Lipidic particles containing ionisable lipids for extrahepatic delivery

Abstract

It is provided a lipid particle comprising an ionisable lipid, a neutral helper lipid, a stealth lipid or a pharmaceutically acceptable salt or stereoisomer or tautomer or isotopic variant of any one of them, for use as an encapsulation agent, optionally comprising a cargo molecule or a pharmaceutically active agent; and a pharmaceutical composition comprising the lipid particle. It is also provided a lipid particle or a pharmaceutical composition comprising thereof for use in medicine, and the use of the lipid particle as an encapsulating agent for extrahepatic targeting.

Description

[0001] Lipidic particles containing ionisable lipids for extrahepatic delivery

[0002] The present application claims priority of European patent application EP24382955 dated 6 September 2024

[0003] Technical Field

[0004] The present disclosure relates to lipidic particles or lipid nanoparticles (LNPs) comprising ionisable lipids. These lipid particles can be used as non-viral vectors for the extrahepatic delivery of active ingredients, including polynucleotides, to non-liver cells, in particular, these lipid particles are able to deliver active ingredients to the spleen.

[0005] Background Art

[0006] Treatment of different conditions such as infectious diseases is rapidly changing nowadays, especially since new polynucleotide delivery systems are being designed and their efficiency optimized to be a useful alternative to traditional therapies.

[0007] Gene delivery systems intended to contain encapsulated polynucleotides, such as RNA for systemic delivery, must be safe and non-toxic, be in the nanometre scale, provide protection against degradation to the polynucleotides, remain intact in the system for a given period of time in order to reach their target, and be easily degraded once they have released the load.

[0008] The use of RNA in gene therapy and vaccination is generally considered safer than the use of DNA, since RNA does not involve the risk of being stably integrated into the genome of the transfected cell. In addition, RNA degrades more easily in vivo, and therefore the risk of generating undesired anti-RNA antibodies that would reduce therapy efficacy and could produce very serious side effects is lower. Two of the main limitations of RNA-based therapies are the low transfection rates and the limited protein production efficiency. To compensate these, the dosage is increased in order to obtain the desirable therapeutic effects. Consequently, a higher dose also means elevated costs and some undesirable side effects observed.

[0009] One of the more potent intracellular delivery technologies for encapsulation and delivery of active molecules, such as for example genetic material (e.g. mRNA) in vaccines, is lipid nanoparticles (LNPs). Currently, LNPs used in commercial mRNA vaccines usually comprise four types of lipids in their composition, that is, a cationic or an ionisable lipid, a structural lipid which is a sterol such as cholesterol, a PEG-modified lipid, and a non-cationic lipid, also known as “helper”, such as a phospholipid. In particular, ionisable lipids are crucial in LNPs. lonisable 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] From 1998 until 2021 , 16 drugs based on nucleic acids have been approved in some areas of US, Europe and Japan, of which 13 were approved since 2016. In 2020, the US Food and Drug Administration authorised two mRNA vaccines against COVID-19; other mRNA-based vaccines for influenza, cytomegalovirus and advanced melanoma are now or have been in clinical trials. On top of that, some drugs comprising siRNA have also been authorized for the treatment of rare medical conditions such as Onpattro®, which comprises siRNA also encapsulated in LNPs formulated with the ionisable lipid DLin-MC3-DMA (also known as MC3). The structures of the ionisable lipids used in these therapies are shown below.

[0011] Despite continuous improvements in such LNPs delivery systems, efficient distribution of active agents to specific tissues is still problematic. In general, mRNA delivery systems such as LNPs incorporating ionisable lipids, are not selective and accumulate through the biological processes in the liver, which reduces the efficacy of the composition if they are intended for delivery into a different target organ. Even though many efforts have been done in developing nanosystems capable to reach different organs such as lungs, spleen, heart, bone marrow, etc. by functionalization with ligands like antibodies, proteins and peptides (known as active targeting), the progress has been limited. This is mainly due to the highly complex biological phenomena involved which remain in part unknown, as well as the high cost of personalized systems. Thus, there is still a need for alternative targeting delivery systems that avoid the disadvantages of the prior art, in particular, showing good stability, high transfection efficiency, safety issues and excellent distribution to target organs.

[0012] Regarding passive targeting strategy, recent studies such as the one published by Siegwart et al. (Nature Nanotechnology, Vol.15, 2020, 313-320) discovered selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery by incorporating a 5thmolecule to established LNP compositions. The authors examined the effect of adding permanently cationic lipids, permanently anionic and ionisable lipids to a degradable dendrimer ionisable cationic lipid. Addition of permanently cationic lipids showed a shift of luminescence activity from the liver to the lung. All the anionic lipids delivered exclusively to the spleen, wherein ionisable lipids such as DODAP or C12-200 enhanced hepatic delivery.

[0013] Since then, many studies have reported similar experiments (such as S.T. LoPresti et al., J. Controlled Release 2022, 345, 819; R.M. Waymouth et al., Bioconjug. Chem. 2023, 34,673; D.G. Anderson et al., Nano Lett. 2018, 18, 6449); the downside of these being that they can induce massive thrombosis in the lungs as well as in other organs as reported by J.S. Brenner et al. in Adv. Mater. 2024 Jun; 36(26), doi:10.1002 / adma.202312026.

[0014] Within the recent past, Su et al. (Nature Communications 2024 15:5659, doi:10.1038 / s41467- 024-50093-7) have described the preparation of 3-component LNPs comprising an ionisable lipid, a permanently cationic lipid (DOTAP) and a PEG-lipid that targets the lungs.

[0015] An intravenous screening of LNPs containing different ionisable lipids that fall into Formula (I) of patent application WO2024110381 A1 was carried out in our labs. Most of the ionisable lipids disclosed in WO2024110381 A1 had shown high percentages of liver protein expression, (typically, over 90%). Surprisingly, the level of protein expressed extra-hepatically observed was very high for a few of them.

[0016] The present inventors developed a very simple 3-component formulation and were pleasantly amazed when comparing the results achieved by the lipid particles of the present disclosure side by side with those formulated in WO2024110381 A1.

[0017] Unexpectedly, the simplified 3-component formulation disclosed herein achieved high levels of protein expression extrahepatically, more particularly, in the spleen. Remarkably enough, although it is scientifically accepted in the literature that LNPs need an apparent pKa that falls in the range of 6-7 to achieve any performance in vivo, the characterization of the pKa for these spleen-targeting LNPs showed data that deviate from these well-established ranges (some of them >7.5).

[0018] The reduction of components in the LNPs simplifies their preparation and decreases the associated cost. Moreover, the 3-component LNPs do not require the use of charged lipids to achieve effective passive targeting to the spleen, hence avoids potential toxicity and tissue inflammation or clotting. The 3-component LNPs offered excellent targeting ability and an outstanding in vivo signal with protein expression luminescence values superior to regular values reported for targeted RNA expression in the spleen using charged lipids.

[0019] Finally, the present disclosure also demonstrates that this 3-component spleen targeting LNP formulation maintains its functionality after lyophilisation and storage under mild refrigerated conditions.

[0020] Summary of the Invention

[0021] The present inventors have developed a new simple 3-component formulation comprising an ionisable lipid, a helper lipid and a lipid-polymer conjugate that allows obtaining lipid particles that can be effectively used as non-viral vectors for delivery of active ingredients, including polynucleotides, to non-hepatic cells. Particularly, the present inventors have found that transfection rates and protein expression in vivo of lipid nanoparticles (LNPs) prepared with the ionisable lipids SM-102, VC-LC-1254, VC-LC-1405, VC-LC-1143 and VC-LC-0866, and comprising a polynucleotide as their active agent are unexpectedly high in extrahepatic tissues compared to other commercial or standard compositions known in the art.

[0022] Thus, from the data provided in the Examples, it is apparent that the 3-component formulation of the present disclosure provides a new tool to overcome some of the limitations of known lipid particles.

[0023] The higher transfection efficiency of the lipid particles of the present disclosure may allow reducing the therapeutic dose of polynucleotide, such as RNA, required in gene therapy and / or vaccination, which may help to reduce the associated secondary effects. At the same time, the lipid particles of the present disclosure show good targeting to specific tissues, in particular, to the spleen which allow accurate delivery of diverse cargoes to the targeted organ including polynucleotides such as RNA.

[0024] Therefore, a first aspect of the present disclosure relates to a lipid particle comprising an ionisable lipid, a neutral helper lipid a lipid-polymer conjugate or a pharmaceutically acceptable salt or stereoisomer or tautomer or isotopic variant of any one of them, wherein the lipid particle does not comprise cholesterol or sterol derivatives thereof, and wherein the lipid particle has a mean particle diameter between 80 and 300 nm, preferably between 100 and 250 nm, as determined by dynamic light scattering. A second aspect of the invention relates to a pharmaceutical composition comprising the lipid particle described in the present disclosure; and one or more pharmaceutically acceptable excipient or carrier.

[0025] A third aspect of the invention relates to a method for targeted delivery of a pharmaceutically active agent to a non-liver organ or a non-liver cell therein in a subject in need thereof, the method comprising administering to said subject said pharmaceutically active agent assembled with a lipid particle as described in the present disclosure, further optionally, wherein the subject is a human.

[0026] A fourth aspect of the invention relates to a pharmaceutical composition as described in the present disclosure for use in the treatment of a disease or a condition.

[0027] Thus, a fifth aspect of the present invention relates to the use of the lipid particle as described herein as an encapsulation agent.

[0028] Detailed description of the invention

[0029] 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.

[0030] 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.

[0031] The term "and / or" means that any one of the options to which it relates are possible or the at least two options take place at the same time.

[0032] The term “moiety” refers to a specific segment or functional group of a molecule or compound.

[0033] As used herein, the term "subject" refers to any mammal, including both human and non-human mammals.

[0034] 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. 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.

[0035] 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.

[0036] 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.

[0037] As used herein, "expression vector" refers to a vector used to introduce a specific nucleic acid, typically a DNA construct, into a target cell forexpression 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.

[0038] The term “oligonucleotide” refers to a short, single-stranded chain of nucleotides (either DNA or RNA), typically between 15 and 27 nucleotides, or between 19 and 25 nucleotides.

[0039] 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.

[0040] 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 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” encompasses also 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.

[0041] The term “nanoparticle” as used herein, refers to a particle with at least two dimensions at the nanometre 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” or“LNP” as used herein, refers to a nanoparticle whose external envelope is totally or partially made of lipids.

[0042] 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.

[0043] 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.

[0044] 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 lipid particle or lipid nanoparticle. Apparent pKa is different to the intrinsic pKa of any individual molecule, and it is an important parameter for the performance of lipid particles and nanoparticles encapsulating RNAs. The apparent pKa of lipid particles can be measured by different techniques known in the art. For example, acidbase titration and 2-(p-toluidino)-6-naphtalene sulfonic acid (TNS) fluorescent methods are widely used in the art. Lipid particles and LNPs with an optimum pKa carry negligible charges at physiological pH, which prevent nonspecific binding and toxicity in the body. The optimum pKa plays an important role in the endosomal escape mechanism and in the release of RNAs in the cytosol to exert therapeutic effect.

[0045] Lipid particle

[0046] As mentioned above, a first aspect of the invention refers to a lipid particle comprising an ionisable lipid, a neutral helper lipid, a lipid-polymer-conjugate or a pharmaceutically acceptable salt or stereoisomer or tautomer or isotopic variant of any one of them, wherein the lipid particle does not comprise cholesterol or sterol derivatives thereof, and wherein the lipid particle has a mean particle diameter or an average size below 300 nm. Preferably, the lipid particle has a mean particle diameter below 250 nm. More preferably, the lipid particle has a mean particle diameter of between 80 and 300 nm, more preferably, the lipid particle has a mean diameter of between 100 and 250 nm determined by dynamic light scattering.

[0047] Typically in solution, the lipid particle has a core-shell structure comprising an inner core and an external shell. Lipid formulations with several lipidic components of different nature comprise several phases that separate into a hydrophobic core region formed by the ionisable lipid and a surrounding shell formed by the neutral-helper lipid and a shielding or lipid-polymer conjugate covering the surface. The term “ionisable lipid” used herein refers to a lipidic molecule capable of modulating their charge depending on the environmental pH through any process by which electrically neutral atoms are converted to electrically charged through gaining or losing electrons.

[0048] The term “neutral helper lipid” used herein refers to a helper lipid or to a class of lipid molecules that increase particle stability and fluidity of lipid particles and LNPs and which have an overall net charge of zero. Examples of neutral helper 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 (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2-di-0-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 Lyso 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 neutral helper lipid is DOPE; and / or wherein the neutral helper lipid is DSPC.

[0049] The term “lipid-polymer conjugate” used herein refers to a molecule where a lipid moiety is covalently attached to a polymer chain, either directly or through a linker, which combines the features of both and creates materials with enhanced properties such as biocompatibility. Lipidpolymer conjugates comprising hydrophilic polymeric moieties are also referred to as “stealth lipids” which have the ability to escape recognition and clearance by the immune system, particularly the reticuloendothelial system (RES). LNPs comprising lipid-polymer conjugates create a protective hydrated layer that prevents opsonin from binding (opsonization) and shields the particle from the immune system, achieving longer circulation times in the bloodstream. Nonlimiting examples of lipid-polymer conjugates include: polyethylene glycol (PEG), polyvinylmethylether, polyhydroxypropyl methacrylate, polymethyloxazoline, polyhydroxypropylmethacrylamide, polyhydroxyethyl acrylate, polyvinylpyrrolidone, polymethacrylamide, polydimethylacrylamide, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polyethyloxazoline, polyglycolic acid, polysarcosine and polyaspartamide.

[0050] The term “sterol derivatives” refers to a type of compound comprising a gonane or sterane skeleton. The terms “gonane” or “sterane” refer to polycyclic hydrocarbon compounds comprising 17 carbon atoms, although optionally they can comprise substituents. Examples of sterols include, without being limited to: cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof; further optionally, the sterol is cholesterol.

[0051] The term “pharmaceutically acceptable salts” used herein encompasses any salt formed from pharmaceutically acceptable non-toxic acids including inorganic or organic acids.

[0052] 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 ionisable 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 ionisable 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.

[0053] Non-limiting 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 ionisable 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.

[0054] The ionisable lipids of the present disclosure may have, at least, one asymmetric centre and, therefore, can give rise to various stereoisomers. As used herein, the term "stereoisomer" refers to all isomers of individual ionisable 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.

[0055] The preparation processes are known in the art and / or commercially available; the preparation processes 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 centres 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.

[0056] The term “tautomer” as used herein refers to structural isomers or constitutional isomers that readily interconvert each other. In other words, a single chemical compound tends to exist in two or more interconvertible structures that differ in the relative position of one atomic nucleus, which is usually hydrogen.

[0057] The term “isotopic variant” as used herein refers to distinct nuclear species of the same chemical element with the same number of protons (same atomic number) but that differ in different number of neutrons in the nuclei (different mass number) and physical properties. 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.

[0058] In all embodiments of the invention referring to the ionisable lipid, neutral helper lipid and lipidpolymer conjugate comprised in the lipid particle of the present disclosure, the pharmaceutically acceptable salts, stereoisomers, tautomers or isotopic variants are always contemplated even if they are not specifically mentioned.

[0059] An embodiment, optionally in combination with one or more features described before, refers to the amount of ionisable lipid in the lipid particle being equal to or between 34.5 mol% and 55.5 mol%, preferably being equal to or between 35 mol% and 53 mol%, more preferably being equal to or between 36.5 mol% and 51.5 mol%, even more preferably being equal to or between 37 mol% and 50 mol%; the amount of lipid-polymer conjugate in the lipid particle being equal to or between 0.45 mol% and 1.6 mol%, more preferably being equal to or between 0.48 mol% and 1 .5 mol%; and the amount of neutral helper lipid in the lipid particle being equal to or between 44.05 mol% and 65.05 mol%, preferably being equal to or between 45 mol% and 64 mol%, more preferably being equal to or 46 mol% and 63.5 mol%; more preferably being equal to or between 48 mol% and 63 mol%, even more preferably being equal to or between 49 mol% and 62.5 mol% of the total number of moles in the lipid particle.

[0060] As used herein, "mol%" refers to a component's molar percentage relative to the total moles of all lipid components in the lipid particle (i.e. , total moles, for example of ionisable lipid, neutral helper lipid and lipid-polymer conjugate).

[0061] An embodiment, optionally in combination with one or more features described previously, refers to the use of the lipid particle described above for extrahepatic passive targeting.

[0062] The term “passive targeting” refers to a method for passive diffusion to a specific organ, tissue or cell in which the external characteristics of the particle to be delivered are tuned in order to improve its specificity without the need for specific targeting agents, as opposed to active targeting which involves incorporating one or more targeting elements into the delivery vehicle to enhance its deliverability to the targeted organ, tissue or cell. Hence, passive targeting takes advantage of the physiological features of the targeted region, the capillarity and permeability of the vascular wall, which facilitates the accumulation in the specific targeted region and maximizes therapeutic impact.

[0063] In another embodiment, optionally in combination with one or more features described previously, the lipid particle of the present disclosure further comprises a pharmaceutically active agent for use in extrahepatic distribution of the pharmaceutically active agent to one or more tissues.

[0064] The term “pharmaceutically active agent” used, also commonly known as “active pharmaceutically ingredient” (API) or “pharmaceutically active ingredient” or ’’active principle” or “therapeutic ingredient”, herein refers to the chemical, biological, mineral or any other entity or component responsible for the therapeutic (pharmacological, physiological, physical, etc.) effects or any other direct effect important in the diagnosis, cure, prevention, mitigation, treatment or prevention of a disease condition in a subject, in particular an animal or a human, or that modifies the structure or function of the body in a product; pharmaceutically active ingredients are distinguishable from inactive pharmaceutical ingredients, known as excipients.

[0065] The term “extrahepatic distribution” or “extrahepatic delivery” used herein refers to the biodistribution of a molecule or a cargo encapsulated to a targeted organ or tissue outside the liver, liver cells or liver tissues. Biodistribution is described as the transfer of chemicals or biologies or any compounds of interest from one location to another within the body, particularly for therapy that is administered by any possible via to a subject (e.g. intramuscularly, intravenously, intranasally, orally, intraperitoneally, intradermally, subcutaneously, topically, intraarticularly) and that ideally accumulates in one or more particular site(s): tissues, cells, etc. The compound of interest is unloaded to expectedly produce an effect. Biodistribution and clearance are the key points of pharmacokinetics that maximise the therapeutic efficacy and minimise side effects. Generally, biodistribution is influenced by several parameters, non-limiting examples of which are: the composition of the lipid particle, morphology and size, coating, surface charge, physiological environment to which they are exposed to and route of administration. Elucidating biodistribution of an API is an essential step in preclinical studies in order to determine the final destination of an administered API.

[0066] Non limiting examples of extrahepatic tissues include lungs, spleen, intestines, pancreas, heart, kidneys, urethra, ureter, prostate, bladder, eye, ear, brain, mucosa, uterus, cervix, ovarian tissue and mammary tissue.

[0067] Optionally, the biodistribution of the lipid particle to the extrahepatic tissue is at least 60%, at least 65%, at least 70%, at least 72%, at least 75%, at least 77%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. Further optionally the extrahepatic tissue to which the lipid particle comprising a pharmaceutically active agent is distributed is the spleen.

[0068] In an embodiment, optionally in combination with one or more features described previously, the lipid particle described above comprises: an ionisable lipid selected from the group consisting of SM-102, VC-LC-1254, VC-LC-1405, VC-LC-1143 and VC-LC-0866, which are depicted in Table 1 below; and a neutral helper lipid 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-0-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 a more preferred embodiment, the neutral helper lipid is DOPE; and a lipid-polymer conjugate selected from a PEG-modified lipid, or an alternative non-immunogenic hydrophilic lipid-polymer conjugate; preferably the lipid-polymer conjugate is a PEG-modified lipid selected from the group consisting of 1 ,2-distearoyl-rac-glycero-3-methylpolyoxyethylene-2000 (DSG-PEG2000), 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-

[0069] PEG2000) and 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-sialic acid (DSPE-PEG2000-sialic acid); in a more preferred embodiment, the lipid-polymer conjugate is DSG-PEG2000.

[0070] Table 1

[0071] The ionisable lipids used in the present disclosure are either commercial (SM-102, CAS Number 2089251-47-6) or, in the case of VC-LC-0866, VC-LC-1143, VC-LC-1254 and VC-LC-1405, have been prepared following the procedures described in WO2024110381 (A1). All reactants and solvents needed for the preparation of the compounds of the present disclosure are commercially available.

[0072] Any person skilled in the art will know which reactants are required to obtain any particular ionisable lipid according to the present disclosure following the available procedures described in the literature or analogous methods thereof.

[0073] The term "PEG-modified lipid” used herein refers to a lipid comprising a polyethylene moiety or, alternatively, a hydrophilic polymer-lipid conjugate with similar structural characteristics which includes a lipid covalently attached to a polymer chain that is hydrophilic, preferably a polyethylene chain. 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.

[0074] The term “non-immunogenic” means that no immune response is triggered by an element or a composition comprising several components. Therefore, the element or the composition do not produce, are involved in or are related to an immune response or to the immune system.

[0075] The term “lipid-polymer conjugate” as used herein refers to lipid molecules that contain other functional groups covalently bonded. Hence, these molecules comprise a lipid moiety characterised by their hydrophobic properties and functional groups that are responsible for characteristic chemical properties. Therefore, a “lipid-polymer conjugate” mentioned here defines a lipid moiety covalently bonded to, at least, one hydrophilic polymer functional group. Addition of neutral, hydrophilic functional groups increases water solubility and bioavailability, prevents various bloodstream interactions and hence can extend the circulation lifetime of a lipid composition or a lipid particle containing thereof. Some non-limiting examples of suitable non- immunogenic hydrophilic conjugated lipids are polysarcosine (pSar), polysaccharides and polyvinylpyrrolidone (PVP), poly(amino acid) based lipopolymers (PAA-based lipopolimers) such as polyglutamic acid (PGA), poly(hydroxyethyl-1 -asparagine) (PHEA) or poly(hydroxyethyl-1- glutamine) (PHEG), biopolymers engineered from repeating amino acid sequences with high structural homogenicity such as XTEN, or poly(thioglycidyl glycerol) (PTTG).

[0076] In an embodiment, optionally in combination with one or more features previously described, the lipid particle comprises a pharmaceutically active agent being a nucleic acid selected from the group consisting of one or more oligonucleotide, polynucleotide or polypeptide; preferably one or more polynucleotide of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA); more preferably is a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide, or an expression vector comprising a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide, or a messenger RNA (mRNA) , a small interfering RNA (siRNA), a self-amplifying RNA (saRNA), a self-replicating RNA (srRNA), a single-stranded RNA (ssRNA), a short hairpin RNA (shRNA), an anti-sense oligonucleotide (ASO), a micro RNA (miRNA), a circular RNA (circRNA), or combinations thereof.

[0077] In a preferred embodiment, the pharmaceutically active agent is a natural or artificial polynucleotide of deoxyribonucleic acid (DNA) or a natural or artificial polynucleotide of ribonucleic acid (RNA), 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-thiou ridine (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, 6-methoxyuridine, 2'-O-methyl uridine and combinations thereof. In particular, the chemical modification is N1 -methylpseudouridine, 5-methoxyuridine ora combination thereof; particularly the chemical modification is N1 -methylpseudouridine.

[0078] In an embodiment, optionally in combination with one or more features previously described in the present disclosure the N / P ratio (mol / mol) of the lipid particle is selected from 3 to 7, preferably the N / P ratio is selected from 3.5 to 6.5, more preferably the N / P ratio is selected from 3, 3.3, 3.5, 3.8, 4, 4.3, 4.5, 4.8, 5, 5.3, 5.5, 5.8, 6, 6.3, 6.5, 6.8 to 7.

[0079] The N / P ratio in lipid nanoparticles is a parameter that defines the molar ratio of amine groups (N) in the ionizable lipid to the phosphate groups (P) in the nucleic acid cargo (DNA or RNA).

[0080] In an embodiment, optionally in combination with one or more features previously described in the present disclosure the lipid particle is lyophilised. Pharmaceutical composition

[0081] As mentioned above, a second aspect of the invention refers to a pharmaceutical composition comprising the lipid particle as described above and one or more pharmaceutically acceptable excipient or carrier. More particularly, a pharmaceutical composition comprising the lipid particle as defined previously, a pharmaceutically active agent and, preferably, one or more pharmaceutically acceptable excipient or carrier, wherein the pharmaceutically active agent is partially or totally encapsulated by the lipid particle.

[0082] For the purposes of the present invention, pharmaceutically active agents include low molecular weight drugs, polynucleotides, peptides, antibodies, proteins, and combinations thereof.

[0083] 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, 6- methoxyuridine or a combination thereof.

[0084] The lipid particles 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.

[0085] Typically, the process for the preparation of lipid particles comprises: i) preparing a first alcoholic mixture comprising an ionisable lipid and, optionally, at least one lipid selected from the group consisting of a neutral helper, and a stealth 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 lipid particles.

[0086] 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. 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. Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, solvents, dispersion media, inert diluents, or other liquid vehicles, dispersing and / or granulating agents, disintegrating agents, suspension aids, surface active agents and / or emulsifiers, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, binding agents, lubricating agents, buffering agents, and / or oils 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. Excipients such as colouring agents, coating agents, sweetening, and flavouring agents can be present in the composition, according to the judgment of the formulator.

[0087] In a particular embodiment, optionally in combination with any of the embodiments provided above, the pharmaceutically active ingredient or pharmaceutically active agent comprised in the pharmaceutical composition described above is selected from the group consisting of one or more oligonucleotide, polynucleotide, or polypeptide. In a more preferred embodiment, it is selected from one or more polynucleotide of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In an even more preferred embodiment, it is selected from 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 or messenger RNA (mRNA), small-interfering RNA (siRNA), self-amplifying RNA (saRNA), self-replicating RNA (srRNA), single-stranded RNA (ssRNA), short hairpin RNA (shRNA), anti-sense oligonucleotides (ASOs), micro RNA (miRNA), circular RNA (circRNA), and combinations thereof.

[0088] The skilled person knows how to produce the oligonucleotide, polynucleotide, polypeptide, DNA, RNA, the DNA construct, or the expression vector, mRNA, siRNA, saRNA, srRNA, ssRNA, shRNA, ASOs, miRNA, or circRNA by routine methods well known in the art, for example, by chemical synthesis or by molecular biology techniques, without exercising any inventive skill.

[0089] In an embodiment, optionally in combination with one or more features described previously, the pharmaceutical active ingredient comprised in the pharmaceutical composition is a natural or an artificial deoxyribonucleic acid (DNA) or a natural or artificial ribonucleic acid (RNA); further optionally, the polynucleotide comprises at least one chemical modification selected from the group described above. In an embodiment, optionally in combination with one or more features described previously, the pharmaceutical composition is for use as a medicament.

[0090] In an embodiment, optionally in combination with one or more features described previously, the pharmaceutical composition is for use in treating, diagnosing or preventing a disease.

[0091] In another embodiment, optionally in combination with one or more features described previously, the pharmaceutical composition is to be used for delivery of a load, in particular to a non-liver cell or non-liver tissue. Preferably the load is one or more of therapeutic, prophylactic or diagnostic agents.

[0092] When the pharmaceutically active agent is a polynucleotide, the lipid particle 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.

[0093] As mentioned above, another aspect of the invention refers to a method for targeted delivery of a pharmaceutically active agent to a non-liver organ or a non-liver cell in a subject in need thereof, wherein the method comprises administering to said subject said pharmaceutically agent assembled with a lipid particle as defined previously, further optionally, the subject is a human.

[0094] In an embodiment, optionally in combination with one or more features described previously, the non-liver organ or non-liver cell of the method described above is the spleen or a spleen cell.

[0095] In an embodiment, optionally in combination with one or more features described previously, the lipid particle or the pharmaceutical composition previously described are lyophilised.

[0096] Another aspect refers to a pharmaceutical composition comprising the lipid particle as previously defined, for use in treating a disease or disorder in a subject in need thereof; non-limiting examples of diseases or disorders are infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases.

[0097] Another aspect of the invention refers to the use of the lipid particle previously described as an encapsulation agent.

[0098] The term “encapsulation agent” used herein refers to an agent capable of enclosing or entrapping something (e.g. a pharmaceutically active agent) in or as if in a capsule or an envelope.

[0099] In an embodiment, optionally in combination with one or more features described previously, the lipid particle described above has a ionisable lipid to RNA ratio (N / P) that ranges from 3:1 to 11 :1. In a more preferred embodiment the (N / P) ratio ranges from 3.2:1 to 7:1. In an even more preferred embodiment, the (N / P) ratio ranges from 3.3:1 to 3.6:1 .

[0100] In an embodiment, optionally in combination with one or more features described previously, the pharmaceutical composition described above is a vaccine which optionally comprises an adjuvant.

[0101] As used herein, the term “adjuvant” refers to a substance, or a combination of substances, which increases or modifies the activity, potency or effects of certain pharmaceutically active agents. Adjuvants are also commonly included in vaccine compositions to enhance the immune system of the subject receiving a vaccine.

[0102] Another embodiment, optionally in combination with one or more features described previously, refers to the lipid particle or the pharmaceutical composition described above 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 a localised response in the spleen, the method comprising administering to the subject a therapeutically effective amount of the lipid particle or of the pharmaceutical composition as defined herein; optionally, the disease or disorder is a spleen disease, a spleen disorder or a spleen related condition; further optionally, the subject is a human or animal.

[0103] 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.

[0104] 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 particles 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 lipid particle 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.

[0105] The skilled in the art would know, depending on the intended use of the composition, which lipid particle use to encapsulate each pharmaceutically active agent. In particular, methods for the synthesis of the compositions formed by the lipid particles 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.

[0106] The skilled person would know, based on its common general knowledge, which excipients, carriers, and adjuvants to include in the composition depending on the intended use.

[0107] 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’.

[0108] 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.

[0109] Examples

[0110] Reagents were purchased from Sigma-Aldrich, Merck, Cayman Chemical, Avanti Polar Lipids, Corden Pharma, Thermo Fischer Scientific, Kamulinbiotechco TCI Chemicals, Fluorochem, Trilink Biotechnologies, Panreac AppliChem or VWR International Eurolab. All possible wise combinations shown previously were prepared following a three-step reaction protocol.

[0111] Example 1 - Preparation and characterisation of 3-component lipid particles of the present disclosure.

[0112] RNA encapsulation into LNPs

[0113] Encapsulation of mRNA comprising in the 5’ to 3’ direction a sequence encoding for luciferase of SEQ ID NO:1 (CleanCap® FLuc mRNA (L-7602), Trilink Biotechnologies) into LNPs was performed either by microfluidics or, alternatively, by manual means.

[0114] For example, 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 ionisable lipid of the inventiomhelper lipid (DOPE, Merck. 850725P-200MG):lipid-polymer conjugate (DSPE-PEG2K-sialic acid, Kamulinbiotechco. LTD R-G-008 or DSG-PEG2000, Merck 880152P-1 G) were dissolved into ethanol at the respective molar percentages of 39:60:1 or 39:59,5:1 ,5, and with a lipid-nitrogen-to-phosphate ratio (N:P) mol ratio of 3,5:1.

[0115] Alternatively, when the LNPs were obtained by manual methods, e.g. by manual / bulk mixing as disclosed in Wang X., 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), the mRNA aqueous solution was carefully added to the ethanol solution, and the resulted solution was homogenised 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™ RNA Assay Kit (Thermo Fisher Scientific R11490) or AGE (Agarose DNA Grade Electran® (100g), VWR International VWRC438792U; SYBR Safe DNA Gel Stain, Thermo Fisher S33102; Gel Loading Buffer II, Thermo Fisher AM8547; TBE (10X) RNase-free, Thermo Fisher AM9863), using a FLUOstar microplate Multimode Reader (BIOGEN CIENTIFICA B-415.101) or an electrophoresis system (Bio-Rad Laboratories, 1640302) following the manufacturer’s instructions.

[0116] 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.

[0117] Finally, the LNP solution was passed through a 0,22 mm filter and the LNPs were stored at -80 °C until required.

[0118] Sialic acids (SA) are a class of alpha-keto acid sugars comprising a nine-carbon backbone, with the most common being N-acetylneuraminic acid, present in the cell surface of some prokaryotes as well as eukaryotes. It is the type of sugar most prevalent found on the surface of mammalian cells, in tissues and organs such as in the human brain. They are normally at the end of sugar chains at the surface of cells or soluble proteins and possess a negative charge which could enhance spleen tropism; moreover, specific SA-receptors exist in organs such as liver or spleen. Hence two formulations were compared side by side comprising 3 components: an ionisable lipid, a neutral helper and a either a (SA) / lipid-polymer conjugate or a lipid-polymer conjugate without SA.

[0119] Table 2 provides the characterisation of several parameters for lipid particles comprising VC-LC- 1254 as the ionisable lipid, DOPE as neutral helper lipid and either DSG-PEG 2000 (Merck Life Science S.L.U. 880152P-1G) or DSPE-PEG-2000 sialic acid (Kamulinbiotechco LTD. R-G-008) as lipid-polymer conjugate:

[0120] Table 2

[0121] As shown in Table 2, each of the lipid particles tested obtained acceptable values in diameter for the purpose sought. The encapsulation efficiency of the lipid particles, measured as the percentage of RNA entrapped in the LNP, ranged from approximately 95% to approximately 93%. Noteworthy, 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.

[0122] SEQ ID. NO. 1 :

[0123] 1 AUGGAGGACG CCAAGAACAU CAAGAAGGGC CCCGCCCCCU UCUACCCCCU GGAGGACGGC ACCGCCGGCG AGCAGCUGCA CAAGGCCAUG AAGCGGUACG 101 CCCUGGUGCC CGGCACCAUC GCCUUCACCG ACGCCCACAU CGAGGUGGAC AUCACCUACG CCGAGUACUU CGAGAUGAGC GUGCGGCUGG CCGAGGCCAU 201 GAAGCGGUAC GGCCUGAACA CCAACCACCG GAUCGUGGUG UGCAGCGAGA ACAGCCUGCA GUUCUUCAUG CCCGUGCUGG GCGCCCUGUU CAUCGGCGUG 301 GCCGUGGCCC CCGCCAACGA CAUCUACAAC GAGCGGGAGC UGCUGAACAG CAUGGGCAUC AGCCAGCCCA CCGUGGUGUU CGUGAGCAAG AAGGGCCUGC 401 AGAAGAUCCU GAACGUGCAG AAGAAGCUGC CCAUCAUCCA GAAGAUCAUC AUCAUGGACA GCAAGACCGA CUACCAGGGC UUCCAGAGCA UGUACACCUU 501 CGUGACCAGC CACCUGCCCC CCGGCUUCAA CGAGUACGAC UUCGUGCCCG AGAGCUUCGA CCGGGACAAG ACCAUCGCCC UGAUCAUGAA CAGCAGCGGC 601 AGCACCGGCC UGCCCAAGGG CGUGGCCCUG CCCCACCGGA CCGCCUGCGU GCGGUUCAGC CACGCCCGGG ACCCCAUCUU CGGCAACCAG AUCAUCCCCG 701 ACACCGCCAU CCUGAGCGUG GUGCCCUUCC ACCACGGCUU CGGCAUGUUC ACCACCCUGG GCUACCUGAU CUGCGGCUUC CGGGUGGUGC UGAUGUACCG 801 GUUCGAGGAG GAGCUGUUCC UGCGGAGCCU GCAGGACUAC AAGAUCCAGA GCGCCCUGCU GGUGCCCACC CUGUUCAGCU UCUUCGCCAA GAGCACCCUG 901 AUCGACAAGU ACGACCUGAG CAACCUGCAC GAGAUCGCCA GCGGCGGCGC CCCCCUGAGC AAGGAGGUGG GCGAGGCCGU GGCCAAGCGG UUCCACCUGC 1001 CCGGCAUCCG GCAGGGCUAC GGCCUGACCG AGACCACCAG CGCCAUCCUG AUCACCCCCG AGGGCGACGA CAAGCCCGGC GCCGUGGGCA AGGUGGUGCC 1101 CUUCUUCGAG GCCAAGGUGG UGGACCUGGA CACCGGCAAG ACCCUGGGCG UGAACCAGCG GGGCGAGCUG UGCGUGCGGG GCCCCAUGAU CAUGAGCGGC 1201 UACGUGAACA ACCCCGAGGC CACCAACGCC CUGAUCGACA AGGACGGCUG GCUGCACAGC GGCGACAUCG CCUACUGGGA CGAGGACGAG CACUUCUUCA

[0124] 1301 UCGUGGACCG GCUGAAGAGC CUGAUCAAGU ACAAGGGCUA CCAGGUGGCC CCCGCCGAGC UGGAGAGCAU CCUGCUGCAG CACCCCAACA UCUUCGACGC

[0125] 1401 CGGCGUGGCC GGCCUGCCCG ACGACGACGC CGGCGAGCUG CCCGCCGCCG UGGUGGUGCU GGAGCACGGC AAGACCAUGA CCGAGAAGGA GAUCGUGGAC

[0126] 1501 UACGUGGCCA GCCAGGUGAC CACCGCCAAG AAGCUGCGGG GCGGCGUGGU GUUCGUGGAC GAGGUGCCCA AGGGCCUGAC CGGCAAGCUG GACGCCCGGA

[0127] 1601 AGAUCCGGGA GAUCCUGAUC AAGGCCAAGA AGGGCGGCAA GAUCGCCGUG UGA

[0128] Example 2 - Biodistribution and protein expression in vivo in mice administered with selected examples of lipid particles of the present invention and containing RNA as active agent.

[0129] Once characterised, the LNPs were tested in vivo in mice to measure their performance and transfection efficiency, as well as biodistribution.

[0130] Administration of the mRNA (LNP) to mice

[0131] 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.

[0132] For Firefly Luciferase activity measurement in mice, LNPs produced as indicated above (ionisable lipidmeutral helper lipidlipid-polymer conjugate at molar ratios 39:60:1 or 39:59,5:1 ,5, with an N / P ratio of 3,5:1 (mokmol) in all cases, and containing 10 pg of the indicated mRNA in 250 pl final volume, were inoculated intravenously via tail vein injection.

[0133] 4 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 potassium salt (DISMED LUC K-250) was injected intraperitoneally at 150 mg / kg, normally ~200 pL ofthe stock at 15 mg / mL in PBS for a 20 g mouse. Mice were euthanized 20 minutes after luciferin injection by cervical dislocation. Liver, spleen, lungs, heart, kidneys and intestine were aseptically removed and ex vivo luciferase images were acquired using the I VIS Lumina XRMS Imaging System following manufacturer's instructions.

[0134] The intracellular transfection efficiency of the mRNA (encoding for the expression of luciferase) is measured as luminescence given as p s-1. The values of luminescence 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 of cells after the endosomal escape, producing protein expression in mice administered intravenously with the selected examples. The results are summarised in Table 3 below.

[0135] Table 3

[0136] Table 3 shows that the three LNPs, comprising the same ionizable lipid and neutral helper, and differing in the shielding lipid being either DSG-PEG or DSPE-PEG2000-sialic acid, efficiently produced cellular transfection after intravenous administration to mice. All of them showed passive tropism to the spleen with luminescence values (measured as total flux) over or very close to E+08 and between 93% and 74% expression in the spleen. Contrary to the expectations, the two LNPs comprising DSPE-PEG2000-sialic acid as the shielding lipid performed slightly worse than the LNPs using DSG-PEG as the shielding lipid that obtained the best results. Hence, it was decided to not use sialic acid conjugated lipids in the rest of the experiments. Example 3 - Characterisation and biodistribution of 3-component lipid particles with different ionisable lipids and molar ratios

[0137] Lipid particles with a constant N / P ratio of 3,34 or 3,5 (mokmol) and comprising VC-LC-1254, VC- LC-1405, VC-LC-0866, VC-LC-1143 or SM-102 as the ionisable lipid, DOPE as neutral helper lipid and DSG-PEG as lipid-polymer conjugate in variable molar ratios, as shown in Table 4 below, were characterized.

[0138] Table 4

[0139] Table 4 shows the measured values for particle size (diameter) which were mostly comprised between 118 and 138 nm although, when the molar ratio of neutral helper lipid was increased from 60 mol% to 62,21 mol% (in LNP-5, compared to LNP-4), the diameter doubled the size up to 242 nm; polydispersity (PDI) between 0,006 and 0,25; negative values between 0.5 and 4 for Z potential, and pKa values between 6.94 and 7.85 in solution.

[0140] After characterisation, the lipid particles were tested in vivo (following the same procedure previously described in Example 2). Hence, they were administered intravenously to mice; liver, spleen, lungs, heart, kidneys and intestine were aseptically removed to acquire ex vivo luciferase images as described previously in order to prove their ability to target extrahepatic tissues. The results corresponding to luminescence in p s-1and percentage of distribution to each of the organs are summarised below in Table 5. Table 5

[0141] As shown in Table 5, the tested 3-component lipid particles LNP-1 to LNP-7 effectively transfected extrahepatic tissues; in particular, these lipid particles targeted mostly the spleen, with percentages ranging between 71 % and 93%, whereas the percentages in the liver ranged between 2.4% and 19%, followed by percentages in the lungs between 0.5% and 6.3% and only residual targeting to kidneys, intestine or heart. Hence, passive targeting to the spleen is shown to be very superior to other tissues in in vivo experiments.

[0142] On top of that, lipid particles comprising 4-components (ionisable lipid:helper lipid:cholesterol:polymer-lipid conjugate) in molar ratios 50:10:38,5:1 ,5 (the gold standard for LNPs present in pharmaceutical compositions such as Onpattro) and N / P=6 (mokmol), particularly VC-LC-1254 or VC-LC-0866 as ionisable lipids, DOPE as helper, cholesterol and DMG-PEG2000 were tested in vivo in mice following the same procedure previously described. The results are summarized in table 6 below.

[0143] Table 6

[0144] The results summarized in table 6 prove that the selectivity to extrahepatic cells or tissues depends on the lipid particle composition (3 or 4 components) and the particular molar ratios, since the same lipids employed for 3-component cholesterol-less formulations targeted extrahepatic tissues (e.g. spleen), whereas in 4-component formulations the lipid particles targeted the liver.

[0145] Example 4 - Effect of N / P ratio in 3-component lipid particles in extrahepatic delivery

[0146] It was investigated whether varying N / P ratios in the 3-component formulations of the present disclosure could have any effect in the delivery. LNPs comprising SM-102, VC-LC-1254, VC-LC- 1405, VC-LC-0866 and VC-LC-1143 as the ionizable lipids, were formulated with DOPE as the neutral helper lipid and DSG-PEG2000 as the lipid-polymer conjugate in a molar ratio of 37,21 :62,21 :0,48. The same procedure previously described in Example 2 of the present disclosure was followed. The results are summarized below in Table 7.

[0147] Table 7

[0148] The results were quite variable, yet it must be considered that in vivo experiments imply different conditions and particularities between the individuals along the study. Observed biodistribution to the spleen fluctuated from 60% to 93%, hence the specific delivery to splenic tissue / cells proved to be still very efficient despite the N / P ratio.

[0149] Example 5 - Comparative example of transfection efficiency and ability of targeting specific tissues of 3-component lipid particles comprising different helper lipids.

[0150] In the light of a recent publication by Su et al. it has to be considered whether tropism could be attributable to the nature of the different ionizable lipids, the nature and physicochemical properties of the helper lipid or the molar ratios of the components in the formulated lipid particles. The published study compared the 5-component lung-targeting strategy (comprising an ionisable lipid, a permanently cationic lipid, a phospholipid, cholesterol and a PEG-lipid, also known as SORT), a so-called “veritable targeting” comprising 4 components (including cholesterol and a phospholipid) and a 3-component composition comprising an ionisable lipid, a permanently cationic lipid (DOTAP) and a PEGylated lipid (DMG-PEG2000). The authors stated that cholesterol and phospholipid are dispensable for LNP functionality, and that non-cholesterol containing compositions prevent the lipid particles from accumulating in hepatic tissues. They found a certain 3-component formulation which enhanced pulmonary delivery that was tested for different ionizable lipids (including commercial lipids SM-102, ALC-0315 and MC3) as well as permanently cationic helper lipids (DOTAP and DDAB).

[0151] Hence, an experiment was designed to elucidate, to some extent, whether the tropism could be attributable to the ionisable lipid nature, the presence or absence of permanently cationic helper lipids, or the molar ratios of the 3 components. Since SM-102 showed good results in both approaches, it was the first ionisable lipid of choice to compare the rest of the variables, and VC- LC-1254 was also included as the second one. The tested compositions, summarised in Table 8, were injected intravenously in mice (following the same procedure previously described in Example 2), observing high transfection efficiency related to the luminescence measured as the total flux (p s-1)and specific targeting to lungs for LNPs comprising a cationic helper lipid (DOTAP), whereas LNPs comprising a neutral helper lipid (DOPE) showed high transfection efficiency and specific targeting to the spleen.

[0152] Table 8

[0153] Therefore, according to the experimental results shown in Table 8, when the permanently cationic lipid DOTAP is switched by the neutral phospholipid DOPE, the spleen is the targeted organ instead of the lungs. Indeed, it has been reported many times that including a positively charged lipid increases expression in the lungs. However, the present 3-component composition achieves selective targeting in the spleen just by substituting the cationic lipid with a neutral phospholipid.

[0154] Example 6 - Comparative example of transfection efficiency and selectivity ability of targeting specific tissues of 3-component lipid particles comprising different ionisable lipids and N / P ratios in in vivo experiments in mice.

[0155] In view of document CN117430541 B by Shenzhen Bay Laboratory, which discloses ionizable lipids with a guanidine-substituted five-membered heterocycles assembled in LNPs to achieve stable and efficient delivery, and to provide three-component cholesterol-free LNPs. Table 9 below shows a comparison of the performance of LNPs formulated with lipids VC-LC-0866 and VC-LC-1254 in a composition of the present disclosure (ionisable lipid:helper lipidJipid-polymer conjugate with molar ratios 37,31 :62,2:0,48), lipid VC-LC-0866 in two compositions disclosed in document CN117430541 B comprising ionisable lipid:helper lipidJipid-polymer conjugate in molar ratios 55,9:43,48:0,62 and 72,84:26,49:0,66 and lipid GaC18-2 of CN117430541 B but in the composition disclosed in the present document (ionisable lipid: helper lipidJipid-polymer conjugate with molar ratios 37,31 :62, 2:0, 48).

[0156] The LNPs were tested in vivo following the same protocol previously described in Example 2: administered intravenously to mice, then liver, spleen, lungs, heart, kidneys and intestine were aseptically removed to acquire ex vivo luminescence (luciferase) images in order to prove their ability to target extrahepatic tissues. The results corresponding to luminescence and percentage of distribution to each of the organs are summarised below in Table 9. Table 9

[0157] As it is apparent from the results shown in Table 9, luciferase expression was significantly high, reaching values as high as 2.02E+08 photons / second in the spleen with the formulation disclosed in the present disclosure. In contrast, CN117430541 B by Shenzhen Bay Laboratory, which only showed in vitro results for HeLa cells, could not prove selectivity in distribution to organs in vivo. In 3-component cholesterol-less formulations with GaCis-2 and MaCis-i experiments, the luminescence values shown in Figures 5 and 12 was less than 1.0E+05, less than 5.0E+04 and less than 3.0E+05. The intensity of luminescence (therefore, transfection rate) appears to be higher due to the nature of the ionisable lipid, resulting in two to three orders of magnitude higher for VC-LC- 0866 and VC-LC-1254 compared to GaCis-2.

[0158] It is also observable that the selectivity to non-hepatic cells or tissues is greater for the formulations disclosed herein than for those disclosed in CN117430541 B.

[0159] Indeed, due to the fact that the formulations cited in CN117430541 B showed high N / P ratios, VC-LC-0866 was tested in different N / P ratios of 6, 9,87 and 12,93 (mokmol) in order to determine if it could have an impact in transfection rates, protein expression and / or selectivity towards non-hepatic cells or tissues. It seems that higher N / P ratios could have an impact in selectivity depending on the nature of the ionisable lipid as well as on the molar ratios of the lipids in the formulation.

[0160] Example 7 - Lyophilised formulations and storage conditions of 3-component compositions for extrahepatic delivery.

[0161] Lyophilisation of the lipid particles can be performed by standard methods known in the art by a skilled person. For instance, a helpful protocol is described in Muramatsu et al. (“Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine” Mol Ther. 2022 May 4;30(5): 1941 -1951 ).

[0162] The 3-component lipid particles of the present disclosure were lyophilised and stored for 10 weeks at T=4 °C. Three different ionisable lipids were tested, keeping the molar ratios and N / P ratio (3,34:1 , mokmol) constant. Physical properties were measured to ensure all the parameters were still appropriate fortheir use in vivo. The measurements are summarised below in Table 10.

[0163] Table 10 As shown in Table 10 the physical properties of the lyophilised and reconstituted LNPs were appropriate for in vivo experiments.

[0164] Therapeutically suitable compositions comprising the reconstituted lyophilised-LNPs were produced by adding 300 pL of ultrapure Milli-Q water to lyophilised LNPs comprising a total amount of 30 pg of mRNA, and were injected intraperitoneally in mice as previously indicated.

[0165] In all cases, the LNPs comprised ionisable lipid (IL), DOPE as the helper lipid and DSG.PEG as the lipid-polymer conjugate in molar ratios of 37,31 / 62,21 / 0,48.

[0166] The organs were aseptically removed, and the luminescence, measured as total flux (p.s-1) and percentage distribution in each organ (liver, kidney, lung, heart, intestine and spleen) are shown in Table 11 below.

[0167] Table 11

[0168] Although variability is usually high in in vivo experiments (due to unique conditions in each of the individuals tested), all of the reconstituted lyophiles achieved good luminescence values as shown in Table 11 , and distribution to the spleen varied between 74% and 96%.

[0169] From the values displayed in Table 11 , it can be concluded that the extrahepatic delivery to this particular tissue proved to be very efficient for lyophilised lipid particles that were stored for 10 weeks at T=4 °C, very gentle conditions considering that other commercial compositions comprising similar LNPs require very low conservation temperatures, sometimes as cold as -80 °C.

[0170] The invention comprises the following clauses:

[0171] 1 . A lipid particle comprising:

[0172] -an ionisable lipid;

[0173] -a neutral helper lipid;

[0174] -a stealth lipid; or a pharmaceutically acceptable salt or stereoisomer or tautomer or isotopic variant of any one of them; wherein the lipid particle has an average particle diameter below 250 nm.

[0175] 2. The lipid particle according to clause 1 for extrahepatic passive targeting.

[0176] 3. The lipid particle according to any of clauses 1 or 2 wherein the lipid particle does not comprise cholesterol or sterol derivatives .

[0177] 4. The lipid particle according to any of clauses 1 to 3 consisting essentially of: -an ionisable lipid;

[0178] -a neutral helper lipid;

[0179] -a stealth lipid; wherein the amount of ionisable lipid in the lipid particle is equal or between 34.5 mol% and

[0180] 55.5 mol%, preferably is equal or between 35 mol% and 53 mol%, more preferably is equal or between 36.5 mol% and 51.5 mol%, even more preferably is equal or between 37 mol% and 50 mol%; the amount of stealth lipid in the lipid particle is equal or between 0.45 mol% and 1.6 mol%, more preferably is equal or between 0.48 mol% and 1 .5 mol%; and the amount of neutral helper lipid in the lipid particle is between 44.05 mol% and 65.05 mol%, preferably is between 45 mol% and 64 mol%, more preferably is between 46 mol% and 63.5 mol%, more preferably is between 48 mol% and 63 mol%, even more preferably is between 49 mol% and

[0181] 62.5 mol% of the total number of moles in the lipid particle.

[0182] 5. The lipid particle according to any of clauses 1 to 3, further comprising a pharmaceutically active agent for use in extrahepatic biodistribution of said pharmaceutically active agent to one or more extrahepatic tissues, further optionally wherein the biodistribution of the lipid particle to the extrahepatic tissue is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and further optionally wherein said extrahepatic tissue is the spleen. The lipid particle according to any of clauses 1 to 5, wherein the ionisable lipid is selected from the group consisting of wherein the neutral helper is selected from the group consisting of:

[0183] 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-0-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; preferably, the neutral helper lipid is DOPE; and wherein the stealth lipid is selected from a PEG-modified lipid, or a non-immunogenic hydrophilic conjugate lipid; preferably the stealth lipid is a PEG-modified lipid selected from the group consisting of 1 ,2-distearoyl-rac-glycero-3-methylpolyoxyethylene-2000 (DSG-PEG2000),

[0184] 1 .2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) and 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-sialic acid (DSPE-PEG2000-sialic acid), more preferably the stealth lipid is DSG-PEG2000.

[0185] 7. A pharmaceutical composition comprising:

[0186] -the lipid particle according to any of clauses 1 to 6; and

[0187] -a pharmaceutically active ingredient; and

[0188] -preferably, one or more pharmaceutically acceptable excipient or carrier, wherein the pharmaceutically active ingredient is partially or totally encapsulated by the lipid particle.

[0189] 8. The pharmaceutical composition according to clause 7, wherein the pharmaceutically active ingredient is a therapeutic nucleic acid selected from the group consisting of one or more oligonucleotide, polynucleotide or polypeptide; preferably one or more polynucleotide of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA); more preferably 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, messenger RNA (mRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), self-replicating RNA (srRNA), single-stranded RNA (ssRNA), short hairpin RNA (shRNA), anti-sense oligonucleotides (ASOs), micro RNA (miRNA), circular RNA (circRNA), and combinations thereof.

[0190] 9. The pharmaceutical composition according to any of clauses 7 or 8 wherein the pharmaceutically active ingredient is a natural or 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'-thiou ridine , 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.

[0191] 10. The pharmaceutical composition according to clauses 7 to 9 for use as a medicament.

[0192] 11. The pharmaceutical composition according to clauses 7 to 10 for use in treating, diagnosing or preventing a disease.

[0193] 12. The pharmaceutical composition according to any of clauses 7 to 11 for use in delivering a load, wherein the load is one or more of therapeutic, prophylactic or diagnostic agents.

[0194] 13. A method for targeted delivery of a therapeutic agent to a non-liver organ or a non-liver cell therein in a subject in need thereof, the method comprising administering to said subject said therapeutic agent assembled with a lipid composition comprising an ionisable lipid, a neutral helper lipid and a stealth lipid, further optionally, wherein the subject is a human.

[0195] 14. The method of clause 13 wherein said non-liver organ is the spleen, or wherein said non- liver cell is a spleen cell. 15. The lipid particle according to any of clauses 1 to 6 or the pharmaceutical composition according to any of claims 7 to 12 wherein the lipid particle or the pharmaceutical composition are lyophilised.

[0196] 16. A pharmaceutical composition according to any of clauses 7 to 12 for use in the treatment of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno- vascular diseases and metabolic diseases.

[0197] 17. Use of the lipid particle according to clauses 1 to 6 as an encapsulation agent.

[0198] 18. The lipid particle according to clauses 1 to 6, wherein the ionisable lipid to RNA ratio (N / P) ranges from 3:1 to 11 :1 , particularly from 3.5:1 to 7:1 , preferably from 3.5:1 to 5:1 .

[0199] 19. The pharmaceutical composition according to clauses 7 to 12 and 16 which is a vaccine, optionally, further comprising an adjuvant.

[0200] 20. A lipid particle as defined in any one of clauses 1 to 6, or a pharmaceutical composition as defined in clauses 7 to 12 and 16, 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 lipid particle composition or of the pharmaceutical composition; optionally, wherein the disease or disorder is selected from a spleen disease, a spleen disorder, a spleen related condition; 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, more preferably for use in a method of inducing a localised response in the spleen.

[0201] Citation List Cheng Q, Wei T, Farbiak L, Johnson LT, Dilliard SA, Siegwart DJ. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat Nanotechnol. 2020 Apr;15(4):313-320. doi: 10.1038 / s41565-020-0669-6. Epub 2020 Apr 6. PMID: 32251383; PMCID: PMC7735425. LoPresti ST, Arral ML, Chaudhary N, Whitehead KA. The replacement of helper lipids with charged alternatives in lipid nanoparticles facilitates targeted mRNA delivery to the spleen and lungs. J Control Release. 2022 May;345:819-831 ; doi: 10.1016 / j.jconrel.2022.03.046. Epub 2022 Mar 26. PMID: 35346768; PMCID: PMC9447088. Timothy R. Blake, Ole A. W. Haabeth, Adrienne Sallets, Rebecca L. McClellan, Trevor J. Del Castillo, Jose G. Vilches-Moure, Wilson C. Ho, Paul A. Wender, Ronald Levy, Robert M. Waymouth Lysine-Derived Charge-Altering Releasable Transporters: Targeted Delivery of mRNA and siRNA to the Lungs. Bioconjugate Chem. 2023, 34, 4, 673-685; doi.org / 10.1021 / acs.bioconjchem.3c00019.

[0202] 4. James C. Kaczmarek, Kevin J. Kauffman, Owen S. Fenton, Kaitlyn Sadtler, Asha K. Patel, Michael W. Heartlein, Frank DeRosa, Daniel G. Anderson Optimization of a Degradable Polymer-Lipid Nanoparticle for Potent Systemic Delivery of mRNA to the Lung Endothelium and Immune Cells. Nano Lett. 2018, 18, 10, 6449-6454; doi.org / 10.1021 / acs.nanolett.8b02917.

[0203] 5. Serena Omo-Lamai, Marco E. Zamora, Manthan N. Patel, Jichuan Wu, Jia Nong, Zhicheng Wang, Alina Peshkova, Aparajeeta Majumder, Jilian R. Melamed, Liam S. Chase, Eno-Obong Essien, Drew Weissman, Vladimir R. Muzykantov, Oscar A. Marcos-Contreras, Jacob W. Myerson, Jacob S. Brenner; Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions. Advanced Materials, Vol. 36, Issue 26, 2312026; doi.org / 10.1002 / adma.202312026.

[0204] 6. Su, K., Shi, L., Sheng, T. et al. Reformulating lipid nanoparticles for organ-targeted mRNA accumulation and translation. Nat Commun 15, 5659 (2024). doi.org / 10.1038 / s41467-024- 50093-7

[0205] 7. IONIZABLE LIPIDS AND LIPID NANOPARTICLES CONTAINING THEREOF, WO2024110381 (A1)

[0206] 8. Hassett KJ, Benenato KE, Jacquinet E, Lee A, Woods A, Yuzhakov O, Himansu S, Deterling J, Geilich BM, Ketova T, Mihai C, Lynn A, McFadyen I, Moore MJ, Senn JJ, Stanton MG, Almarsson O, Ciaramella G, Brito LA. Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines. Mol Ther Nucleic Acids. 2019 Apr 15; 15:1-11 . doi: 10.1016 / j.omtn.2019.01.013. Epub 2019 Feb 7. PMID: 30785039; PMCID: PMC6383180.

[0207] 9. 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. Nat Protoc 18, 265-291 (2023). https: / / doi.org / 10.1038 / s41596-022-00755-x

[0208] 10. Muramatsu H, Lam K, Bajusz C, Laczko D, Kariko K, Schreiner P, Martin A, Lutwyche P, Heyes J, Pardi N. Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine. Mol Ther. 2022 May 4;30(5): 1941 -1951 . doi: 10.1016 / j.ymthe.2022.02.001. Epub 2022 Feb 4. PMID: 35131437; PMCID: PMC8815268.

[0209] 11. CN117430541 B Shenzhen Bay Laboratory

Claims

CLAIMS1 . A lipid particle comprising:-an ionisable lipid;-a neutral helper lipid;-a lipid-polymer conjugate; or a pharmaceutically acceptable salt or stereoisomer or tautomer or isotopic variant of any one of them, and wherein the lipid particle does not comprise cholesterol or sterol derivatives thereof, and wherein the lipid particle has a mean particle diameter between 80 and 300 nm, preferably between 100 and 250 nm, as determined by dynamic light scattering.

2. The lipid particle according to claim 1 wherein the amount of ionisable lipid in the lipid particle is equal to or between 34.5 mol% and55.5 mol%, preferably is equal to or between 35 mol% and 53 mol%, more preferably is equal to or between 36.5 mol% and 51 .5 mol%, even more preferably is equal to or between 37 mol% and 50 mol%; the amount of lipid-polymer conjugate in the lipid particle is equal to or between 0.45 mol% and 1 .6 mol%, more preferably is equal to or between 0.48 mol% and 1 .5 mol%; and the amount of neutral helper lipid in the lipid particle is equal to or between 44.05 mol% and 65.05 mol%, preferably is equal to or between 45 mol% and 64 mol%, more preferably is equal to or between 46 mol% and 63.5 mol%, more preferably is equal to or between 48 mol% and 63 mol%, even more preferably is equal to or between 49 mol% and62.5 mol% of the total number of moles in the lipid particle.

3. The lipid particle according to any of claims 1 to 2, wherein the ionisable lipid is selected from the group consisting ofwherein the neutral helper 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-0-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; preferably, the neutral helper lipid is DOPE; and wherein the lipid-polymer conjugate is selected from a PEG-modified lipid, or a non- immunogenic hydrophilic conjugate lipid; preferably the lipid-polymer conjugate is a PEG- modified lipid selected from the group consisting of 1 ,2-distearoyl-rac-glycero-3- methylpolyoxyethylene-2000 (DSG-PEG2000), 1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000) and 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-polyethylene glycol-sialic acid (DSPE-PEG2000-sialic acid), more preferably the lipid-polymer conjugate is DSG- PEG2000.

4. The lipid particle according to any of claims 1 to 3, further comprising a pharmaceutically active agent.

5. The lipid particle according to claim 4 wherein the pharmaceutically active agent is a nucleic acid selected from the group consisting of one or more oligonucleotide, polynucleotide or polypeptide; preferably one or more polynucleotide of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA); more preferably is a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide, or an expression vector comprising a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide, or a messenger RNA (mRNA) , a small interfering RNA (siRNA), a self-amplifying RNA (saRNA), a self-replicating RNA (srRNA), a single-stranded RNA (ssRNA), a short hairpin RNA (shRNA), an anti-sense oligonucleotide (ASO), a micro RNA (miRNA), a circular RNA (circRNA), or combinations thereof.

6. The lipid particle according to claim 5, wherein the polynucleotide is a natural or artificial deoxyribonucleic acid (DNA) or a natural or artificial ribonucleic acid (RNA), 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, 6-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.

7. The lipid particle according to any of claims 5 or 6 wherein the N / P ratio (mol / mol) is selected from 3 to 7, preferably the N / P ratio (mol / mol) is selected from 3.5 to 6.5.

8. The lipid particle according to any of claims 1 to 7 wherein the lipid particle is lyophilised.

9. A pharmaceutical composition comprising:-the lipid particle as defined in any of claims 1 to 8; and -one or more pharmaceutically acceptable excipient or carrier.

10. The pharmaceutical composition according to claim 9 for use as a medicament.11 . The pharmaceutical composition according to claim 9 for use in treating, diagnosing or preventing a disease.

12. The pharmaceutical composition according to claim 9 for use in delivering a load to a non-liver cell, wherein the load is one or more of therapeutic, prophylactic or diagnostic agents.

13. A method for targeted delivery of a pharmaceutically active agent to a non-liver organ or a nonliver cell therein in a subject in need thereof, the method comprising administering to said subject said pharmaceutically active agent assembled with a lipid particle according to any of claims 1 to 3, further optionally, wherein the subject is a human.

14. The method of claim 13 wherein said non-liver organ is the spleen, or wherein said non-liver cell is a spleen cell.

15. A pharmaceutical composition according to any of claims 9 to 12 for use in the treatment of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno- vascular diseases andmetabolic diseases.

16. Use of the lipid particle according to claims 1 to 3 as an encapsulation agent.

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