, chloroform-d) δ 173.6 (ester C=O), 163.3 (carbamate C=O), 155.8 (carbamate C=O), 74.2 (C27), 74.1, 62.9, 58.1, 53.4, 45.1, 37.9, 34.7, 34.6, 34.1, 31.9, 29.6, 29.5, 29.3, 29.2, 29.1, 26.4, 25.3, 25.1, 24.9, 22.6, 14.1 (C36, C43, C56 & C68) ppm. LRMS (ESI) (m / z): [M+H] + (C 58 H 113 N3O8S2) calculated value 1044.8, measured value: 1044.7

[0289]

[0290] Yield: 13% colorless oil. 1H-NMR (400 MHz, chloroform-d) δ4.87 (qu, J=6.3, 2H, H26&H46), 4.37-4.27 (m, 4H, H2&H7), 3.33-3.25 (brd, 2H, H14), 3.24-3.14 (brd, 4H, H21&H22), 3.03-2.81 (m, 10H, H3, H6, H71, H74, H15), 2.34-2.24 (m, 4H ,H64,H67),1.87-1.74(brd,4H,H71&H74),1.72-1.58(m,8H,H63,H66,H72,H73),1.57-1.46(m,12 H,H16,H18,H28,H29,H48,H49),1.38-1.19(m,56H),0.89(t,J=6.8Hz,12H,H36,H43,H56&H68)ppm. 13 C-NMR (100 MHz, chloroform-d) δ 173.6 (ester C=O), 163.1 (carbamate C=O), 154.7 (carbamate C=O), 74.2 (C27), 74.1, 62.9, 55.3, 53.4, 45.1, 37.9, 34.7, 34.6, 34.1, 31.9, 29.6, 29.5, 29.3, 29.2, 29.1, 26.4, 25.3, 25.1, 24.9, 22.6, 14.1 (C36, C43, C56 & C68) ppm. LRMS (ESI) (m / z): [M+H] + (C 62 H 119 N3O8S2) calculated value 1098.9, measured value: 1.098.8

[0291] Example: LNP Synthesis

[0292] mRNA synthesis:

[0293] All mRNAs were made in vitro from linearized DNA templates (peTheRNA vectors) by T7-mediated transcription, which introduced 5' and 3' UTRs and a poly A tail. The final mRNA utilized Cap 1 and 100% replacement of uridine with N1-methyl-pseudouridine.

[0294] The ionizable lipids of the invention were prepared according to the reaction scheme defined herein above. As a comparative example, ETG-23 corresponding to S-Ac7-DOg (see WO2022136641) was used and prepared according to the reaction scheme defined in '641.

[0295] LNP Synthesis:

[0296] By using NanoAssemblr Benchtop (Precision Nanosystems), mRNA solution (100mM, pH 4) and lipid solution in sodium acetate buffer were mixed with 2:1 volume ratio microfluidic at a speed of 16mL / min to prepare lipid-based nanoparticles.LNPs were prepared with the standard molar ratio of ionizable lipid / DSPC (Avanti Polar Lipids) / cholesterol (Sigma) / DMG-PEG2000 (Avanti Polar Lipids) of about 50 / 10 / 38.5 / 1.5.eGFP mRNA was encapsulated in all LNPs as a reporter mRNA with a mRNA / ionizable lipid molar ratio of 1 / 10.LNPs were dialyzed with TBS (TBS volume was 10000 times larger than LNP volume) using slide-a-lyzer dialysis cassette (20KMWCO, 3mL, ThermoFisher).Size, polydispersity and ζ potential were measured with Zetasizer Nano (Malvern). mRNA encapsulation was measured by a standard Ribogreen RNA assay (Invitrogen).

[0297] Data Analysis:

[0298] All raw data were analyzed using Graph Pad Prism version 9 software.

[0299] Table 1

[0300] Ionizable lipids size PDI % Encapsulation Comparative Example ETG-23 75,97 0,082 97 ETG-51 111,97 0,119 96 ETG-52 90,70 0,099 97 ETG-53 103,23 0,085 97 ETG-54 90,30 0,150 98 ETG-55 93,73 0,123 96 ETG-56 86,26 0,120 98 ETG-57 109,10 0,114 95 ETG-58 101,50 0,086 98

[0301] Example 1: In vivo FLUC MRNA expression after intravenous injection of MRNA LNPs

[0302] LNPs were prepared at a standard molar ratio of about 50 / 10 / 38.5 / 1.5 of ionizable lipid / DSPC / cholesterol / DMG-PEG2000. Firefly luciferase (Fluc) mRNA was encapsulated in all LNPs at a mRNA / ionizable lipid molar ratio of 1 / 10.

[0303] All experimental groups (BALB / c, female, 6-8 week old mice) received 1 intravenous injection of the corresponding mRNA LNP (5 μg mRNA, 100 μL volume; TBS buffer) or control buffer. After intraperitoneal injection of the substrate D-luciferin, Fluc mRNA expression was assessed via in vivo bioluminescence measurements at 4 hours and 24 hours after injection. High expression levels in vivo ( Figure 1 ).

[0304] Example 2: Evaluation of antibody responses to influenza hemagglutinin HA

[0305] LNPs were prepared at a standard molar ratio of about 50 / 10 / 38.5 / 1.5 of ionizable lipid / DSPC / cholesterol / DMG-PEG2000. Hemagglutinin (HA) mRNA was encapsulated in all LNPs at a mRNA / ionizable lipid molar ratio of 1 / 10.

[0306] On days 0 and 21, BALB / c mice (female, 6 weeks old) were injected intramuscularly via bilateral injections of 50 μl of the corresponding mRNA LNPs (2 μg mRNA, TBS buffer) or control buffer. Blood was collected on days 21 (primary immunization) and 35 (boost), and the IgG1 antibody titer against HA was determined using an in-house ELISA assay ( Figure 2 ).

[0307] Example 3: In vivo HEPO expression following intravenous injection of mRNA LNPs

[0308] LNPs were prepared at a standard molar ratio of about 50 / 10 / 38.5 / 1.5 of ionizable lipid / DSPC / cholesterol / DMG-PEG2000. Human erythropoietin (hEPO) mRNA was encapsulated in all LNPs at a mRNA / ionizable lipid molar ratio of 1 / 10.

[0309] BALB / c mice (female, 6 weeks old) were injected intravenously with 200 μl of the corresponding mRNA LNP (10 μg mRNA, TBS buffer) or control buffer in the tail vein. Blood was collected from the submandibular vein at different time points, and hEPO concentration was determined using the hEPO ELISA assay kit (catalog number # BMS2035-2-Thermofisher Scientific) according to the manufacturer's instructions. Compared with the ETG-23 control, ETG-53 showed higher hEPO expression ( Figure 3 A and B).

[0310] References

[0311] 1. Sabnis, S.; Kumarasinghe, S.; Salerno, T.; et al. A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non - human Primates. Molecular Therapy, 2018, 26, 1509 - 1519

[0312] 2. Rajappan, K.; Tanis, S.P.; Mukthavaram, R. et al. Property - Driven Design and Development of Lipids for Efficient Delivery of siRNA. J. Med. Chem., 2020, 63, 21, 12992 - 13012

[0313] 3. Amano, Y.; Umezawa, N.; Sato, S.; Watanabe, H.; Umehara, T.; Higuchi, T. Activation of lysine - specific demethylase 1 inhibitor peptide by redox - controlled cleavage of a traceless linker. Bioorg. Med. Chem. 2017, 25, 1227 - 1234.

Claims

1. An ionizable lipid represented by formula (I) in R1 and R2 are each independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Each of the alkynyl groups may be optionally substituted with 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl substitution; wherein the total number of C atoms in R1 and R2 together is at least 8; wherein R1 and R2 are different from each other; R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH; Each occurrence of R7 is independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution; m and n are each independently an integer selected from 1, 2, 3 and 4; - Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl; R5 is selected from -NH- and -O-; and R6 is -C 1-6 Alkylene-.

2. An ionizable lipid as defined in claim 1 and represented by any one of formulas (Ia), (Ib) and (Ic) in R1 and R2 are each independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Each of the alkynyl groups may be optionally substituted with 1 to 3 -O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 Alkenyl and -C 2-20 Alkynyl substitution; wherein the total number of C atoms in R1 and R2 together is at least 8; wherein R1 and R2 are different from each other; R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH; Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution; m and n are each independently an integer selected from 1, 2, 3 and 4; Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl; R5 is selected from -NH- and -O-; and R6 is -C 1-6 Alkylene-.

3. An ionizable lipid as defined in claim 1 and represented by any one of formula (II) or (III) in R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH; R7 and R7" are independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution; and the total number of C atoms in R7 and R7" together is at least 6; 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; X is selected from -O-, -S-, -SS-, -O-(C=O)-, -O-(C=O)-O-, -(C=N-NH2)-, -O-CR8R9-O- and -S-CR8R9-S-; Each R8 and R9 is independently selected from -H, -C 1-6 Alkyl and -C 3-6 Cycloalkyl; R5 is selected from -NH- and -O-; R6 is -C 1-6 Alkylene-; and wherein o and p are different from each other; or wherein R7 and R7″ are different from each other.

4. An ionizable lipid as defined in claim 1 and represented by formula (IIa) in R3 and R4 are each independently -C 1-6 or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may also optionally contain one or more additional N atoms, and / or may be optionally substituted by 1-3 substituents selected from the following: -C 1-6 Alkyl and -OH; R7 and R7" are independently selected from -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl; wherein the -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Each of the alkynyl groups may be optionally 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 substitution; and the total number of C atoms in R7 and R7" together is at least 6; 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; R5 is selected from -NH- and -O-; R6 is -C 1-6 Alkylene-; and wherein o and p are different from each other; and / or wherein R7 and R7″ are different from each other.

5. An ionizable lipid as defined in any one of claims 1 to 4 and selected from the list comprising:

6. An ionizable lipid as defined in any one of claims 1 or 2, wherein the total number of C atoms in R1 and R2 together is at least 14.

7. An ionizable lipid as defined in any one of claims 3 or 4, wherein the total number of C atoms in R7 and R7" together is at least 12.

8. An ionizable lipid as defined in any one of claims 1 to 3 or 5, wherein m and n are the same and are integers selected from 1, 2, 3 and 4; preferably 2.

9. An ionizable lipid as defined in any one of claims 1 to 3 or 5, wherein R5 is -NH-.

10. A lipid nanoparticle or lipid nanoparticle composition comprising an ionizable lipid as defined in any one of claims 1 to 9.

11. The lipid nanoparticle or lipid nanoparticle composition according to claim 10, further comprising a phospholipid, a sterol and / or a PEG lipid.

12. The lipid nanoparticle or lipid nanoparticle composition according to any one of claims 10 to 11, further comprising an active agent, in particular a nucleic acid, preferably mRNA.

13. Use of an ionizable lipid as defined in any one of claims 1 to 9 in the preparation of lipid nanoparticles or lipid nanoparticle compositions.

14. A pharmaceutical composition comprising a lipid nanoparticle or a lipid nanoparticle composition as defined in any one of claims 10 to 12 and a pharmaceutically acceptable agent.

15. A pharmaceutical composition as defined in claim 14 for use in medicine.

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