Ionizable lipids and lipid nanoparticles containing thereof

Abstract

An ionizable lipid of formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer of any one of them; a lipid nanoparticle comprising the ionizable lipid, particularly, as an encapsulation agent, optionally comprising a pharmaceutically active agent; and a pharmaceutical composition comprising the lipid nanoparticle. A lipid nanoparticle or a pharmaceutical composition comprising thereof for use in medicine, and the use of the lipid nanoparticles as an encapsulating agent.

Description

[0001] Ionizable lipids and lipid nanoparticles containing thereof

[0002] Technical Field

[0003] The present disclosure relates to ionizable lipids comprising at least one thioester moiety and one amide moiety and to lipid nanoparticles (LNPs) comprising said ionizable lipids. These LNPs can be used as non-viral vectors for the delivery of active ingredients, including polynucleotides, to cells.

[0004] Background Art

[0005] Nowadays, different conditions such as infectious diseases are experiencing fast changes in their treatment approaches, especially since new delivery systems of polynucleotides are being designed and optimized in order to increase their efficiency, being a useful alternative to traditional therapies. Nucleic acid therapies are excellent modalities for rapid vaccination designs. On top of that, they avoid pathogen culture.

[0006] Polynucleotides, such as RNA for systemic delivery, can be successfully encapsulated in gene delivery systems containing them. Said delivery systems must fulfil a few requisites such as to be safe and non-toxic, be on the nanometer scale, provide protection to avoid degradation of the polynucleotides, remain intact in the system for the sufficient period of time in order to reach their target, and be easily degraded once they have released the load.

[0007] Generally, the use of RNA in gene therapy and vaccination is considered safer than the use of DNA. On one hand, RNA does not involve the risk of being stably integrated into the genome of the transfected cell. On the other hand, RNA degrades more easily in vivo, and therefore there is a lower risk of generating undesired anti-RNA antibodies that would reduce therapy efficacy and could produce very serious side effects. There are two main limitations regarding RNA-based therapies: the low transfection rates and the limited protein production efficiency. For these reasons, the dosage is increased in order to obtain the desirable therapeutic effects although, consequently, a higher dose also means elevated costs and, more importantly, some undesirable side effects observed.

[0008] LNPs have become one of the more potent intracellular delivery technologies for encapsulation and delivery of active principles, such as for example genetic material (e.g. mRNA) in vaccines. Presently, LNPs used in commercial mRNA vaccines usually comprise four types of lipids in their composition, in particular, an ionizable or a cationic lipid, a structural lipid which is a sterol such as cholesterol, a PEG-modified lipid, and a non-cationic lipid such as a phospholipid. Notably, ionizable lipids are crucial in LNPs.

[0009] Ionizable lipid properties have a great impact on the protection of the genetic material encapsulated inside the LNP, as they allow the structure and physicochemical properties of the genetic material to be maintained until the LNP reaches the target (e.g. a tissue, a cell) where the genetic material is to be released.

[0010] Ionizable lipids usually present the following general structure:

[0011] Lipophilic moiety— -linking group— -bridge— - Hydrophilic moiety

[0012] Currently there are many known examples where the hydrophilic moiety comprises an ionizable tertiary amine, a functional group where a change in pH influences its formal charge. Besides, it has also been disclosed that the presence of ester groups, easily hydrolysable by enzymes, facilitates the degradation of the ionizable lipid once the genetic material has been released into the tissue / cell of interest, what improves its biocompatibility and biodegradability. The proximity of ester groups to the hydrophilic moiety has also been described to have a great impact on the potency of the lipid.

[0013] As illustrative examples, the lipid commercially known as SM-102 lipid and the lipid known as ALC-0315, depicted below, are respectively comprised in the commercial SARS-CoV-2 vaccine formulations Spikevax (Moderna) and Comirnaty (Pfizer). In both cases, the structures comprise a tertiary ionizable amine as well as two ester groups.

[0014] SM-102

[0015] Molla MR, et al. (cf. Molla MR, et al. "One-Pot Parallel Synthesis of Lipid Library via Thiolactone Ring Opening and Screening for Gene Delivery"; Bioconjug. Chem. 2018, vol. 29(4), pp. 992-999. doi: 10.1021 / acs.bioconjchem.8b00007) discloses a combinatorial library of lipidoids with hydrophobic tails containing reducible disulfide groups and allowing to obtain stable liposomes.

[0016] Despite continuous improvements in such LNPs delivery systems, efficient and specific delivery of targeted agents is still problematic. Biodegradability and toxicity are also two inter-related aspects to bear in mind in order to offer alternative ionizable lipids and / or LNPs which overcome some of the disadvantages of the ones of the prior art, in particular, showing good stability, high transfection efficiency, and safety.

[0017] Summary of Invention

[0018] The present inventors have developed a new ionizable lipid that allows obtaining lipid nanoparticles (LNPs) that can be effectively used as non-viral vectors for delivery of active ingredients, including polynucleotides, to cells. Particularly, LNPs comprising the ionizable lipid of the present disclosure show enhanced / improved transfection rates and biodegradability without compromising their stability.

[0019] The present inventors found that ionizable lipids of Formula (I) as defined herein below: Formula (I) comprising a polar head, at least one thioester moiety, at least one stereogenic center, at least one amide moiety, and a moiety X selected from amide, ester or thioester, wherein Q, X, R1, R2, m, p and t are as defined in the present disclosure, are particularly useful in the preparation of LNPs, which are capable of encapsulating an active agent. Moreover, the present inventors have found that transfection rates in vivo of LNPs prepared with the ionizable lipids of the present disclosure and comprising an active agent are unexpectedly high compared to other commercial or standard compositions known in the art.

[0020] Thus, from the data provided in the Examples, it is apparent that the ionizable lipids of the present disclosure provide a new tool to overcome some of the limitations of known LNPs.

[0021] The higher transfection efficiency of the LNPs of the present disclosure may allow reducing the therapeutic dose of polynucleotide, such as RNA, required in gene therapy and vaccination. The high degradability of the thioester moiety could be detrimental for the stability of the ionizable lipids of the present disclosure. However, the present inventors have found that not only the biodegradability, and hence cytotoxicity are better than other known alternatives, but also the stability of the ionizable lipids of Formula (I) is unexpectedly good which may provide easier handling and storage conditions.

[0022] Therefore, a first aspect of the present disclosure relates to an ionizable lipid of formula or a pharmaceutically acceptable salt thereof, or a stereoisomer of any one of them, wherein

[0023] R’ is selected from the group consisting of H, methyl, and ethyl;

[0024] X is selected from the group consisting of -NH-C(O)-, -C(O)-NH-, -C(O)-O-, -O-C(O)-, -S- C(O)-, -C(O)-S-; m is selected from 0, 1 , 2, 3, 4, 5, and 6; p is selected from 0, 1 2, 3, 4, 5, and 6; t is selected from 1 , 2 and 3;

[0025] Q is a heterocycle comprising at least one N atom, or alternatively Q is: wherein Ra and Rb are independently linear or branched C1-C6 alkyl, optionally substituted with a hydroxyl group; and wherein Ri is selected from linear C11-C30 alkyl or branched C6-C30 alkyl, C6-C30 alkenyl and C6-C30 alkynyl and R2 is selected from linear or branched C6-C30 alkyl, C6- C30 alkenyl or C6-C30 alkynyl, wherein R1 and R2 are optionally substituted with one or more substituents selected from the group consisting of -OH, -COOR4, and -C(=O)SR4, wherein R4 is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl.

[0026] Another aspect of the invention relates to a LNP comprising the ionizable lipid of formula (I) as defined herein.

[0027] In another aspect, the LNP of the present disclosure can further comprise a pharmaceutically active agent and, as such, be formulated in a pharmaceutical with excipients and carriers. Thus, another aspect of the present invention relates to a pharmaceutical composition comprising the LNP comprising a pharmaceutically active agent as defined herein and a pharmaceutically acceptable excipient or carrier.

[0028] The LNP comprising a pharmaceutically active agent or the pharmaceutical composition of the present disclosure may be used in medicinal applications.

[0029] Hence, another aspect of the invention relates to the LNP comprising a pharmaceutically active agent or the pharmaceutical composition of the present disclosure for use in medicine, particularly for use in a method for treating a disease or disorder in a subject in need thereof; or for use in a method of inducing an immune response in a subject, for use in a method for the therapeutic immunization of a subject, for use as a vaccine, or for use in gene therapy. The method comprises administering to the subject a therapeutically effective amount of the nanoparticle composition or of the pharmaceutical composition.

[0030] Another aspect of the invention relates to the use of the LNP as defined herein as an encapsulation agent for an active ingredient.

[0031] Brief Description of Drawings

[0032] Figure 1. Related to Example 4, depicts protein expression in mice administered with LNPs prepared with the following lipids of the invention: VC-LC-1272, VC-LC-1285 and VC-LC-1289 containing mRNA for luciferase as the active ingredient.

[0033] Figure 2. Related to Example 5, depicts a comparative hydrolytic cleavage of thioester and ester bonds, of the ionizable lipids VC-LC-1282, SM-102, and VC-LC-0729.

[0034] Detailed description of the invention

[0035] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

[0036] As used herein, the indefinite articles “a” and “an” are synonymous with “at least one” or “one or more.” Unless indicated otherwise, definite articles used herein, such as “the” also include the plural of the noun.

[0037] The term "and / or" means that any one of the options to which it relates is possible or the at least two options take place at the same time.

[0038] The term “moiety” refers to a specific segment or functional group of a molecule or compound. As used herein, the term "subject" refers to any mammal, including both human and nonhuman mammals.

[0039] As used herein, the term "C1-C# alkyl" refers to saturated linear or branched hydrocarbon which contains from 1 to # carbon atoms, and which is optionally substituted. Non-limiting examples of alkyl groups include, without being limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, neopentyl, n-hexyl, decyl, isodecyl, undecyl, dodecyl, tetradecyl, and hexadecyl.

[0040] The term “C2-C# alkenyl" refers to an unsaturated linear or branched hydrocarbon chain which comprises from 2 to # carbon atoms and at least one or more double bonds, and which is optionally substituted. Examples of alkenyl groups may include without limitation ethenyl (i.e. vinyl), allyl, propenyl, butenyl, pentenyl and hexenyl, dodecenyl, tetradecenyl and hexadecenyl.

[0041] The term “C2-C# alkynyl" refers to an unsaturated linear or branched hydrocarbon chain which comprises from 2 to # carbon atoms and at least one or more triple bonds, and which is optionally substituted. Examples of alkynyl groups include, without being limited to, ethynyl, prop-1 -ynyl, prop-2-ynyl, 1- methylprop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3- ynyl.

[0042] The term "heterocycle comprising at least one N atom" refers to an optionally monosubstituted or multi-substituted cyclic system including one or more rings, where at least one ring includes at least one nitrogen.

[0043] The term "optionally substituted" means that the number of substituents can be equal to or different from zero. Unless otherwise indicated, it is possible that optionally substituted groups are substituted with as many optional substituents as can be accommodated by replacing a hydrogen atom with a non-hydrogen substituent on any available carbon or nitrogen atom.

[0044] The term "polynucleotide" is interchangeably used with "nucleic acid" and refers to a polymer of nucleotides, either ribonucleotides or deoxyribonucleotides. A polynucleotide formed by ribonucleotides may be referred to as "RNA polynucleotide", "ribonucleic acid" or simply "RNA"; and a polynucleotide formed by deoxyribonucleotides may be referred to as "DNA polynucleotide", "deoxyribonucleic acid" or simply "DNA". The polynucleotide may be single- or double-stranded, optionally incorporating synthetic, non-natural, or altered nucleotides capable of incorporation into DNA or RNA. "Artificial polynucleotide" refers to a polynucleotide with a sequence that does not occur in nature or that has been altered by human intervention.

[0045] As used herein, the term “messenger RNA”, abbreviated as "mRNA", refers to any RNA polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, or ex vivo. Typically, an mRNA is single stranded and comprises an ORF in its structure.

[0046] As used herein “open reading frame” or “ORF” refers to a sequence of several nucleotide triplets that encodes a polypeptide, that is, that can be translated into a polypeptide sequence.

[0047] As used herein, "DNA construct" refers to an artificial polynucleotide including a sequence of interest operatively linked to an expression promoter, said promoter controlling expression of the sequence of interest.

[0048] As used herein, "expression vector" refers to a vector used to introduce a specific nucleic acid, typically a DNA construct, into a target cell for expression of the nucleic acid by the cell. Examples of suitable expression promoters and expression vectors include those conventionally used in molecular biology and known to the skilled person.

[0049] The term "polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds (i.e., peptide isosteres). "Polypeptide" refers to both short chains, commonly referred as peptides, oligopeptides, or oligomers, and to longer chains generally referred to as proteins.

[0050] The expression "therapeutically effective amount" as used herein, refers to the amount of a compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the symptoms of the disease to which it is addressed. The particular dose of compound administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, the particular circumstances of the individual subject to be treated, and similar considerations. The term “pharmaceutical” also encompasses the concept of “veterinary composition”. Thus, they relate to compositions that are therapeutically effective when administered by any desired or applicable route to any animal, including humans.

[0051] According to the present invention, the term "antigen" is a compound that can be recognized by immunoglobulin receptors of B cells, or by the T-cell receptor when complexed with MHC. Preferably, an "antigen" is a polypeptide.

[0052] The term “nanoparticle” as used herein, refers to a particle with at least two dimensions at the nanometer scale, particularly with all three dimensions at the nanoscale, where the nanoscale is in the range of about 1 nm to about 500 nm. Particularly, when the nanoparticle is substantially rod-shaped with a substantially circular cross-section, such as a nanowire or a nanotube, the "nanoparticle" refers to a particle with at least two dimensions at the nanoscale, these two dimensions being the cross-section of the nanoparticle. As used herein, "size" or "mean size" in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition. The term “lipid nanoparticle” as used herein, refers to a nanoparticle whose external envelope is totally or partially made of lipids.

[0053] As used herein, the "polydispersity index (PDI)" is a ratio that describes the homogeneity of the particle size distribution of a system. A small value, e.g., less than 0.3, indicates a narrow particle size distribution.

[0054] As used herein, the term "zeta potential" is the electrokinetic potential of a lipid, e.g., in a particle composition. Z potential is also defined as the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle. It is generally accepted as a quantification of the magnitude of the charge, and it is often the only available path for characterization of double-layer properties.

[0055] As used herein, “apparent pKa” refers to an experimentally determined value resulting from the average ratio of all the ionized to deionized groups in a nanoparticle. Apparent pKa is different to the intrinsic pKa of any individual molecule, and it is an important parameter for the performance of nanoparticles encapsulating RNAs. The apparent pKa of nanoparticles can be measured by different techniques known in the art. For example, acid-base titration and 2-(p-toluidino)-6-naphtalene sulfonic acid (TNS) fluorescent methods are widely used in the art. Nanoparticles with an optimum pKa carry negligible charges at physiological pH, which prevent nonspecific binding and toxicity in the body. The optimum pKa of nanoparticles plays an important role in the endosomal escape mechanism and in the release of RNAs in the cytosol to exert therapeutic effect.

[0056] Ionizable lipids

[0057] As mentioned above, a first aspect of the invention refers to an ionizable lipid of formula

[0058] (I):

[0059] Formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer of anyone of them, as defined above.

[0060] The term “pharmaceutically acceptable salts” used herein encompasses any salt formed from pharmaceutically acceptable non-toxic acids including inorganic or organic acids. There is no limitation regarding the salts, except that if used for therapeutic purposes, they must be pharmaceutically acceptable. The preparation of pharmaceutically acceptable salts of the ionizable lipids of the present disclosure can be carried out by methods known in the art. For instance, they can be prepared from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of the ionizable lipids of the present disclosure with a stoichiometric amount of the appropriate pharmaceutically acceptable base or acid in water or in an organic solvent or in a mixture of them.

[0061] Examples of pharmaceutically acceptable salts include acid addition salts formed with inorganic acids e.g. hydrochloric, hydrobromic, sulfuric, nitric, or phosphoric acid; and organic acids e.g. succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, malic, lactic, formic, propionic, glycolic, camphorsulfuric, mandelic, benzenesulfonic, p-toluenesulfonic, oxalic, methanesulfonic or naphthalenesulfonic acid; and base addition salts formed with alkali metals and alkaline earth metals and organic bases such as N,N-dibenzylethylene- diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, lysine, and procaine. The ionizable lipids of the present disclosure and their salts may differ in some physical properties, but they are equivalent for the purposes of the present invention.

[0062] The ionizable lipids of the present disclosure have asymmetric centers and, therefore, can give rise to various stereoisomers. As used herein, the term "stereoisomer" refers to all isomers of individual ionizable lipids that differ only in the orientation of their atoms in space. The term stereoisomer includes enantiomers, racemates, racemic mixtures, geometric isomers (cis / trans or syn / anti or E / Z), and diastereomers. The present invention relates to each of these stereoisomers and also mixtures thereof.

[0063] The preparation processes described herein can be modified to give enantiopure compounds as well as mixtures of stereoisomers. It is possible to prepare specific stereoisomers or specific mixtures by various processes including the use of stereospecific reagents or by introducing chiral centers into the compounds during its preparation process. In addition, it is possible to separate stereoisomers once the compound has been prepared by standard resolution techniques known to the skilled person.

[0064] In all embodiments of the invention referring to the ionizable lipids of the present disclosure, the pharmaceutically acceptable salts, or stereoisomer of the ionizable lipids or of their pharmaceutically acceptable salts are always contemplated even if they are not specifically mentioned.

[0065] The ionizable lipids of the present disclosure also include isotopes of the structure depicted. "Isotopes" refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes include, without being limited to, tritium, deuterium,13C or14C, or15N. Further, a compound or salt of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.

[0066] The ionizable lipids of the present disclosure are characterized by their retention time, mass spectrometry, size distribution, polydispersity index, and Z-potential. These parameters can be measured by methods well-known in the art. Some of them are indicated in more detail in the examples below.

[0067] In an embodiment, Ri is selected from linear C11-C30 alkyl or branched C6-C30 alkyl, C6-C30 alkenyl and C6-C30 alkynyl and R2 is selected from linear or branched C6-C30 alkyl, C6-C30 alkenyl or C6-C30 alkynyl, wherein R1 and R2 are optionally substituted with one or more substituents selected from the group consisting of -OH, -COOR4, and - C(=O)SR4, wherein R4 is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl.

[0068] According to an embodiment of Formula (I), optionally in combination with one or more features of the various embodiments described above, R’ is selected from the group consisting of H and methyl and m is selected from 0, 1 , 2, 3 and 4.

[0069] In an embodiment, the ionizable lipid of Formula (I) is a compound of Formula (IA)

[0070] Formula (IA) wherein R', X, A, R1, R2, and m are as defined above.

[0071] According to another embodiment, optionally in combination with one or more features of the various embodiments described above, X is -NH-C(O)- or -C(O)-NH-; or X is -C(O)-O- or -O-C(O)-; or X is -C(O)-S- or -S-C(O)-.

[0072] According to another embodiment, optionally in combination with one or more features of the various embodiments described above, Q is: wherein Ra and Rb are independently linear or branched C1-C6 alkyl, optionally substituted with a hydroxyl group.

[0073] According to another embodiment, optionally in combination with one or more features of the various embodiments described above, Q is a 5-membered ring or a 6-membered ring comprising one N atom and, optionally, a second heteroatom selected preferably from N and O.

[0074] In another embodiment, optionally in combination with one or more features of the various embodiments described above, Q is selected from the following structures:

[0075] In a preferred embodiment of Formula (I), Q is N(Me)2; m=t=2; p=0; R’ is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is selected from the group consisting of C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 branched alkyl; and R2 is selected from the group consisting of C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 , C22, C23 and C24 branched alkyl, and C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 alkenyl or alkdienyl. In a more preferred embodiment of Formula (I), Q is N(Me)2; m=t=2; p=0; R’ is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is selected from the group consisting of C11 , C15 and C19 branched alkyl; and R2 is selected from the group consisting of C11, C15 and C17 branched alkyl, and C17 alkenyl or alkdienyl.

[0076] In another preferred embodiment of Formula (I), Q is N(Me)2; m=t=2; p=0; R’ is H; X is selected from -C(O)-O- and -O-C(O)-; R1 is selected from the group consisting of C8, C9, C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 branched alkyl; and R2 is selected from the group consisting of C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 , C22, C23 and C24 branched alkyl and C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 linear alkenyl or alkdienyl.

[0077] In a more preferred embodiment, of Formula (I), Q is N(Me)2; m=t=2; p=0; R’ is H; X is selected from -C(O)-O- and -O-C(O)-; R1 is selected from the group consisting of C12, C16 and C24 branched alkyl; and R2 is selected from the group consisting of C15 branched alkyl, and C17 linear alkenyl. In another preferred embodiment, of Formula (I), Q is N(Me)2; m=t=2; p=0; R’ is H; X is selected from -C(O)-S- and -S-C(O)-; R1 is selected from the group consisting of C8, C9, C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 , C22, C23 and C24 branched alkyl; and R2 is C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 branched alkyl.

[0078] In a more preferred embodiment of Formula (I), Q is N(Me)2; m=t=2; p=0; R’ is H; X is selected from -C(O)-S- and -S-C(O)-; R1 is C8 branched alkyl; and R2 is C15 branched alkyl.

[0079] In another preferred embodiment of Formula (I), Q is N(Et)2; m=t=2; p=0; R’ is H; X is selected from -C(O)-O- and -O-C(O)-; R1 is selected from the group consisting of C8, C9, C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 , C22, C23 and C24 branched alkyl; and R2 is selected from the group consisting of C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 branched alkyl and C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 linear alkenyl or alkdienyl.

[0080] In a more preferred embodiment of Formula (I), Q is N(Et)2; m=t=2; p=0; R’ is H; X is selected from -C(O)-O- and -O-C(O)-; R1 is C15 branched alkyl; and R2 is C17 alkenyl.

[0081] In another preferred embodiment of Formula (I), Q is a heterocycle comprising at least one N atom, m=t=2; p=0; R’ is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is selected from C the group consisting of C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 , C22, C23 and C24 branched alkyl; and R2 is selected from the group consisting of C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 , C22, C23 or C24 branched alkyl and C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 , C22, C23 and C24 linear alkenyl or alkdienyl.

[0082] In a more preferred embodiment of Formula (I), Q is a five membered-heterocycle or a six-membered heterocycle comprising one or two N atoms, m=t=2; p=0; R’ is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is C15 branched alkyl; and R2 is C17 linear alkenyl. Examples of ionizable lipids of Formula (I) include the compounds depicted in Table 1 below. Table 1

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] In an embodiment, the ionizable lipid of the present disclosure is a compound selected from the group consisting of: (VC-LC-1272), (VC-LC-1284), (VC-LC-1285), (VC-LC-1286), (VC-LC-1287), (VC-LC-1288), (VC-LC-1289), (VC-LC-1293), (VC-LC-1297), (VC-LC- 1311), (VC-LC-1369), (VC-LC-1371), (VC-LC-1373), (VC-LC-1374), (VC-LC-1376), (VC- LC-1377), (VC-LC-1378), (VC-LC-1539), (VC-LC-1540), (VC-LC-1541), (VC-LC-1543), (VC-LC-1545).

[0090] In another embodiment, the ionizable lipid of Formula (IA) is a compound selected from the group consisting of: (VC-LC-1272), (VC-LC-1284), (VC-LC-1285), (VC-LC-1286), (VC- LC-1287), (VC-LC-1288), (VC-LC-1289), (VC-LC-1293), (VC-LC-1297), (VC-LC-1311), (VC-LC-1369), (VC-LC-1371), (VC-LC-1373), (VC-LC-1374), (VC-LC-1376), (VC-LC- 1377), (VC-LC-1378), (VC-LC-1539), (VC-LC-1540), (VC-LC-1541), (VC-LC-1543), (VC- LC-1545) described herein; or a pharmaceutically acceptable salt thereof, or a stereoisomer of any one of them.

[0091] Preparation processes

[0092] Processes for the preparation of the ionizable lipids of Formula (I) are also part of the invention.

[0093] All reactants and solvents needed for the preparation of the compounds of the present disclosure are commercially available.

[0094] As a way of example, the ionizable lipid of Formula (I) can be prepared according to any of the following synthetic schemes. Any person skilled in the art will know which reactants are required to obtain any particular ionizable lipid according to the present disclosure following the synthetic methods depicted in the schemes below or an analogous method thereof.

[0095] Ionizable lipids of Formula (I) wherein X is an amide moiety can be prepared by the synthetic method, or an analogous one, depicted in scheme 1 below. Scheme 1

[0096] Ionizable lipids of Formula (I) wherein X is an ester moiety can be prepared by the synthetic method, or an analogous one, depicted in scheme 2 below.

[0097] Scheme 2 Ionizable lipids of Formula (I) wherein X is a thioester moiety can be prepared by the synthetic method, or an analogous one, depicted in scheme 3 below.

[0098] HO^R1SOCI2

[0099] Y

[0100] 060 °C

[0101] Acyl chloride intermediate

[0102] (R1COCI)

[0103] Acyl chloride i t di t

[0104] Scheme 3

[0105] Lipid nanoparticles

[0106] As mentioned above, the ionizable lipid of the present disclosure can form LNPs in solution. Therefore, the present invention also relates to a LNP comprising an ionizable lipid of formula (I) as defined herein.

[0107] All embodiments indicated for the ionizable lipid of formula (I) also apply to LNP.

[0108] Typically, LNPs have a core-shell structure comprising an inner core and an external shell. LNP formulations with several lipidic components of different nature, such as an ionizable lipid, a sterol, a PEGylated lipid, and a non-cationic lipid (also referred to as “helper lipid”) such as a phospholipid, comprise several phases that separate into a hydro- phobic core region formed by the ionizable lipid and cholesterol, and a surrounding shell formed by the helper lipid, cholesterol and PEGylated lipids covering the surface. More particularly, the ionizable lipid of formula (I) forms part of the internal shell of the LNP and, optionally, a pharmaceutically active agent is encapsulated or loaded in the inner core.

[0109] In an embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP further comprises at least one lipid selected from the group consisting of a non-cationic lipid; a sterol, a steroid precursor or steroid derivative; and a PEG-modified lipid.

[0110] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises the ionizable lipid of the present disclosure and a non-cationic lipid. Examples of non-cationic lipids include, without being limited to, 1 ,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-stearoyl-2- oleoyl-sn-glycero-3-phosphocoline (SOPC), 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1 ,2-diundecanoyl-sn- glycero-phosphocholine (DUPO), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPO), 1 ,2-di-O-octadecenyl- sn -glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl- 2-cholesterylhemisuccinoyl- sn -glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), 1 ,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 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-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In an embodiment, the non-cationic lipid is DSPC. In a particular embodiment, the non-cationic lipid is DOPE. In another particular embodiment, the lipid- containing particle comprises DSPC and DOPE. In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP further comprises a sterol or a sterol precursor.

[0111] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises the ionizable lipid of the present disclosure and a sterol, a steroid precursor, or a steroid derivative.

[0112] Examples of sterols include, without being limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alphatocopherol, and mixtures thereof. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above, the sterol is cholesterol. Examples of sterol precursors include, without being limited to, a triterpene, a triterpenoid, or a steroid precursor of this kind. Non-limiting examples of triterpenes, triterpenoids and other steroid precursors include squalene, achilleol, polypodatetrane, lanostane, cucurbitacin, hopane, oleanane, chamaecydin, lupine, and mixtures thereof. The term “steroid derivative” refers to a derivative of the simplest steroid containing the nucleus gonane, also known as cyclopentanoperhydrophenantrene, which contains seventeen carbon atoms arranged in four fused rings. A steroid derivative can include different modifications such as different functional groups attached to the four-ring core or the oxidation state of the rings. Examples of steroid derivatives include, without being limited to cholic acid, lanosterol and p-sitosterol.

[0113] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises the ionizable lipid of the present disclosure, a non-cationic lipid as defined above, and a sterol, a steroid precursor, or a steroid derivative as defined above.

[0114] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP further comprises a PEG-modified lipid. The term "PEG-modified lipid refers to a lipid comprising a polyethylene moiety. Examples of PEG- modified lipids include, without being limited to, a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified phosphatidylcholine, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG- modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, PEG-DPG, or a combination thereof.

[0115] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP further comprises a conjugated lipid.

[0116] Examples of conjugated lipid include, without being limited to, polysarcosine (pSar) lipids and derivatives such as, for example, N-tetradecyl-pSar25, N-hexadecyl-pSar25, N- octadecyl-pSar25, N-dodecyl-pSar25, DMG-pSar25, 18:1 PE (DOPE) pSar25, N- TETAMINE-pSar25, N-TETAMINE-pSar35, N-TETAMINE-pSar45, N-TETAMINE-pSar45- Maleimide.

[0117] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises the ionizable lipid of the present disclosure, a non-cationic lipid as defined above, and a PEG-modified lipid or a conjugated lipid as defined above.

[0118] In an embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises a lipid component comprising or consisting of the ionizable lipid as disclosed herein, a non-cationic lipid, a sterol, and a PEG-modified lipid.

[0119] In a particular embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises a lipid component comprising the ionizable lipid as disclosed herein and at least one of distearolyphosphatidycholine (DSPC), cholesterol, and DMG-PEG2000. In a more particular embodiment, the lipid component comprises or consists of the ionizable lipid as disclosed herein, DSPC, cholesterol, and DMG-PEG2000.

[0120] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises from 25 to 60 mol% of a ionizable lipid; from 0.1 to 10 mol% of a PEG-modified lipid or, alternatively, of a conjugated lipid, particularly of a PEG-modified lipid; from 10 to 45 mol% non-cationic lipid; and from 10 to 40 mol% sterol. Particularly, the LNP comprises from 32 to 50 mol% ionizable lipid, from 1 to 8 mol% PEG-modified lipid or a conjugated lipid, particularly PEG-modified lipid; from 12.5 to 42 mol% non-cationic lipid; and from 15 to 38.5 mol% sterol. More particularly, the LNP comprises from 35 to 47 mol% ionizable lipid, from 1 to 4 mol% PEG-modified lipid, from 30 to 38 mol% non-cationic lipid, and from 15 to 25 mol% sterol.

[0121] In another embodiment of the LNP, optionally in combination with one or more features of the various embodiments described above, the ionizable lipid is in an amount from 25 to 64 mol%, the PEG-modified lipid is in an amount from 0.1 to 1.5 mol%; and the sterol is in an amount from 35 to 74 mol%.

[0122] As used herein, "mol%" refers to a component's molar percentage relative to the total moles of all lipid components in the LNP (i.e., total mols of ionizable lipid, PEG-modified lipid; non-cationic lipid; and sterol).

[0123] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the molar ratio of ionizable lipid to the non-cationic lipid ranges from 6:1 to 1:2, or from 2:1 to 1 :1.

[0124] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the molar ratio of ionizable lipid to the sterol ranges from 5:1 to 1:2, or from 2:1 to 1 :1.

[0125] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the molar ratio of ionizable lipid to the PEG-modified lipid ranges from 120:1 to 2:1 , or from 100:1 to 10:1.

[0126] Active agents

[0127] As mentioned above, the LNPs of the invention may comprise one or more pharmaceutically active agents.

[0128] As used herein, the term “pharmaceutically active agent” refers to an agent that has pharmacological activity and is used for curing, mitigating, treating or preventing a disease in a subject, in particular a human.

[0129] For the purposes of the present invention, pharmaceutically active agents include low molecular weight drugs, polynucleotides, peptides, antibodies, proteins, and combinations thereof.

[0130] As used herein, the term "polynucleotide" refers to a natural or an artificial deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The polynucleotide may comprise at least one chemical modification selected from the group consisting of pseudouridine, N1 -methylpseudouridine (also referred to as 1 -methylpseudouridine or ml^P), N6- methyladenosine (also referred to as m6A), 2-thiouridine (also referred to as s2U), 4'- thiouridine, 5-methylcytosine (also referred to 5mC), 2-thio-1-methyl-1-deaza- pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine (also referred to as m5U), 5-methoxyuridine, 2'-O-methyl uridine, and combinations thereof. In particular, the chemical modification is N1 -methylpseudouridine, 5-methoxyuridine or a combination thereof; particularly the chemical modification is N1 -methylpseudouridine.

[0131] The polynucleotide is partially modified with at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%, of N1-methylpseudouridine or fully modified with N1-methylpseudouridine, 5- methoxyuridine or a combination thereof. In a particular embodiment, optionally in combination with any of the embodiments provided above, the active agent is selected from the group consisting of a polynucleotide, a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide, and an expression vector comprising a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide.

[0132] In a particular embodiment, optionally in combination with any of the embodiments provided above, the polynucleotide is a ribonucleic acid (RNA).

[0133] In particular, the RNA is selected from the group consisting of a short interfering RNA (siRNA), a self-replicating RNA (srRNA), circular RNA (circRNA), a self-amplifying RNA (saRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a small interfering RNA (siRNA), a small RNA (sRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and mixtures thereof.

[0134] In a particular embodiment, optionally in combination with any of the embodiments provided above, the RNA is an mRNA.

[0135] The skilled person knows how to produce the polynucleotide, the DNA construct, or the expression vector by routine methods well known in the art, for example, by chemical synthesis or by molecular biology techniques, without exercising any inventive skill.

[0136] In a particular embodiment, optionally in combination with any of the embodiments provided above, the ionizable lipid to RNA ratio (N / P; where N represents the moles of amine present in the ionizable lipid and P represents the moles of phosphate present in the polynucleotide backbone) in the LNP ranges from 20:1 to 2:1 , particularly from 10:1 to 3:1.

[0137] In a particular embodiment, optionally in combination with any of the embodiments provided above, the polynucleotide is an isolated artificial polynucleotide.

[0138] In an embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises an ionizable lipid as defined herein, a non-cationic lipid, a sterol, a PEG-modified lipid, and a polynucleotide.

[0139] The LNPs containing one or more polynucleotides may be prepared by standard methods, for instance, microfluidic mixing as disclosed in Hassett, K. J. et al., ("Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines", 2019, Mol. Ther. Nucleic Acid, vol. 15, pp. 1-11), or by manual / bulk mixing as disclosed in Wang X., Liu S., Sun Y., et al. (“Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery”, 2022, Nat. Protoc., doi:10.1038 / s41596-022-00755-x). Both methods are known in the art and the skilled person would know how to proceed in each specific case.

[0140] Typically, the process for the preparation of LNPs comprises: i) preparing a first alcoholic mixture comprising the ionizable lipid of the present disclosure and, optionally, at least one lipid selected from the group consisting of a non-cationic lipid, a sterol, and a PEG- modified lipid in a suitable alcohol such as for example ethanol; ii) preparing a second aqueous composition comprising a polynucleotide and an acidification buffer; and iii) mixing i) with ii) in a microfluidic mixer. The microfluidic mixer allows thorough and rapid mixing of the lipid phase and the polynucleotide phase in a microscale device. Depending on the process parameters, and in particular on the total flow rate, the skilled person will be able to modulate the size of the LNPs.

[0141] In a particular embodiment, optionally in combination with any of the embodiments provided above, the polynucleotide encodes a polypeptide, particularly, wherein the polypeptide is an antigen. More particularly, the antigen is selected from the group consisting of a viral protein, a bacterial protein, and a tumor-associated antigen.

[0142] In a particular embodiment, optionally in combination with any of the embodiments provided above, the polypeptide is an antibody or a fragment thereof. In a more particular embodiment, the antibody or a fragment thereof is a therapeutic antibody or a fragment thereof.

[0143] In another embodiment, optionally in combination with any of the embodiments provided above, the antigen is a SARS-CoV-2 antigen, particularly a SARS-CoV-2 spike antigen.

[0144] Preparation methods for the above lipids, LNPs and pharmaceutical compositions are described herein and / or known in the art. The skilled in the art would know, depending on the intended use of the composition, which LNP use to encapsulate each pharmaceutically active agent. In particular, methods for the synthesis of the compositions formed by the LNP encapsulating RNA are well-known by the skilled person in the art and duly established in the protocols for molecular biology. Particular conditions are indicated in the examples.

[0145] As above indicated, another aspect of the invention relates to a pharmaceutical composition comprising the LNP as defined herein and a pharmaceutically acceptable excipient or carrier.

[0146] The expression "pharmaceutically acceptable excipient or carrier" refers to pharmaceutically acceptable materials, compositions, or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and non-human animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0147] Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

[0148] The relative amounts of the pharmaceutically active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered.

[0149] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Excipients such as colouring agents, coating agents, sweetening, and flavouring agents can be present in the composition, according to the judgment of the formulator.

[0150] In an embodiment, optionally in combination with any of the embodiments provided above, the pharmaceutical composition disclosed herein is administered orally, intranasally, intravenously, intraperitoneally, intramuscularly, intradermally, subcutaneously, topically, or by intra-articular administration.

[0151] The pharmaceutical compositions of the present disclosure may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining the lipid nanoparticles of the invention with sterile, distilled water or other carrier so as to form a solution. Some excipients or carriers can be added to ease the formation of a homogeneous solution or suspension. As mentioned above, the LNP comprising a pharmaceutically active agent, or the pharmaceutical composition of the present disclosure may be used in therapeutic applications. In particular, they may be used as non-viral vectors of general use for biomedical applications, such as vaccines or gene therapy, being effective for transfection of genetic material into eukaryotic cells. Thus, an aspect of the invention relates to a LNP or a pharmaceutical composition as defined herein for use in a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the nanoparticle composition or of the pharmaceutical composition as defined herein.

[0152] Another aspect of the invention relates to a LNP or a pharmaceutical composition as defined herein for use in a method of inducing an immune response in a subject, for use in a method for the therapeutic immunization of a subject, for use as a vaccine, or for use in gene therapy. Particularly, the subject is a human.

[0153] In an embodiment, optionally in combination with any of the embodiments provided above, the disease or disorder is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases.

[0154] When the pharmaceutically active agent is a polynucleotide, the LNP or the pharmaceutical composition described herein can be used in vaccine therapy, in the enhancement of the efficacy of a conventional vaccine and / or as a novel vaccine form for use against infectious pathogens, such as viruses, bacteria, fungi, protozoa, prions, and helminths (worms); or for use in treating diseases such as cancer and proliferative diseases.

[0155] In an embodiment, optionally in combination with any of the embodiments provided above, the pharmaceutical composition is a vaccine. In a more particular embodiment, the pharmaceutical composition is a vaccine and further comprises an adjuvant. The skilled person would know, based on its common general knowledge, which excipients, carriers, and adjuvants to include in the vaccine depending on the intended use.

[0156] In case that one of the structures shown in the present document and its chemical name do not correlate, the chemical structure prevails over the chemical name in all cases.

[0157] Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of”.

[0158] The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein. Examples

[0159] Reagents were purchased from Sigma-Aldrich, TCI Chemicals, Fluorochem, or VWR. All possible wise combinations shown previously were prepared following a three-step reaction protocol.

[0160] Example 1 - Synthesis of lipid VC-LC-1272

[0161] VC-LC-9018

[0162] Scheme 4 Synthetic route for Step 1 in the preparation of lipid VC-LC-1272.

[0163] DL-homocysteine thiolactone hydrochloride (270.4 mg, 1.76 mmol) was dissolved in 5 mL of anhydrous dichloromethane at room temperature. Then triethylamine (252 pL, 1 .8 mmol) was added followed by N-(3-dimethylaminopropyl)-Nz-ethylcarbodiimide hydrochloride (249.2 mg, 1.3 mmol), 4-(dimethylamino)pyridine (24.4 mg, 0.2 mmol) and 2-hexyldecanoic acid (256.42 mg, 1 mmol). The reaction mixture was stirred overnight under an argon atmosphere at room temperature. Then, the reaction crude was washed twice using distilled water (2 x 10 mL) and finally using saturated brine (10 mL). The organic layer was dried with anhydrous MgSO4, filtered, and evaporated under reduced pressure. The resulting residue was purified by flash chromatography (gradient of hexane / ethyl acetate: 100 / 0 to 0 / 100) to afford VC-LC-9018 as a yellowish oil (89 % yield).

[0164] The product was characterised by HPLC-ELSD-MS. Retention time: 6.02 min, MS (ES): experimental m / z [M+H]+ 356.44 {theoretical m / z [M+H]+ 356.26}.

[0165] Step 2. Synthesis of intermediate VC-LC-9121

[0166] VC-LC-9018

[0167] VC-LC-9121

[0168] Scheme 5. Synthetic route for Step 2 in the preparation of lipid VC-LC-1272.

[0169] The product from step 1 (VC-LC-9018, 249 mg, 0.7 mmol) was dissolved in anhydrous tetrahydrofuran (1.5 mL) and was added to a solution of N,N-Dimethylethylenediamine (780 pL, 7.03 mmol) in anhydrous tetrahydrofuran (1.0 mL) under argon atmosphere. The solution was then stirred at room temperature for 30 minutes, after which the solvent was evaporated under reduced pressure. The dry mixture was redissolved in ethyl acetate (20 mL) and washed with a solution of 0.1 M HCI (aq.) (20 mL, 3 consecutive washes), then distilled water (20 mL, 3 consecutive washes) and finally with saturated brine (20 mL, 3 consecutive washes; NaCkwater prepared by adding 35 g of NaCI in 100 mL of water). The combined organic fractions were dried with MgSO4 (anhydrous) and subsequently evaporated under reduced pressure to yield VC-LC-9121 as a yellowish oil (quantitative yield)

[0170] The product was characterised by HPLC-ELSD-MS. Retention time: 3.58 min, MS (ES): experimental m / z [M+H]+ 444.55 {theoretical m / z [M+H]+ 444.36}.

[0171] Step 3. Final step in the synthesis of lipid VC-LC-1272

[0172] Scheme 6. Synthetic scheme for the final step in the synthesis of lipid VC-LC-1272.

[0173] The product from the previous step (VC-LC-9121, 177.5 mg, 0.4 mmol), N-(3- dimethylaminopropyl)-Nz-ethylcarbodiimide hydrochloride (101.7 mg, 0.52 mmol), 4- (dimethylamino)pyridine (9.8 mg, 0.08 mmol) and oleic acid (153.8 pL, 0.48 mmol) were dissolved in 2.5 mL of anhydrous dichloromethane at room temperature. The reaction mixture was stirred at room temperature overnight under argon atmosphere. Then, the reaction crude was redissolved in 40 mL of dichloromethane and washed two times using distilled water (2 x 10 mL) and finally with saturated brine (10 mL). The organic layer was dried with anhydrous MgSO4, filtered, and evaporated under reduced pressure. The resulting residue was purified by flash chromatography (gradient of dichloromethane / eluent A: 100 / 0 to 0 / 100). After removing the solvent under reduced pressure, VC-LC-1272 is afforded as an oil (31 % yield).

[0174] *Eluent A: dichloromethane / methanol / ammonium hydroxide (80:20:1)

[0175] The product was characterised by HPLC-ELSD-MS. Retention time: 8.33 min, MS (ES): experimental m / z [M+H]+ 708.80 {theoretical m / z [M+H]+ 708.61 }. VC-LC-1272 was characterised by HPLC-ELSD.

[0176] General procedure for the characterization of the afforded products by HPLC-LSD-MS (HPLC Water Alliance ELSD 2424 and column X bridge BEH C8, 4.6 mm, 50 mm, 2.5 pm. Injection volume is 2 pL and the temperature in the column is 65 °C

[0177] Gradient:

[0178] Sample preparation for the reaction monitoring: 5 pL of the reaction crude are dissolved in 100 pL of HPLC-MS-grade acetonitrile in a HPLC sample tube. The tube is vortexed manually for 5 seconds and placed inside the HPLC sample chamber.

[0179] Final product sample preparation: 1 pL of dry lipid are dissolved in 100 pL of HPLC-MS- grade acetonitrile in a HPLC sample tube.

[0180] Successful detection of the analyte was performed by an evaporative light dispersion detector (Waters ELSD 2424) and by a simple quadrupole mass detector in positive mode with a spectrophotometer Waters Acquity QDa. Retention time: 8.33 minutes, MS (ES): experimental m / z [M+H]+ 708.7955 {theoretical m / z [M+H]+ 710.6071 }.

[0181] Example 2 - Synthesis of lipid VC-LC-1285

[0182] VC-LC-9099

[0183] Scheme 7. Synthetic route for Step 1 in the preparation of lipid VC-LC-9099

[0184] 2-Oxotetrahydrothiophene-3-carboxylic acid (730 mg, 5 mmol) was dissolved in 13 mL of anhydrous methylene chloride at room temperature. Then N-(3-dimethylaminopropyl)- Nz-ethylcarbodiimide hydrochloride (870 mg, 4.51 mmol) was added followed by, dimethylaminopyridine (104 mg, 0.85 mmol) and 2-hexyl-1 -decanol (1.03 g, 4.25 mmol). The reaction was stirred at room temperature overnight, after which it was checked for completion by TLC chromatography. The resulting product was purified by flash chromatography using a gradient of hexane / ethyl acetate (100 / 0 to 0 / 100). The fractions containing the product were combined and evaporated under reduced pressure yielding VC-LC-9099 as a yellowish oil (1.33 g, 84%).

[0185] The product was characterised by HPLC-ELSD (retention time: 8.21 min) and thin layer chromatography (hexane:ethyl acetate).

[0186] Step 2 and 3. Final steps in the synthesis of lipid VC-LC-1285 Oleic acid

[0187] VC-LC-9099

[0188] VC-LC-1285

[0189] Scheme 8. Combined synthetic route for Steps 2 and 3 in the preparation of lipid VC-LC-

[0190] 1285.

[0191] In order to obtain VC-LC-1285, steps 2 and 3, which are analogous to the corresponding steps followed for the synthesis of lipid VC-LC-1272 described in Example 1 , were carried out. This allowed for the obtention of a combined 21% yield (quantitative and 21% yield for steps 2 and 3, respectively).

[0192] VC-LC-1285 was characterised by HPLC-ELSD-MS. Retention time: 9.37 min, MS (ES): experimental m / z [M+H]+ 723.85 {theoretical m / z [M+H]+ 723.61.

[0193] VC-LC-1285 was characterised by HPLC-ELSD-MS following the general procedure detailed above and successful detection of the analyte was performed by an evaporative light dispersion detector and by a simple quadrupole mass detector in positive mode with an spectrophotometer Waters Acquity QDa.

[0194] Retention time: 9,37 minutes, MS (ES): experimental m / z [M+H]+723.8485 {theoretical m / z [M+H]+ 723.6068.

[0195] Example 3 - Synthesis of lipid VC-LC-1311 Complete synthetic procedure is shown in scheme 9 below:

[0196] Scheme 9. Complete synthetic route for the preparation of VC-LC-1311

[0197] Step 1. Synthesis of intermediate VC-LC-9240

[0198] VC-LC-9240

[0199] Scheme 10. Synthetic scheme for the preparation of intermediate VC-LC-9240

[0200] 2-Hexyldecanoic acid (534 pL, 1.7 mmol) was dissolved in excess thionyl chloride (2 mL, 27.5 mmol) and stirred for 2 hours at 60°C under argon atmosphere. After checking the reaction mix for completion using TLC, 2 mL of toluene were added to the crude and then evaporated under reduced pressure. Once dried, the product was redissolved in 2 mL of dichloromethane and evaporated using reduced pressure till dryness to afford VC-LC- 9240 in a quantitative yield. No characterization of VC-LC-9240 is performed.

[0201] VC-LC-9248 Scheme 11. Synthetic scheme for the preparation of intermediate VC-LC-9248.

[0202] 2-Oxotetrahydrothiophene-3-carboxylic acid (640 mg, 4.66 mmol) was dissolved in 1.4 mL of anhydrous tetrahydrofuran under argon atmosphere. Then N,N- dimethylethylenediamine (776 pL, 7 mmol) was added and stirred for 3 hours, after which it was checked for completion using HPLC-ELSD and the solvent was evaporated under reduced pressure. The product, obtained in quantitative yield, was used without further purifications in step 3.

[0203] VC-LC-9248 was characterised by HPLC-ELSD-MS. Retention time: 0.58 min MS (ES): 235.23 experimental m / z [M+H]+ {theoretical m / z [M+H]+ 235.11}

[0204] Step 3. Synthesis of intermediate VC-LC-9261

[0205] VC-LC-9248

[0206] VC-LC-9261

[0207] Scheme 12. Synthetic scheme for the preparation of intermediate VC-LC-9261.

[0208] Trifluoracetic acid (1 mL, 1.3 mmol) was added to the product VC-LC-9248 (492 mg, 2.1 mmol) and left stirring under argon atmosphere for 30 minutes at 0°C. Consequently, VC- LC-9240 (577 mg, 2.1 mmol) was added to the reaction mixture and stirred at room temperature overnight. Then, the trifluoroacetic acid was evaporated under reduced pressure and the resulting product was purified by flash chromatography using a gradient of dichloromethane / eluent B (100 / 0 to 0 / 100). The fractions containing the product were combined and evaporated under reduced pressure to afford pure VC-LC-9261 (yield 21%)

[0209] *Eluent B: methanol / ammonium hydroxide (99:1)

[0210] VC-LC-9261 was characterised by HPLC-ELSD-MS. Retention time: 4.17 min, MS (ES): 473.65 experimental m / z [M+H]+ {theoretical m / z [M+H]+ 473.34}

[0211] Step 4. Final step in the synthesis of lipid VC-LC-1311 - -

[0212] Scheme 13. Synthetic scheme for the final step in the preparation of lipid VC-LC-1311

[0213] The product from the previous step (VC-LC-9261, 137 mg, 0.29 mmol), EDC hydrochloride (74 mg, 0.38 mmol), 4-(dimethylamino)pyridine (7.1 mg, 0.06 mmol) and 2- ethylhexanethiol (103 pL, 0.58 mmol) were dissolved in 2 mL of anhydrous dichloromethane at room temperature. The reaction mixture was stirred at room temperature overnight under an argon atmosphere. Then, the reaction crude was redissolved in 40 mL of dichloromethane and washed two times using distilled water (2 x 10 mL) and finally with saturated brine (10 mL). The organic layer was dried with anhydrous MgSO4, filtered, and evaporated under reduced pressure. The resulting residue was purified by flash chromatography (gradient of dichloromethane / eluent A: 100 / 0 to 0 / 100). After removing the solvent under reduced pressure, VC-LC-1311 is afforded as an oil (10 % yield).

[0214] Eluent A: dichloromethane / methanol / ammonium hydroxide (80:20:1)

[0215] VC-LC-1311 was characterised by HPLC-ELSD-MS. Retention time: 6.98 min, MS (ES): 601.56 experimental m / z [M+H]+ {theoretical m / z [M+H]+ 601.44}

[0216] *Eluent A: dichloromethane / methanol / ammonium hydroxide (80:20:1)

[0217] VC-LC-9241 was characterised by HPLC-LSD-MS following the general procedure detailed above. Retention time: 6.98 min, MS (ES): 601.56 experimental m / z [M+H]+ {theoretical m / z [M+H]+ 601.44}

[0218] RNA encapsulation into LNPs

[0219] Encapsulation of mRNA comprising in the 5’ to 3’ direction a sequence encoding for luciferase of SEQ ID NO:1 into LNPs was performed in some cases by microfluidics or, alternatively, by manual means.

[0220] A microfluidic mixer was used with the same procedure described in Hassett, K. J. et al., “Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines”, 2019, Mol. Ther. Nucleic Acid, vol. 15, pp. 1-11. Briefly, the purified mRNAs were first diluted in sodium citrate buffer at pH=4 at a final concentration of 266 pg / ml. Separately, a mixture of the ionizable lipid of the invention: DSPC (Merk 850365P):cholesterol (Sigma C3045):DMG-PEG2000 (Cayman 33945-1) were dissolved into ethanol at the respective molar percentages of 50:10:38.5:1.5, and with a lipid-nitrogen-to-phosphate ratio (N:P) ratio of 5,5:1.

[0221] Alternatively, when the LNPs were obtained by manual methods, e.g. by manual / bulk mixing as disclosed in Wang X., Liu S., Sun Y., et al. (“Preparation of selective organtargeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissuespecific mRNA delivery”, 2022, Nat. Protoc., doi:10.1038 / s41596-022-00755-x), the mRNA aqueous solution was carefully added to the ethanol solution, and the resulted solution was homogenized by pipetting up and down for 4-5 times. The resulting LNPs were immediately diluted 1 :1 with Tris buffer and dialyzed overnight against Tris buffer containing 15% sucrose. The resulting LNP solution was then collected, and the encapsulated mRNA was assessed by Quant-IT® Ribogreen (Invitrogen R11490) following the manufacturer’s instructions.

[0222] The LNP solution was then adjusted to a final concentration of mRNA of 100 pg / ml. Size distribution, polydispersity and Z-potential were measured by dynamic light scattering (DLS) using Malvern Zetasizer Advance Lab Blue Label. RNA encapsulation was assessed by Quant-IT® Ribogreen following the manufacturer’s instructions.

[0223] Finally, the LNP solution was passed through a 0.22 mm filter and the LNPs were stored at -80 °C until required.

[0224] Table 6 provides the results of several parameters for LNPs comprising the specific ionizable lipids shown therein in the following standard formulation: ionizable lipidmon-cationic lipid:sterol:PEG-modified lipid 50:10:38.5:1.5.

[0225] Table 6

[0226] As shown in Table 6, each of the compounds tested had an acceptable particle size for the purpose sought. The encapsulation efficiency, measured as the percentage of RNA entrapped in the LNP, ranged from 100% to approximately 15%. Nevertheless, this is only an indicative of how easily the components of the nanoparticle interact between them to form a LNP able to entrap RNA. It does not indicate the intracellular transfection efficiency. The intracellular transfection efficiency of the mRNA (encoding for the expression of luciferase) is measured as luminescence given as total flux (p s-1). The total flux is a bioluminescence measurement in photons per second or average radiance in each pixel integrated over the region of interest.

[0227] For clarification, the encapsulation efficiency means the approximate percentage of the RNA that was encapsulated when contacted with the components of the LNP. The value of the total flux observed indicated that the LNPs were not immediately degraded in vivo after injection, and that transfection of the cells was indeed very successful, hence the contents encapsulated inside the LNPs effectively reached the cytosol after the endosomal escape.

[0228] Example 4 - Protein expression in mice muscle administered with selected examples of LNPs prepared with the lipids of the invention and containing mRNA as active ingredient.

[0229] Administration of the mRNA (LNP) to mice

[0230] Female BALB / c mice (Charles River Laboratories), 8-10-week-old, weighting 18-23 g, were acclimatized to new conditions upon arrival to experimental facilities for 3-7 days. Housing conditions were room temperature 20-24 °C, humidity 50-70 %, and light intensity 60 lux with a light-dark cycle of 12 hours.

[0231] For Firefly Luciferase activity measurement in mice, LNPs produced as indicated above (ionizable lipid:helper lipid: sterol: PEG-modified lipid 50:10:38.5:1.5) and containing 1 pg of the indicated mRNA in 30 l final volume, were injected intramuscularly.

[0232] From 4 to 72 hours after RNA-LNP inoculation mice were anaesthetized by inhalation with 4% of isoflurane using a vaporizer. The maintenance of the anaesthesia was performed at 1.5% of isoflurane. Then, D-luciferin (Quimigen, 12507) was injected intraperitoneally at 150 mg / kg, normally -200 pL of the stock at 15 mg / mL in PBS for a 20 g mouse.

[0233] Luciferase images were acquired 10 minutes after luciferin inoculation using the I VIS Lumina XRMS Imaging System following manufacturer's instructions.

[0234] Table 7 below shows selected examples of LNPs comprising ionizable lipids of Formula (I) with different combinations of R1 and R2 in order to prove that the general structure of Formula (I) produces the desired effect, independently of the selected substituents. Figure

[0235] 1 depicts protein expression in mice of compounds depicted in Table 7.

[0236] Table 7

[0237] Table 7 demonstrates that LNPs comprising ionizable lipids of Formula I effectively encapsulate polynucleotides and produce high levels of cellular transfection in vivo after administration.

[0238] Figure 1 depicts images of the total flux measured in mice which had been administered LNPs formulated with ionizable lipids VC-LC-1272, VC-LC-1285 and VC-LC-1289. Example 5 - Comparative study of the hydrolytic cleavage of selected functional groups.

[0239] In order to monitor the occurrence of the hydrolytic cleavage of thioester and ester bonds, three ionizable lipids at the same concentration were incubated separately for over 4 hours in similar acidic conditions to those found inside endosomes (pH=5.2). Thus, the acidic cleavage of the ionizable lipids VC-LC-1282, SM-102, VC-LC-0729 took place following the proposed scheme of hydrolysis, yielding fragments A, B and C respectively, as shown in scheme 14 below:

[0240]

[0241] Scheme 14. Proposed hydrolysis scheme for VC-LC-1284, SM-102 and VC-LC-0729.

[0242] With the aim of measuring the occurrence of the hydrolysis reaction, the appearance of Fragments A, B and C was monitored using HPLC-MS single ion recording with the following theoretical m / z. Hence, 50 mM stock solutions of lipids VC-LC-1284, SM-102 and VC-LC-0729 were prepared in DMSO (TCI, >99% pure). Then, 20 pL of the stock solution were added to a vial containing 60 pL of pH=5.2, 2.4 M aqueous buffer (sodium acetate, Sigma-Aldrich, >99%) and 20 pL of DMSO (TCI, >99% pure). The measurements started immediately after the mixing and then the vial was left stirring during the complete experiment whilst HPLC injections were extracted every 42 minutes. Experimental method for the synthesis of VC-LC-0729

[0243] Step 1. Synthesis of intermediate VC-LC-9018

[0244] VC-LC-9018 Scheme 15 Synthetic route for Step 1 in the preparation of lipid VC-LC-0729.

[0245] DL-homocysteine thiolactone hydrochloride (270.4 mg, 1.76 mmol) was dissolved in 5 mL of anhydrous dichloromethane at room temperature. Then triethylamine (1620 pL, 1.8 mmol) was added followed by EDC hydrochloride (249.2 mg, 1.3 mmol), 4- (dimethylamino)pyridine (24.4 mg, 0.2 mmol) and 2-hexyldecanoic acid (256.42 mg, 1 mmol). The reaction mixture was stirred at room temperature overnight under an argon atmosphere. Then, the reaction crude was washed two times using distilled water (2 x 10 mL) and finally with saturated brine (10 mL). The organic layer was dried with anhydrous MgSC filtered, and evaporated under reduced pressure. The resulting residue was purified by flash chromatography (gradient of hexane / ethyl acetate: 100 / 0 to 0 / 100) to afford VC-LC-9018 as a yellowish oil (89 % yield).

[0246] The product was characterised by HPLC-ELSD-MS. Retention time: 6.02 min, MS (ES): experimental m / z [M+H]+356.44 {theoretical m / z [M+H]+356.26}.

[0247] Step 2. Synthesis of intermediate VC-LC-9121

[0248] VC-LC-9121

[0249] Scheme 16. Synthetic route for Step 2 in the preparation of lipid VC-LC-0729

[0250] The product from step 1 (VC-LC-9018, 249 mg, 0.7 mmol) was dissolved in anhydrous tetrahydrofuran (1.5 mL) and was added to a solution of N,N-Dimethylethylenediamine (756 pL, 7.03 mmol) in anhydrous tetrahydrofuran (1.0 mL) under argon atmosphere. The solution was then stirred at room temperature for 30 minutes, after which the solvent was evaporated under reduced pressure. The dry mixture was redissolved in ethyl acetate (20 mL) and washed with a solution of 0.1 M HCI (aq) (20 mL, 3 consecutive washes), then distilled water (20 mL, 3 consecutive washes) and finally with saturated brine (20 mL, 3 consecutive washes; NaCkwater prepared by adding 35 g of NaCI in 100 mL of water). The combined organic fractions were dried with MgSC>4 (anhydrous) and subsequently evaporated under reduced pressure to yield VC-LC-9121 as a yellowish oil (quantitative yield)

[0251] The product was characterised by HPLC-ELSD-MS. Retention time: 3.58 min, MS (ES): experimental m / z [M+H]+444.55 {theoretical m / z [M+H]+444.36}.

[0252] Step 3. Synthesis of intermediate VC-LC-9076

[0253] VC-LC-9076

[0254] Scheme 17. Synthetic route for Step 3 in the preparation of lipid VC-LC-0729.

[0255] Acryloyl chloride (0.335 mL, 4 mmol) was added dropwise to a cooled (0 °C) solution of 2- hexyl-1 -decanol (1.315 mL, 4.4 mmol) and triethylamine (0.843 mL, 6 mmol) in anhydrous dichloromethane (15 mL). The resulting solution was protected from light and stirred overnight under an argon atmosphere, allowing it to warm to room temperature. The reaction mixture was followed to completion by TLC chromatography (hexane:ethyl acetate, 6:4). Then, the reaction mixture was first washed with water (2 x 6 mL) and finally with brine (6 mL). The organic phase was dried with anhydrous MgSCL, filtered and evaporated under reduced pressure. It was used in the next step without further purification.

[0256] Step 4. Final step of the synthesis of lipid VC-LC-0729 Scheme 18. Synthetic route for Step 4 in the preparation of lipid VC-LC-0729.

[0257] To a solution of the product from step 2 (VC-LC-9121, 133.12 mg, 0.3 mmol) in tetrahydrofuran (1 mL), triethylamine (42 pL, 0.3 mmol) followed by VC-LC-9076 ( 88.94 mg, 0.3 mmol) were added. The reaction mixture was stirred overnight at room temperature after which it was checked for completion by TLC chromatography. The resulting product was purified by column chromatography using a gradient of dichloromethane / dichloromethane:methanol:Ammonium hydroxide (80:20:1) (100 / 0 to 0 / 100). The fractions containing the product were combined and evaporated under reduced pressure yielding VC-LC-0729 as an oil (77% yield).

[0258] The product was characterised by HPLC-ELSD-MS. Retention time: 8.19 min, MS (ES): experimental m / z [M+H]+740.85 {theoretical m / z [M+H]+740.63 }.

[0259] HPLC Water Alliance ELSD 2424

[0260] Column: X Bridge - Waters - BEH C8, 4.6 mm, 50 mm, 2.5 pm

[0261] Gradient: (A: Water with 0.01% TFA; B: Acetonitrile with 0.01% TFA), 15 min, constant flow rate= 1 mL / min)

[0262] SM-102 is commercially available and was purchased from BOCSI (95% purity, Catalogue Number B2699-358154).

[0263] Sample preparation for the reaction monitoring: In a HPLC sample tube, 5 pL of the reaction crude are dissolved in 100 pL of HPLC-MS-grade tetra hydrofuran. Then, the sample tube is vortexed manually for 5 seconds and placed inside the HPLC sample chamber.

[0264] Final product sample preparation: In a HPLC sample tube, 1 pL of dry lipid is dissolved in 100 pL of HPLC-MS-grade tetra hydrofuran. Then, the sample tube is vortexed manually for 5 seconds and placed inside the HPLC sample chamber. Injection volume: 2 pL

[0265] Column temperature: 65 °C.

[0266] Detection: Evaporative light scattering detector plus simple quadrupole mass detector in positive mode (Waters Acquity QDa). ELSD: Evaporative light dispersion detector (Waters ELSD 2424).

[0267] Characterization (MS): Simple quadrupole mass detector (Waters Acquity QDa).

[0268] General procedure for the characterization of the afforded fragments by HPLC-ELSD-MS (HPLC Water Alliance ELSD 2424 and column X bridge BEH C8, 4.6 mm, 50 mm, 2.5 pm. Injection volume is 2 pL and the temperature in the column is 65 °C. Gradient: (A: Water with 0.01% TFA; B: Acetonitrile with 0.01% TFA), 15 min, constant flow rate= 1 mL / min)

[0269] Table 8

[0270] Table 9

[0271] Tables 8 and 9 summarize the data obtained for the appearance of the expected fragments produced during the hydrolysis of ionizable lipids using selected ion recording (SIR) HPLC-MS

[0272] The peak area for each fragment obtained in this experiment is represented in Figure 2. It is therefore apparent that the fragment corresponding to hydrolysis of VC-LC-1284 appears at much higher concentration compared to SM-102 and VC-LC-0729, which demonstrates that VC-LC-1284 hydrolyses faster. This demonstrates that the ionizable lipids of the present disclosure, containing at least one amide moiety and one thioester moiety, have a superior degradability compared to SM-102 and VC-LC-0729, which do not contain amide and thioester (SM-102), or which contain an amide moiety but do not contain a thioester moiety (VC-LC-0729); importantly, VC-LC-1284 demonstrates that the ionizable lipids of the present disclosure do not degrade until they reach their target and release their cargo in the system, but when they do their degradability occurs faster which is advantageous in order to avoid several problems such as a strong immunity response.

[0273] Example 6 - Study of protein expression in mice muscle administered with selected examples of LNPs prepared with ionizable lipids comprising shorter R1, excluded from Formula (I).

[0274] Two ionizable lipids comprising shorter R1 alkyl substituents than those defined by Formula (I) or Formula (IA): linear butyl and linear nonyl, were synthesized according to the process described in the present disclosure and administered to mice following the same procedure described previously in Example 4.

[0275] The structures of ionizable lipids VC-LC-1544 and VC-LC-1546, and their characteristic retention times are summarized in Table 10 below.

[0276] Table 10

[0277] Encapsulation of mRNA comprising in the 5’ to 3’ direction a sequence encoding for luciferase of SEQ ID NO:1 into LNPs was performed in by microfluidics in the same manner as previously described in Example 3.

[0278] Table 11 provides the results of several parameters for LNPs comprising the specific ionizable lipids shown therein in the following standard formulation: ionizable lipidmon-cationic lipid:sterol:PEG-modified lipid 50:10:38.5:1.5.

[0279] Table 11

[0280] As shown in Table 11 , the compounds tested had acceptable particle size, P.D.I., Zeta potential, apparent pKa and encapsulation efficiency over 90% both for VC-LC-1544 and VC-LC-1546.

[0281] For Firefly Luciferase activity measurement in mice, the LNPs produced as indicated above (ionizable lipid:helper lipid: sterol: PEG-modified lipid 50:10:38.5:1.5) and containing 1 pg of the indicated mRNA in 30 pl final volume, were injected intramuscularly in the same manner as described in Example 4. Table 12

[0282] Table 12 shows that LNPs comprising ionizable lipids VC-LC-1544 and VC-LC-1546, comprising linear butyl or linear nonyl as R1 substituents, effectively encapsulate polynucleotides, but produce very low levels of cellular transfection in vivo after administration. The total flux observed indicates that the cellular transfection was 3 orders or magnitude lower compared to that observed for LNPs comprising ionizable lipids of Formula (I) and Formula (IA).

[0283] Example 7 - Biodegradability tests in vivo of LNPs comprising an ionizable lipid of Formula (I) encapsulating mRNA.

[0284] In order to assess the biodegradability of LNPs comprising the ionizable lipids of the present disclosure and a commercially available one for comparison, LNPs formulated with ionizable lipid VC-LC-1272, or SM-102, in the standard composition (ionizable lipid:helper lipid:sterol:PEG-modified lipid 50:10:38.5:1.5) and 25 pg of the previously indicated mRNA (Firefly luciferase) were injected intravenously to BALB / c mice (n=3, female), aged 8 weeks. The LNPs were diluted in Tris buffer containing 15% sucrose and administered via tail vein injection in a final volume of 250 pL using a 27 G syringe. Mice were euthanized by CO2inhalation, and liver samples were collected at various time points post-injection; in particular t = 0, 1-, 4-, 24- and 120-hours post-injection.

[0285] 0.4 mg of liver tissue was diluted in 300 pL of isopropanol / ethanol, adding an internal control. Liver tissue was dissociated using a GentleMACS Dissociator (Miltenyi). The sample was sonicated for 15 minutes and centrifuged at 10000g for 15 minutes. Supernatats were collected and concentrated using a SpeedVac system and subsequently diluted with a 1 :1 mixture of isopropanol and ethanol and analyzed against calibration standards. Chromatographic separation and quantification were accomplished with a liquid chromatography (LC)-MS system. Samples were injected and separated in a Bridge_waters-BEH C8 column equilibrated with 95% solvent A containing 0.01% trifluoroacetic acid in acetonitrile (CAN:TFA 99:1). A simple quadrupole MS system (Waters Acquity QDa) operated in positive ion mode under SIR conditions was used for signal detection.

[0286] Quantification of the results obtained for VC-LC-1272, metabolite M1 (detectable after hydrolytic cleavage of VC-LC-1272, shown below), as well as SM-102, are summarized in table 13 below. Table 13

[0287] The results obtained demonstrate that, on one hand, the ionizable lipid VC-LC-1272 is only detectable at the very beginning of the experiment at t = 0; it was undetectable just 1 hour post injection. On the other hand, metabolite M1 , which results from the hydrolytic cleavage of the thioester moiety in VC-LC-1272, was immediately detectable (t=0) and up to 4 hours after injection, hence proving that physiological conditions have an immediate effect and that the LNPs degrade very fast in vivo.

[0288] For comparison, LNPs formulated with commercial ionizable lipid SM-102 were also monitored, the measurements showing that SM-102 was detectable from t=0, and then the concentration increased for the first 4 hours; after 24 hours, considerable amounts of SM-102 were still appreciated in mice. Finally, at t=120 hours post injection SM-102 was not detected or only in neglectable amounts.

[0289] In view of the results shown in Table 13, it is evident that biodegradability of LNPs formulated with ionizable lipids of Formula (I) occurs much faster than biodegradability of LNPs formulated with ionizable lipids commercially available that do not comprise thioester moieties in their structure.

[0290] Example 8 - Stability of LNPs over time.

[0291] LNPs comprising an ionizable of Formula (I) of the present disclosure (ionizable lipid VC- LC-1272:helper lipid: sterol: PEG-modified lipid 50:10:38.5:1.5) encapsulating mRNA for Firefly Luciferase were prepared as previously described. The resulting LNP solution was collected, and either lyophilized or kept in solution. In both cases, the samples were stored at T = 4 °C. The encapsulated mRNA was assessed by Quant-IT® Ribogreen (Invitrogen R11490) following the manufacturer’s instructions or injected intramuscularly in mice, in the same manner as described in Example 4.

[0292] The LNPs were characterised at t=0 and t=30 days together with the activity / transfection; the results observed are summarized in Table 14 below. Table 14

[0293] As shown in Table 14, the LNPs kept their properties with only a slight increase in the particle size, and transfection and protein expression were maintained after 30 days at T = 4°C independently of the storage conditions, either in solution or lyophilized. The experiment demonstrates that the LNPs of the present disclosure, despite comprising an ionizable lipid of Formula (I) comprising a thioester moiety (a functional group more likely to experiment hydrolysis), prove to have a long shelf life when stored at T = 4°C. This is a surprisingly long lifespan when compared to the fast biodegradability tests shown in Example 7.

[0294] Example 9 - Toxicity of LNPs comprising ionizable lipids of Formula (I)

[0295] Concentration of liver enzymes alkaline phosphatase (ALP), alanine transaminase (ALT) and aspartate aminotransferase (AST), as indicators of liver malfunction, and albumin and urea, as markers of liver synthetic function and renal function, were measured in vivo in mice to determine toxicity of ionizable lipids of Formula (I) of the present disclosure.

[0296] LNPs comprising an ionizable of Formula (I) of the present disclosure (ionizable lipid VC- LC-1272:helper lipid: sterol: PEG-modified lipid 50:10:38.5:1.5) encapsulating mRNA for Firefly Luciferase were prepared as previously described.

[0297] BALB / c mice (n=3, female) aged 8 weeks were injected intravenously with 50 pg of FLuc mRNA / mouse encapsulated in LNPs or naked buffer for comparison. The LNPs were diluted in Tris buffer containing 15% sucrose and administered through the tail vein in a final volume of 250 pL using a 27 G syringe.

[0298] Blood samples were collected from the submandibular vein 48 h post-inoculation. Blood was allowed to clot and then centrifuged at 6500 g and 4 °C for 10 min for sera collection. Serum biochemical analyses were performed using Cobas c-311 automatic analyzer (Roche Diagnostics). The results are summarised in table 15 below.

[0299] Table 15

[0300] The results show that only a small increase in the measurements was observed in some cases. Fluctuating results are also expected due to variability in mice. However, none of the LNPs compositions produced significant values suggestive of abnormal liver activity or damage and all of the obtained values are within the established reference limits for this mouse strain. Hence, these tests confirm that the LNPs are safe for systemic administration in vivo.

[0301] The invention comprises the following clauses:

[0302] 1. An ionizable lipid of formula (I): or a pharmaceutically acceptable salt thereof, or a stereoisomer of any one of them, wherein

[0303] R’ is selected from the group consisting of H, methyl, and ethyl;

[0304] X is selected from the group consisting of -NH-C(O)-, -C(O)-NH-, -C(O)-O-, -O-C(O)-, -S- C(O)-, -C(O)-S-; m and p are independently selected from 0, 1 , 2, 3, 4, 5, and 6; t is selected from 1 , 2 and 3;

[0305] Q is a heterocycle comprising at least one N atom, or alternatively Q is: wherein Ra and Rb are independently linear or branched 01 -06 alkyl, optionally substituted with a hydroxyl group; and wherein R1 and R2 are independently linear or branched C1-C30 alkyl, C2-C30 alkenyl or C2-C30 alkynyl, wherein R1 and R2 are optionally substituted with one or more substituents selected from the group consisting of -OH, -COOR4, and -C(=O)SR4, wherein R4 is C1 -C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl; provided that the compounds:

[0306] S-oleoyl-N-acetyl-L-cysteine-3-(dimethylaminopropylamine)-amide, and

[0307] S-oleoyl-N-acetyl-L-cysteine-3(dimethylaminopropylamine)-amide hydrochloride are excluded.

[0308] 2. The ionizable lipid of clause 1 , or a pharmaceutically acceptable salt thereof, or a stereoisomer of any one of them, wherein Ri is selected from linear C11-C30 alkyl or branched C6-C30 alkyl, C6-C30 alkenyl and C6-C30 alkynyl and R2 is selected from linear or branched C6-C30 alkyl, C6-C30 alkenyl or C6-C30 alkynyl, wherein R1 and R2 are optionally substituted with one or more substituents selected from the group consisting of - OH, -COOR4, and -C(=O)SR4, wherein R4 is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl.

[0309] 3. The ionizable lipid of clauses 1 or 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer of any one of them, wherein R’ is selected from the group consisting of H and methyl, and m is selected from 1 , 2, 3 and 4.

[0310] 4. The ionizable lipid of clause 1 , wherein the compound of Formula (I) is a compound is of formula (IA): Formula (IA) wherein Q, R’, X, Ri, R2, and m are as defined in clause 1.

[0311] 5. The ionizable lipid of clause 4, wherein X is -NH-C(O)- or -C(O)-NH-.

[0312] 6. The ionizable lipid of clause 4, wherein X is -C(O)-O- or -O-C(O)-.

[0313] 7. The ionizable lipid of clause 4, wherein X is -S-C(O)- or -C(O)-S-.

[0314] 8. The ionizable lipid according to any one of clauses 5 to 7, wherein Q is: wherein Ra and Rb are as disclosed in clause 1.

[0315] 9. The ionizable lipid according to any one of clauses 5 to 7, wherein Q is a 5-membered ring or a 6 membered ring comprising one N atom and, optionally, a second heteroatom selected preferably from N and O; or, alternatively, wherein Q is selected from the following structures:

[0316] 10. The ionizable lipid according to any one of clauses 1 to 9, wherein Q is selected from the following structures: 11. The ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Me)2; m=t=2; p=0; R’ is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is selected from the group consisting of C8-C24 branched alkyl; and R2 is selected from the group consisting of C10-C24 branched alkyl, and C10-C24 alkenyl or alkdienyl;

[0317] 12. The ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Me)2; m=t=2; p=0; R’ is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is selected from C11, C15 and C19 branched alkyl; and R2 is selected from C11, C15 and C17 branched alkyl, and C17 alkenyl or alkdienyl.

[0318] 13. The ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Me)2; m=t=2; p=0; R’ is H; X is selected from -C(O)-O- and -O-C(O)-; R1 is selected from the group consisting of C8-C24 branched alkyl; and R2 is selected from the group consisting of C10-C24 branched alkyl and C10-C24 linear alkenyl or alkdienyl;

[0319] 14. The ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Me)2; m=t=2; p=0; R’ is H; X is selected from -C(O)-O- and -O-C(O)-; R1 is selected from the group consisting of C12, C16 and C24 branched alkyl; and R2 is selected from the group consisting of C15 branched alkyl, and C17 linear alkenyl.

[0320] 15. The ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Me)2; m=t=2; p=0; R’ is H; X is selected from -C(O)-S- and -S-C(O)-; R1 is selected from the group consisting of C8-C24 branched alkyl; and R2 is C10-C24 branched alkyl.

[0321] 16. The ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Me)2; m=t=2; p=0; R’ is H; X is selected from -C(O)-S- and -S-C(O)-; R1 is C8 branched alkyl; and R2 is C15 branched alkyl.

[0322] 17. The ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Et)2; m=t=2; p=0; R’ is H; X is selected from -C(O)-O- and -O-C(O)-; R1 is selected from the group consisting of C8-C24 branched alkyl; and R2 is selected from the group consisting of C10-C24 branched alkyl and C10-C24 linear alkenyl or alkdienyl.

[0323] 18. The ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Et)2; m=t=2; p=0; R’ is H; X is selected from -C(O)-O- and -O-C(O)-; R1 is C15 branched alkyl; and R2 is C17 alkenyl.

[0324] 19. The ionizable lipid according to any one of clauses 4 to 10, wherein Q is a heterocycle comprising at least one N atom, m=t=2; p=0; R’ is H; X is selected from -NH-C(O)- and - C(O)-NH-; R1 is selected from the group consisting of C8-C24 branched alkyl; and R2 is selected from the group consisting of C10-C24 branched alkyl and C10-C24 linear alkenyl or alkdienyl.

[0325] 20. The ionizable lipid according to any one of clauses 4 to 10, wherein Q is a five membered-heterocycle or a six-membered heterocycle comprising one or two N atoms, m=t=2; p=0; R’ is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is C15 branched alkyl; and R2 is C17 linear alkenyl.

[0326] 21. The ionizable lipid of clause 1, which is a compound selected from the group consisting of: S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecanamido)-4-oxobutyl) (Z)-octadec-9-enethioate (VC-LC-1272), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2- hexyldecanamido)-4-oxobutyl) 2-hexyldecanethioate (VC-LC-1284), 2-hexyldecyl 2-((2- (dimethylamino)ethyl)carbamoyl)-4-(oleoylthio)butanoate (VC-LC-1285), 2-hexyldecyl 2- ((2-(dimethylamino)ethyl)carbamoyl)-4-((2-hexyldecanoyl)thio)butanoate (VC-LC-1286), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecanamido)-4-oxobutyl) (9Z,12Z)- octadeca-9,12-dienethioate (VC-LC-1287), 2-butyloctyl 2-((2-

[0327] (dimethylamino)ethyl)carbamoyl)-4-(oleoylthio)butanoate (VC-LC-1288), 2-butyloctyl 2-((2- (dimethylamino)ethyl)carbamoyl)-4-((2-hexyldecanoyl)thio)butanoate (VC-LC-1289), 2- decyltetradecyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-((2-hexyldecanoyl)thio)butanoate (VC-LC-1293), 2-decyltetradecyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-

[0328] (oleoylthio)butanoate (VC-LC-1297), S-(4-((2-(dimethylamino)ethyl)amino)-3-(((2- ethylhexyl)thio)carbonyl)-4-oxobutyl) 2-hexyldecanethioate (VC-LC-1311), S-(4-((2- (dimethylamino)ethyl)amino)-3-(2-hexyldecanamido)-4-oxobutyl) 2-butyloctanethioate (VC-LC-1369), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecanamido)-4-oxobutyl) 2-heptylundecanethioate (VC-LC-1371), S-(3-(2-butyloctanamido)-4-((2- (dimethylamino)ethyl)amino)-4-oxobutyl) 2-hexyldecanethioate (VC-LC-1373), S-(3-(2- butyloctanamido)-4-((2-(dimethylamino)ethyl)amino)-4-oxobutyl) (Z)-octadec-9-enethioate (VC-LC-1374), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-octyldodecanamido)-4-oxobutyl) (Z)-octadec-9-enethioate (VC-LC-1376), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2- octyldodecanamido)-4-oxobutyl) (9Z,12Z)-octadeca-9,12-dienethioate (VC-LC-1377), S- (4-((2-(dimethylamino)ethyl)amino)-3-(2-octyldodecanamido)-4-oxobutyl) 2- hexyldecanethioate (VC-LC-1378), S-(3-(2-hexyldecanamido)-4-oxo-4-((2-(pyrrolidin-1- yl)ethyl)amino)butyl) (Z)-octadec-9-enethioate (VC-LC-1539), S-(3-(2-hexyldecanamido)- 4-oxo-4-((2-(piperidin-1-yl)ethyl)amino)butyl) (Z)-octadec-9-enethioate (VC-LC-1540), S- (4-((2-(1 H-imidazol-1-yl)ethyl)amino)-3-(2-hexyldecanamido)-4-oxobutyl) (Z)-octadec-9- enethioate (VC-LC-1541), S-(3-(2-hexyldecanamido)-4-((1-methylpiperidin-4-yl)amino)-4- oxobutyl) (Z)-octadec-9-enethioate (VC-LC-1543), S-(4-((2-(diethylamino)ethyl)amino)-3- (2-hexyldecanamido)-4-oxobutyl) (Z)-octadec-9-enethioate (VC-LC-1545).

[0329] 22. A lipid nanoparticle comprising an ionizable lipid of formula (I): or a pharmaceutically acceptable salt thereof, or a stereoisomer of any one of them, wherein

[0330] R’ is selected from the group consisting of H, methyl, and ethyl;

[0331] X is selected from the group consisting of -NH-C(O)-, -C(O)-NH-, -C(O)-O-, -O-C(O)-, -S- C(O)-, -C(O)-S-; m and p are independently selected from 0, 1 , 2, 3, 4, 5, and 6; t is selected from 1 , 2 and 3;

[0332] Q is a heterocycle comprising at least one N atom, or alternatively Q is: wherein Ra and Rb are independently linear or branched C1-C6 alkyl, optionally substituted with a hydroxyl group; and wherein Ri and R2 are independently linear or branched C1-C30 alkyl, C2-C30 alkenyl or C2-C30 alkynyl, wherein R1 and R2 are optionally substituted with one or more substituents selected from the group consisting of -OH, -COOR4, and -C(=O)SR4, wherein R4 is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl; or the ionizable lipid as defined in any one of clauses 2 to 21.

[0333] 23. The lipid nanoparticle according to claim 22, wherein from the ionizable lipid of formula (I) the compounds S-oleoyl-N-acetyl-L-cysteine-3-(dimethylaminopropylamine)-amide, and S-oleoyl-N-acetyl-L-cysteine-3(dimethylaminopropylamine)-amide hydrochloride are excluded.

[0334] 24. The lipid nanoparticle according to clauses 22 or 23, further comprising: i) a non-cationic lipid; optionally, wherein the non-cationic lipid is selected from the group consisting of: 1 ,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero- phosphocholine (DM PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-stearoyl-2- oleoyl-sn-glycero-3-phosphocoline (SOPC), 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-diundecanoyl-sn- glycero-phosphocholine (DLIPC), 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- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), 1 ,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 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-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof; optionally, wherein the non-cationic lipid is DOPE; and / or wherein the non-cationic lipid is DSPC; ii) a sterol, a steroid precursor, or a steroid derivative; optionally, wherein the sterol is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, squalene, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof; further optionally, wherein the sterol is cholesterol; and iii) a conjugated lipid or, alternatively, a PEG-modified lipid; optionally, wherein the PEG-modified lipid is selected from the group consisting of a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified phosphatidylcholine, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG- modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.

[0335] 25. The lipid nanoparticle according to clause 24, wherein the ionizable lipid is in an amount from 25 to 60 mol%, the PEG-modified lipid or the conjugated lipid is in an amount from 0.1 to 10 mol%; the non-cationic lipid is in an amount from 10 to 45 mol%; and the sterol is in an amount from 10 to 40 mol%; or, alternatively, wherein the ionizable lipid is in an amount from 25 to 64 mol%, the PEG-modified lipid or the conjugated lipid is in an amount from 0.1 to 1.5 mol%; and the sterol, steroid precursor, or steroid derivative is in an amount from 35 to 74 mol%. 26. A lipid nanoparticle according to any one of clauses 22 to 25, further comprising a pharmaceutically active agent.

[0336] 27. The lipid nanoparticle according to clause 26, wherein the pharmaceutically active agent is selected from the group consisting of a polynucleotide, a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide, and an expression vector comprising a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide.

[0337] 28. The lipid nanoparticle according to clause 27, wherein the polynucleotide is a natural or an artificial deoxyribonucleic acid (DNA) or a natural or artificial ribonucleic acid (RNA); further optionally, wherein the polynucleotide comprises at least one chemical modification selected from the group consisting of pseudouridine, N1 -methylpseudouridine (also referred to as 1 -methylpseudouridine or ml^P), N6-methyladenosine (also referred to as m6A), 2-thiouridine (also referred to as s2U), 4'-thiouridine, 5-methylcytosine (also referred to 5mC), 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methyluridine (also referred to as m5U), 5-methoxyuridine, 2'-O-methyl uridine, and combinations thereof. In particular, the chemical modification is N1 -methylpseudouridine, 5-methoxyuridine or a combination thereof; particularly the chemical modification is N1- methylpseudouridine further optionally, wherein the polynucleotide is a ribonucleic acid (RNA); further optionally, wherein the RNA is selected from the group consisting of a short interfering RNA (siRNA), a self-replicating RNA (srRNA), circular RNA (circRNA), a selfamplifying RNA (saRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a small interfering RNA (siRNA), a small RNA (sRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and mixtures thereof; particularly the RNA is an mRNA.

[0338] 29. A pharmaceutical composition comprising the lipid nanoparticle as defined in any one of clauses 22 to 28 and a pharmaceutically acceptable excipient or carrier.

[0339] 30. A lipid nanoparticle as defined in any one of clauses 22 to 28, or a pharmaceutical composition as defined in clause 17, for use in a method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the nanoparticle composition or of the pharmaceutical composition; optionally, wherein the disease or disorder is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases; further optionally, wherein the subject is a human; or, for use in a method of inducing an immune response in a subject, for use in a method for the therapeutic immunization of a subject, for use as a vaccine, or for use in gene therapy; or, for use in the prevention or in the treatment of COVID-19.

[0340] 31. Use of the lipid nanoparticle as defined in any one of clauses 22-25 as an encapsulation agent.

[0341] Citation List

[0342] 1. Molla MR, et al. (cf. Molla MR, et al. "One-Pot Parallel Synthesis of Lipid Library via Thiolactone Ring Opening and Screening for Gene Delivery". Bioconjug Chem. 2018, vol. 29(4), pp. 992-999. doi: 10.1021 / acs.bioconjchem.8b00007.

[0343] 2. Churusova, S. et al. (2021). Palladium^ I) Pincer Complexes of Functionalized Amides with S-Modified Cysteine and Homocysteine Residues: Cytotoxic Activity and Different Aspects of Their Biological Effect on Living Cells. Inorganic Chemistry. 2021 , vol. 60, pp. 9880-9898.

[0344] 3. US4929736.

[0345] 4. Garbiras, B.J.; Marburg, S. "Preparation of Carboxythiolactones and Their Active Derivatives". Synthesis, 1999, vol. 2, pp. 270-274; doi: 10.1055 / s-l 999-3377.

[0346] 5. Hassett, K. J. et al., "Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines", 2019, Mol. Ther. Nucleic Acid, vol. 15, pp. 1-11, D0l:10.1016 / j.omtn.2019.01.013.

[0347] 6. Wang X., Liu S., Sun Y., et al. “Preparation of selective organ-targeting (SORT) lipid nanaoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery”, Nat. Protoc., 2022, doi:10.1038 / s41596-022-00755-x.

Claims

Claims1. An ionizable lipid of formula (I):or a pharmaceutically acceptable salt thereof, or a stereoisomer of any one of them, whereinR’ is selected from the group consisting of H, methyl, and ethyl;X is selected from the group consisting of -NH-C(O)-, -C(O)-NH-, -C(O)-O-, -O-C(O)-, -S- C(O)-, -C(O)-S-; m and p are independently selected from 0, 1 , 2, 3, 4, 5, and 6; t is selected from 1 , 2 and 3;Q is a heterocycle comprising at least one N atom, or alternatively Q is:wherein Ra and Rb are independently linear or branched C1-C6 alkyl, optionally substituted with a hydroxyl group; and wherein Ri is selected from linear C11-C30 alkyl or branched C6-C30 alkyl, C6-C30 alkenyl and C6-C30 alkynyl and R2 is selected from linear or branched C6-C30 alkyl, C6- C30 alkenyl or C6-C30 alkynyl, wherein R1 and R2 are optionally substituted with one or more substituents selected from the group consisting of -OH, -COOR4, and -C(=O)SR4, wherein R4 is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl.

2. The ionizable lipid of claim 1 , or a pharmaceutically acceptable salt thereof, or a stereoisomer of any one of them, wherein R’ is selected from the group consisting of H, and methyl; and m is selected from 1 , 2, 3 and 4.

3. The ionizable lipid of claim 1 , wherein the compound of Formula (I) is a compound ofFormula (IA)Formula (IA)wherein Q, R’, X, Ri, R2, and m are as defined in claim 1.

4. The ionizable lipid of claim 3, wherein X is -NH-C(O)- or -C(O)-NH-; or wherein X is - C(O)-O- or -O-C(O)-; or wherein X is -C(O)-S- or -S-C(O)-.

5. The ionizable lipid according to claim 4, wherein Q is:wherein Ra and Rb are independently linear or branched C1-C6 alkyl, optionally substituted with a hydroxyl group.

6. The ionizable lipid according to claims 4 or 5, wherein Q is a 5-membered ring or a 6 membered ring comprising one N atom and, optionally, a second heteroatom selected preferably from N and O; or, alternatively, wherein Q is selected from the following structures:

7. The ionizable lipid of claim 1 , which is a compound selected from the group consisting of: S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecanamido)-4-oxobutyl) (Z)-octadec- 9-enethioate (VC-LC-1272), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecanamido)-4-oxobutyl) 2-hexyldecanethioate (VC-LC-1284), 2-hexyldecyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-(oleoylthio)butanoate (VC-LC-1285), 2-hexyldecyl 2- ((2-(dimethylamino)ethyl)carbamoyl)-4-((2-hexyldecanoyl)thio)butanoate (VC-LC-1286),5-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecanamido)-4-oxobutyl) (9Z,12Z)- octadeca-9,12-dienethioate (VC-LC-1287), 2-butyloctyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-(oleoylthio)butanoate (VC-LC-1288), 2-butyloctyl 2-((2- (dimethylamino)ethyl)carbamoyl)-4-((2-hexyldecanoyl)thio)butanoate (VC-LC-1289), 2- decyltetradecyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-((2-hexyldecanoyl)thio)butanoate(VC-LC-1293), 2-decyltetradecyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-(oleoylthio)butanoate (VC-LC-1297), S-(4-((2-(dimethylamino)ethyl)amino)-3-(((2- ethylhexyl)thio)carbonyl)-4-oxobutyl) 2-hexyldecanethioate (VC-LC-1311), S-(4-((2- (dimethylamino)ethyl)amino)-3-(2-hexyldecanamido)-4-oxobutyl) 2-butyloctanethioate (VC-LC-1369), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecanamido)-4-oxobutyl) 2-heptylundecanethioate (VC-LC-1371), S-(3-(2-butyloctanamido)-4-((2- (dimethylamino)ethyl)amino)-4-oxobutyl) 2-hexyldecanethioate (VC-LC-1373), S-(3-(2- butyloctanamido)-4-((2-(dimethylamino)ethyl)amino)-4-oxobutyl) (Z)-octadec-9-enethioate (VC-LC-1374), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-octyldodecanamido)-4-oxobutyl) (Z)-octadec-9-enethioate (VC-LC-1376), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2- octyldodecanamido)-4-oxobutyl) (9Z,12Z)-octadeca-9,12-dienethioate (VC-LC-1377), S- (4-((2-(dimethylamino)ethyl)amino)-3-(2-octyldodecanamido)-4-oxobutyl) 2- hexyldecanethioate (VC-LC-1378), S-(3-(2-hexyldecanamido)-4-oxo-4-((2-(pyrrolidin-1- yl)ethyl)amino)butyl) (Z)-octadec-9-enethioate (VC-LC-1539), S-(3-(2-hexyldecanamido)- 4-oxo-4-((2-(piperidin-1-yl)ethyl)amino)butyl) (Z)-octadec-9-enethioate (VC-LC-1540), S- (4-((2-(1 H-imidazol-1-yl)ethyl)amino)-3-(2-hexyldecanamido)-4-oxobutyl) (Z)-octadec-9- enethioate (VC-LC-1541), S-(3-(2-hexyldecanamido)-4-((1-methylpiperidin-4-yl)amino)-4- oxobutyl) (Z)-octadec-9-enethioate (VC-LC-1543), S-(4-((2-(diethylamino)ethyl)amino)-3- (2-hexyldecanamido)-4-oxobutyl) (Z)-octadec-9-enethioate (VC-LC-1545).

8. A lipid nanoparticle comprising the ionizable lipid as defined in any one of claims 1 to 7.

9. The lipid nanoparticle according to claim 8, further comprising: i) a non-cationic lipid; optionally, wherein the non-cationic lipid is selected from the group consisting of: 1 ,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-stearoyl-2- oleoyl-sn-glycero-3-phosphocoline (SOPC), 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-diundecanoyl-sn- glycero-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- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), 1 ,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 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-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof; optionally, wherein the non-cationic lipid is DOPE; and / or wherein the non-cationic lipid is DSPC; ii) a sterol, a steroid precursor, or a steroid derivative; optionally, wherein the sterol is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, squalene, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof; further optionally, wherein the sterol is cholesterol; and iii) a conjugated lipid or, alternatively, a PEG-modified lipid; optionally, wherein the PEG-modified lipid is selected from the group consisting of a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified phosphatidylcholine, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG- modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.

10. The lipid nanoparticle according to claim 9, wherein the ionizable lipid is in an amount from 25 to 60 mol%, the PEG-modified lipid or the conjugated lipid is in an amount from 0.1 to 10 mol%; the non-cationic lipid is in an amount from 10 to 45 mol%; and the sterol, the steroid precursor, or the steroid derivative is in an amount from 10 to 40 mol%; or, alternatively, wherein the ionizable lipid is in an amount from 25 to 64 mol%, the PEG-modified lipid or the conjugated lipid is in an amount from 0.1 to 1.5 mol%; and the sterol, steroid precursor, or steroid derivative is in an amount from 35 to 74 mol%.

11. A lipid nanoparticle according to any one of claims 8 to 10, further comprising a pharmaceutically active agent.

12. The lipid nanoparticle according to claim 11 , wherein the pharmaceutically active agent is selected from the group consisting of a polynucleotide, a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide, and an expression vector comprising a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide; optionally, wherein the polynucleotide is a natural or an artificial deoxyribonucleic acid (DNA) or a natural or artificial ribonucleic acid (RNA);further optionally, wherein the polynucleotide comprises at least one chemical modification selected from the group consisting of pseudouridine, N1 -methylpseudouridine (also referred to as 1 -methylpseudouridine or ml^P), N6-methyladenosine (also referred to as m6A), 2-thiouridine (also referred to as s2U), 4'-thiouridine, 5-methylcytosine (also referred to 5mC), 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methyluridine (also referred to as m5U), 5-methoxyuridine, 2'-O-methyl uridine, and combinations thereof. In particular, the chemical modification is N1 -methylpseudouridine, 5-methoxyuridine or a combination thereof; particularly the chemical modification is N1- methylpseudouridine further optionally, wherein the polynucleotide is a ribonucleic acid (RNA); further optionally, wherein the RNA is selected from the group consisting of a short interfering RNA (siRNA), a self-replicating RNA (srRNA), circular RNA (circRNA), a selfamplifying RNA (saRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a small interfering RNA (siRNA), a small RNA (sRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and mixtures thereof; particularly the RNA is an mRNA.

13. A pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 11 to 12 and a pharmaceutically acceptable excipient or carrier.

14. The lipid nanoparticle as defined in any one of claims 11 to 12, or the pharmaceutical composition as defined in claim 13, for use in a method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the nanoparticle composition or of the pharmaceutical composition; optionally, wherein the disease or disorder is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases; further optionally, wherein the subject is a human; or, for use in a method of inducing an immune response in a subject, for use in a method for the therapeutic immunization of a subject, for use as a vaccine, or for use in gene therapy;or, for use in the prevention or in the treatment of COVID-19.

15. Use of the lipid nanoparticle as defined in any one of claims 8-14 as an encapsulation agent.

Images