Lipid nanoparticles with nucleic acid load

Abstract

The present invention discloses a lipid nanoparticle (LNP) encapsulating a nucleic acid load, preferably comprising a messenger ribonucleic acid (mRNA). The LNP comprises at least a cationic lipid fraction and a stabilizer fraction. The stabilizer fraction preferably comprises at least one polyethylene glycol (PEG) lipid. Furthermore, the LNP comprises at least one dialkylglycerol tetraether (GDGT) lipid obtained from, for example, archaea of the genus Sulfolobus, and optionally other ether lipids. Also disclosed are pharmaceutical compositions, such as mRNA vaccines, comprising the LNP.

Description

Technical Field

[0001] The field of the present invention relates to lipid nanoparticles (LNPs) with nucleic acid cargo, in particular lipid nanoparticles for messenger RNA (mRNA) vaccines. Background Art

[0002] LNPs have recently come into the spotlight as LNP-based mRNA vaccines against SARS-CoV-2, primarily developed by Moderna Inc. Elasomeran, marketed by Biontech SE / Pfizer Inc. Tozinameran, marketed as a novel RNA vaccine, has been administered to hundreds of millions of people. Aldosari et al. (2021) reviewed LNPs in general as delivery systems for RNA-based vaccines. Hou et al. (2021) also discussed LNPs for mRNA delivery in detail.

[0003] In general, LNP with nucleic acid load (or useful load) comprises lipid layer and lipid and the microstructure domain of nucleic acid encapsulated.They have the median diameter between 10nm to 1000nm (for example, determined by dynamic light scattering (DLS)), and can adopt such as spherical or polyhedral shape.They can be multilayer, and this depends on their specific lipid composition.LNP comprises cationic lipid (particularly protonated lipid when low pH, i.e. when in endosome, also referred to as ionizable lipid).Usually, LNP also comprises stabilizer, such as polyethylene glycol (PEG) lipid that reduces LNP aggregation.In addition, LNP generally comprises other types of lipid (commonly referred to as " helper lipid "), such as phosphatidylcholine or phosphatidylethanolamine, to improve characteristic, such as delivering efficacy, tolerance or biodistribution.Finally, LNP can contain cholesterol or other sterols to regulate membrane integrity and rigidity.

[0004] LNPs are also disclosed in, for example, US Pat. Nos. 7,404,969, 8,058,069, 9,364,435 and 9,404,127.

[0005] WO 2017 / 099823 A1 discloses an accelerated-blood-clearance-insensitive LNP comprising a cationic lipid, a polyethylene glycol (PEG)-lipid, a sterol and a helper lipid, wherein the helper lipid does not contain phosphatidylcholine.

[0006] WO 2020 / 061284 A1 also relates to LNPs with PEG lipids.

[0007] WO 2020 / 219941 A1 discloses additional LNPs and formulations containing LNPs.

[0008] WO 2021 / 123332 A1 relates to cationic lipids and LNPs comprising the same that can be used to deliver nucleic acids into living cells.

[0009] Despite recent advances in this area, there remains a need for improved LNPs, particularly with respect to storage stability and / or transformation (transfection) efficiency. For example, LNP-based SARS-CoV-2 vaccines They must generally be stored at -90°C to -60°C (Summary of Product Characteristics, version of 13 September 2022, EMEA / H / C / 005735-II / 0143, European Medicines Agency – EMA). In addition, increased conversion efficiency would allow for lower doses, thereby reducing potential side effects.

[0010] It is therefore an object of the present invention to provide improved LNPs with nucleic acid cargo (eg mRNA cargo), in particular LNPs with increased conversion efficiency and / or higher storage stability, in particular at non-refrigerated temperatures (eg room temperature). Summary of the invention

[0011] The present invention provides LNPs encapsulating nucleic acid cargo. The LNPs comprise at least one cationic lipid fraction (which preferably comprises at least one ionizable lipid) and a stabilizer fraction (preferably comprising PEG lipids). The LNPs comprise at least one glycerol dialkyl glycerol tetraether (GDGT) lipid.

[0012] The present invention also provides a pharmaceutical composition (particularly a vaccine) comprising LNP. The pharmaceutical composition generally comprises an additional excipient. It is preferably used to prevent or treat a disease or condition in a (human) patient, particularly as a vaccine for preventing (or ameliorating) a disease (e.g., an infectious disease) or as a cancer vaccine.

[0013] In the course of the present invention, experiments were conducted to improve the LNPs known in the prior art (e.g. Surprisingly, it was shown that the addition of GDGT lipids to the LNP formulation increased the conversion efficiency in target cells and the storage stability of the LNP.

[0014] Importantly, LNPs (as described in more detail above) are distinct from other lipid-containing delivery vehicles, such as liposomes, lipid (poly)complexes, or tetraether liposomes (archaesomes), and present unique advantages but also challenges, particularly in the context of packaging mRNA. For example, Midoux & Pichon (2014) reviewed various lipid-based mRNA vaccine delivery systems and distinguished between lipoplexes, lipid polyplexes, LNPs, and cationic nanoemulsions. More broadly, drug delivery of RNA therapeutics (e.g., small interfering RNA and mRNA) is achieved through lipid-based carriers (e.g., micelles, liposomes, and LNPs) (Paunovska et al. (2022)).

[0015] In stark contrast to LNPs, liposomes are spherical lipid bilayer vesicles (lipid esters) surrounding an aqueous space. They are vehicles for administering drugs, vaccines, genes, proteins, small molecules, antibiotics and nutrients. Liposomes are made of phospholipids (mainly phosphatidylcholine) and cholesterol, but may also include other lipids, such as phosphatidylethanolamine. Liposomes are produced by a number of different methods (e.g., reviews by van Hoogevest (2017); Szoka et al. (1980)), for example by dispersing phospholipids in an aqueous medium, for example using mechanical treatment (e.g., in a homogenizer, preferably by high-pressure homogenization) or ultrasound. Their diameter varies between 0.02 and 10 μm.

[0016] Tetraether liposomes represent a special class of liposomes based on membrane lipids isolated from archaea. Tetraether liposomes consist of lipid ethers, i.e., diether structures (e.g., archaeol) and tetraether structures (e.g., GDGT, such as archaeol); see, e.g., Kaur et al. (2016), Patel et al. (1999). The diether structure is typically composed of a glycerol moiety carrying two phytyl chains (20 to 40 carbons in length) at the sn-2,3 positions. The tetraether structure typically carries two diphytyl chains, which are connected to two glycerol residues in an antiparallel manner (archaeol) or in a parallel manner (isophytyl tetraether). In addition, one or several cyclopentane rings may be present.

[0017] As used herein, "caldarchaeol" refers to the entire group of isoprenoid GDGT lipids with 0 to 8 cyclopentane moieties. In particular, the nomenclature suggested by Schouten et al. (2013) is used herein: "GDGT-x", where x represents the number of cyclopentane moieties, i.e., GDGT-0, GDGT-1, GDGT-2, GDGT-3, GDGT-4, GDGT-5, GDGT-6, GDGT-7, and GDGT-8. All of these belong to the caldarchaeol group.

[0018] Depending on the composition (eg the amount of bisphytane diglycerol relative to bisphytane diglycerol tetraether), the lipid layer of the tetraether liposomes is a monolayer or a bilayer or a mixture thereof.

[0019] WO 2020 / 187526 A1 discloses tetraether liposomes comprising archaeal lipids from Sulfolobus cell culture, which are mainly used for oral acute or oral sustained-release delivery.

[0020] WO 2014 / 143806 A1 relates to acid-stable liposome compositions. These lipid preparations are considered superior to conventional liposomes due to their stability, thereby avoiding the need for intravenous delivery of bioactive agents.

[0021] Zavec et al. (2014) disclosed that tetraether liposomes can efficiently deliver different types of cargoes into epithelial cells in vitro.

[0022] Vishakarma et al. (2019) reviewed various lipid-based carriers for lymphatic transport, including liposomes and tetraether liposomes.

[0023] In addition, with respect to tetraether liposomes, Daswani et al. (2021) disclosed that polar lipid fraction E from Sulfolobus acidocaldarius can be used as a liposomal drug stabilizer to reduce the leakage of the antivascular drug combretastatin a4 disodium phosphate from tetraether / diester hybrid tetraether liposomes.

[0024] In stark contrast to tetraether liposomes, LNPs contain cationic lipids (particularly ionizable lipids).

[0025] In a preferred embodiment of the invention, the cationic lipid is an ionizable lipid (i.e., a lipid that carries an overall positive charge at endosomal pH, e.g., a pH between 5.0 and 6.5, such as pH 5.0, 5.5, 6 or 6.5, but is neutral at a higher pH (e.g., pH 7.0)).

[0026] In another preferred embodiment, the cationic lipid fraction comprises at least one cationic lipid (e.g., ionizable lipid) selected from the group consisting of [(4-hydroxybutyl) azanediyl] di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethylamine (KL10), N1-[2-(didodecylamino)ethyl]N1,N4,N4-triadecyl-1,4-piperazine diethylamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-triadecane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleic acid-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptacotriacont-6,9,28,31-tetraen-19-yl-4-(dimethyl)- 1,2-Dioleyl-N,N-dimethylaminopropane (DODMA), 2-({8[(3β)-cholest-5-en-3-yloxy]octyl}oxy)N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA), (2R)-2- ({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), (2S)2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), and mixtures thereof. Alternatively or in addition, the cationic lipid is preferably selected from the group consisting of: (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhexicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-14,17-dien-14,17-dien-14,17-dien-14,17-dien-14,17-dien-14,17-dien-14,17-dien-14,17-dien-14,17-dien-1417-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosac-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosac ... -dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosan-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethyltriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacosan-18-en-10-amine, (17Z)-N,N-dimethylhexacosan-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosan-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosan-20,23 -dien-10-amine, 1-[(11Z,14Z)-1-nonyleicos-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-10-amine, (15Z)-N,N-dimethylheptacos-15-en-10-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltricariac-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethyltriacontac-22-en-10-amine, (16Z) -N,N-dimethylpentacosa-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylheneicosah-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonylheneicosah-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]heneicosah-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N,N-dimethyl-1-[(1R,2S)-2-undecylcyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-dimethyl-1-[ (1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, RN,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)propane-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)propane-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy ] propane-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, N,N-dimethyl-1-[(9Z)- Octadec-9-en-1-yloxy]-3-(octyloxy)propane-2-amine, (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadec-6,9,12-trien-1-yloxy]-3-(octyloxy)propane-2-amine, (2S)-1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propane-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethylpropane-2-amine, 1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propane-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propane-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropane-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropane-2-amine, 1-[(13Z)-docosa-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropane-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propane-2-amine, (2R)-N,N-dimethyl-H (1-formyloctyl)oxy]-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)- 2-[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propane-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propane-2-amine and (11E,20Z,23Z)-N,N-dimethylnonacoustic-11,20,23-trien-10-amine, and pharmaceutically acceptable salts and stereoisomers thereof, and mixtures thereof.

[0027] Other suitable cationic lipids (particularly ionizable lipids) are disclosed, for example, in Hou et al. (2021), U.S. Patents US 7,404,969, US 8,058,069, US 9,364,435 and US 9,404,127, WO2017 / 099823 A1, WO 2020 / 061284 A1, WO 2020 / 219941 A1 and WO 2021 / 123332 A1. These documents are included herein by reference in their entirety.

[0028] Even further suitable cationic lipids are disclosed, for example, in WO 2017 / 049245 A1, WO 2017 / 112865 A1, WO 2012 / 040184, WO 2011 / 153120 A1, WO 2011 / 149733 A1, WO 2011 / 090965 A1, WO 2011 / 043913 A1, WO 2011 / 022460 A1, WO 2012 / 061259 A1, WO 2012 / 054365 A1, WO 2012 / 044638 A1, WO 2010 / 080724 A1, WO 2010 / 21865 A1, WO 2008 / 103276 A1, WO 2013 / 086373 A1 and WO 2013 / 086354 A1, U.S. Patents 7,893,302, 7,404,969, 8,283,333 and 8,466,122 and U.S. Patent Publications US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541 and US20130225836. These documents are incorporated herein by reference in their entirety.

[0029] The stabilizer fraction of the LNPs of the present invention is generally suitable for achieving one or more of the following: reducing LNP aggregation, increasing the average particle size or hydrodynamic radius, increasing the in vivo half-life of the LNP (e.g., in humans, particularly in the blood circulation), and modulating zeta potential.

[0030] Particularly preferably, the stabilizer fraction comprises at least one PEG lipid. Suitable PEG lipids are, for example: 2-[(polyethylene glycol)-2000]-N,N-ditetradecyl acetamide (ALC-0159), PEGylated diacylglycerol lipids (PEG-DAG), PEGylated ceramide lipids (PEG-Cer), PEGylated phosphatidylethanolamine lipids (PEG-PE), PEGylated succinate diacylglycerol lipids (PEG-S-DAG), PEGylated dialkoxypropylcarbamate lipids, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol ("PEG-DMG" or "DMG-PEG"), in particular PEG2000-DMG, 1,2-didecyl-rac-glycero-3-methoxypolyethylene glycol (Ci 0-diacylglycerol PEG), N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]} (containing N-octanoyl-D-erythro-sphingosine (d18:1 / 8:0), also known as PEG-ceramide 8), or PEG lipids disclosed in, for example, WO 2018 / 126084 A1, WO 2020 / 093061 A1, or WO2020 / 219941A1 (all three references are incorporated by reference in their entirety); and any combination thereof.

[0031] Other suitable PEG lipids are disclosed, for example, in Hou et al. (2021), WO 2017 / 099823, WO 2020 / 061284 A1, WO 2020 / 219941 A1, and WO 2021 / 123332 A1. All of these documents are incorporated herein by reference in their entirety.

[0032] Alternatively or in addition, the stabilizer fraction can include at least one non-PEG part that can be conjugated or not conjugated with lipid, such as XTEN peptide. XTEN peptide can form a hydrated shell around LNP due to its hydrophilicity. Compared with LNP lacking (or not containing) stabilizer fraction, it further contributes to increase the half-life of LNP. XTEN amino acid sequences are known in the art, including those reported in, for example, U.S. Patent 9,062,299 (incorporated herein by reference in its entirety). Alternatively or in addition, in some embodiments, the stabilizer fraction can include a non-PEG part (which can be conjugated or not conjugated with lipid), such as PAS peptide. PAS peptide is a peptide mainly (if not exclusively) comprising proline, alanine and serine. Like PEG and XTEN peptides, PAS peptide can form a hydrated shell around LNP. Compared with LNP lacking (or not containing) stabilizer part, it also contributes to increase the half-life of LNP. PAS amino acid sequences are known in the art and include, for example, those reported in WO 2008 / 155134A1 (incorporated herein by reference in its entirety).

[0033] GDGT lipids (also referred to herein as "GDGTs") are found as membrane lipids of extremophilic archaea, but have recently also been found to be membrane components of some bacteria (see Schouten et al. (2013)). Many phylogenetic groups in archaea synthesize GDGTs. They form a monolayer in the cell membrane instead of a bilayer. In the course of the present invention, it was demonstrated that GDGT lipids are particularly suitable for improving the properties of LNPs with nucleic acid cargo (e.g., storage stability and conversion efficiency).

[0034] Thus, as described above, the LNPs of the present invention comprise at least one GDGT lipid. The GDGT lipid can be, for example, an isoprenoid GDGT lipid, such as GDGT-0, GDGT-1, GDGT-2, GDGT-3, GDGT-4, GDGT-5, GDGT-6, GDGT-7, and GDGT-8, or crenarchaeol (unsubstituted, see Schouten et al. (2013)). Figure 1 , or substituted), or branched GDGT (such as GDGT-I, GDGT-II or GDGT-III), or any mixture thereof. In particular, the GDGT lipid may comprise any GDGT disclosed in Schouten et al. (2013) (in particular Figure 1 ), Kaur et al. (2016) (particularly Figure 2 , Figure 3 and Figure 4) and WO 2020 / 187526A1 (all of which are incorporated herein by reference in their entirety). The GDGT can be substituted (e.g., substituted with a hexose moiety or a phosphatidylinositol moiety) or unsubstituted.

[0035] Bisphytane diglycerol tetraether (e.g. obtained from Sulfolobus) has been shown to be particularly suitable for use in the present invention, improving the stability and conversion efficiency of LNPs (see also the Examples and Figures). According to a preferred embodiment, at least one GDGT lipid (of the ether lipid fraction of the LNP) therefore comprises at least one bisphytane diglycerol tetraether, which is preferably selected from the group consisting of unsubstituted bisphytane diglycerol tetraether, phosphatidylinositol (PI)-bisphytane diglycerol tetraether, dihexose (2Hex)-bisphytane diglycerol tetraether and 2Hex-PI-bisphytane diglycerol tetraether (in particular as disclosed in Figure 4 of WO 2020 / 187526A1), hexose (Hex)-bisphytane diglycerol tetraether and sulfo-trihexose (3Hex)-bisphytane diglycerol tetraether, sulfo-3Hex-PI-bisphytane diglycerol tetraether and any mixture thereof.

[0036] Particularly preferred Hex-bisphytane diglycerol tetraether is:

[0037]

[0038] Particularly preferred sulfo-3Hex-PI-bisphytane diglycerol tetraether is:

[0039]

[0040] The LNPs of the present invention are particularly suitable for use as RNA cargoes. Thus, in a preferred embodiment, the nucleic acid cargo comprises at least one (therapeutic) RNA. Examples of suitable RNA payloads are described, for example, in Paunovska et al. (2022) (particularly Figure 1 In various embodiments, the cargo may be a small interfering RNA (siRNA), an antisense oligonucleotide, an adenosine deaminase acting on RNA (ADAR) oligonucleotide, or mRNA.

[0041] RNA can be chemically modified, for example to improve its chemical stability. For example, it can include nucleoside analogs (e.g., analogs with chemically modified bases or sugars) and backbone modifications. In some embodiments, the RNA can include nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite bonds). Additional modifications are known to those skilled in the art. Suitable modifications are disclosed in, for example, WO 2017 / 099823 A1 and WO 2020 / 061284 A1 (each of which is incorporated herein by reference in its entirety).

[0042] In the process of the present invention, it is found that GDPD lipid improves LNP characteristics, particularly for the characteristics of mRNA payload. According to a particularly preferred embodiment, the nucleic acid payload of LNP therefore comprises mRNA. mRNA can be, for example, therapeutic mRNA or mRNA encoding vaccine antigens. mRNA can be codon optimized. For example, in WO 2017 / 099823 A1 and WO2020 / 061284 A1 (each incorporated by reference in its entirety), in particular, WO 2020 / 061284 A1, paragraphs

[00178] -

[00180] provide examples of suitable mRNA.

[0043] According to another preferred embodiment, LNP comprises at least one additional ether lipid, preferably a diether lipid (particularly bisphytane glycerol diether). In particular, the diether lipid may comprise any diether lipid disclosed in Kaur et al. (2016) and WO 2020 / 187526A1 (all of which are incorporated herein by reference in their entirety). The diether lipid (particularly bisphytane glycerol diether) may be substituted (e.g., substituted with a hexose moiety or a phosphatidylinositol moiety) or unsubstituted. Particularly preferred substituted bisphytane glycerol diether is phosphatidylinositol-bisphytane glycerol diether (PI-Arc).

[0044] According to another preferred embodiment, the LNP comprises an ether lipid fraction, which comprises at least one GDGT lipid (particularly "at least one GDGT lipid" as disclosed above, especially bisphytane diglycerol tetraether) and preferably at least one additional ether lipid (particularly a diether lipid as disclosed above).

[0045] It is particularly preferred that the ether lipid fraction comprises ether lipids obtainable by extraction from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acidothermophilus culture. Suitable growth conditions and extraction methods are disclosed, for example, in WO 2020 / 187526A1 (incorporated herein by reference in its entirety). In particular, the entire ether lipid fraction can be obtained by extraction from the culture.

[0046] In various embodiments, the archaeal culture can also be, for example, a culture of S. acidocaldarius, M. hungatei, M. voltae, M. concilii, M. smithii, M. espanolae, T. acidophilum, M. mazei, M. espanole, T. acidophilum, H. salinarum, M. smithii, M. stadtmanae, M. sphaeroides ... ), H. halobium, H. morrhuae, M. jannaschii, S. islandicus, S. solfataricus, S. shibatae, S. tokodaii, S. metallicus, S. solfataricus, S. tokodaii, S. metallicus, S. sedula, H. hispanica, or H. volcanii, or a mixture thereof (co-culture).

[0047] For example, total bisphytanyl glycerol diether lipids can be extracted from, for example, freeze-dried or spray-dried biomass by organic solvent extraction using chloroform / methanol / water. Polar lipids and neutral lipids can then be separated by precipitation using acetone. The resulting lipid extract can be used directly in the preparation of the ether lipid fraction for LNP production, or can be further purified by chromatography to separate the specific class of ether lipids to be used in LNP production.

[0048] Methods for preparing lipids from archaea or culturing archaea are also disclosed in, for example, US 2017 / 0152533A1, US 6,316,260 B1, EP 1 999 137 B1, EP 0 883 624 B1, EP 2 109459B1, Siliakus et al. (2017), WO 2020 / 187526 A1, Jain et al. (2014); each is incorporated herein by reference in its entirety.

[0049] The LNPs themselves can be produced by microfluidic mixing of LNP components (fractions) comprising GDGT lipids (e.g., in an isolated form or in an ether lipid fraction comprising several ether lipids). Suitable LNP production methods are disclosed, for example, in WO 2017 / 099823 A1, WO 2020 / 061284A1, and WO 2021 / 123332A1; each of which is incorporated herein by reference in its entirety. Additional LNP preparation methods are available to those skilled in the art.

[0050] According to another preferred embodiment, LNP also comprises sterol lipid fraction.In LNP, mixing sterol lipid alleviates the gathering of other lipids in LNP.Sterol lipid is preferably selected from the group consisting of the following items: cholesterol, fecal sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, phytosterol and mixture thereof.According to preferred definition, " sterol lipid " comprises the whole subgroup of the steroid consisting of steroid alcohols.

[0051] In particular, the sterol lipid fraction comprises cholesterol.

[0052] According to another preferred embodiment, the LNP further comprises a helper lipid fraction. The helper lipid that can be used in the present invention comprises (preferably consists of) a non-cationic lipid. In particular, the helper lipid can be a phospholipid.

[0053] Preferably, the helper lipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DOPC), phosphatidylcholine (PC) and mixtures thereof. Other suitable helper lipids are, for example, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholestyl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diacetatetraenoyl-sn-glycero-3-phosphocholine, 1,2-didocotetraenoyl-sn-glycero-3-phosphocholine, 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocotetraenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof.

[0054] Other suitable helper lipids and sterol lipids are disclosed, for example, in WO 2017 / 099823 A1 and WO 2020 / 061284 A1, which are incorporated herein by reference.

[0055] Certain (molar) ratios in LNP compositions have proven to be particularly suitable for achieving improved LNPs, in particular with regard to storage stability and / or conversion efficiency:

[0056] Thus, it is preferred that the ether lipid fraction (as defined above) constitutes 1 mol% to 20 mol% of the total lipids of the LNP, preferably 2 mol% to 15 mol% of the total lipids, even more preferably 4 mol% to 12 mol% of the total lipids, in particular 6 mol% to 10 mol% of the total lipids, or even 7 mol% to 9 mol% of the total lipids.

[0057] Alternatively or additionally, it is preferred that the molar ratio of the ether lipid fraction to the helper lipid fraction is from 20:1 to 1:20, preferably from 15:1 to 1:10, more preferably from 12:1 to 1:3, even more preferably from 8:1 to 4:1, especially from 7:1 to 5:1.

[0058] Furthermore, alternatively or additionally, it is preferred that the molar ratio of the ether lipid fraction to the sterol lipid fraction is from 0.25:1 to 1:30, preferably from 0.5:1 to 1:20, more preferably from 1:1 to 1:10, even more preferably from 1:2 to 1:6, especially from 1:3 to 1:5.

[0059] Particularly good results were obtained with the following LNPs, which therefore form another preferred embodiment of the present invention (total lipids of the LNPs, ie 100 mol %):

[0060] (a) 40 mol% to 70 mol% cationic lipid fraction,

[0061] (b) 5 mol% to 20 mol% of a helper lipid fraction,

[0062] (c) 20 mol% to 40 mol% of a sterol lipid fraction,

[0063] (d) 0.1 mol% to 4 mol% of a stabilizer fraction, and

[0064] (e) 1 mol% to 20 mol% ether lipid fraction (particularly 2 mol% to 15 mol%, even more preferably 4 mol% to 12 mol%, especially 6 mol% to 10 mol%, or even 7 mol% to 9 mol%).

[0065] As will be clear to a person skilled in the art after reading this application, these ratios and mole % given above can be regarded as average values ​​for the entire population of LNPs (eg, all LNPs present in a pharmaceutical composition).

[0066] The pharmaceutical composition of the present invention (which comprises a plurality of LNPs of the present invention) is preferably provided with at least one excipient. After having read this specification, excipients suitable for the pharmaceutical composition of the present invention are known to those skilled in the art, such as water (particularly water for injection), saline, Ringer's solution, glucose solution, buffer, Hank's solution, 5% glucose in saline, substances that enhance isotonicity and chemical stability, buffer and preservatives. The pharmaceutical composition can be administered (as a drug) to patients or individuals in need (i.e., patients or individuals suffering from the disease or condition mentioned herein or at risk of developing the disease or condition) via appropriate procedures known to those skilled in the art (after having read this specification). The preferred route of administration of the pharmaceutical composition is parenteral administration, in particular by intraperitoneal, subcutaneous, intramuscular and / or intravenous administration. The dosage and method of administration depend on the individual patient or individual to be treated. The pharmaceutical composition can be administered in any suitable dosage known from other biological dosage schemes or in a dosage specifically evaluated and optimized for a given individual. For example, the nucleic acid load can be present in the pharmaceutical composition in an amount of 1 mg to 10 g, preferably 50 mg to 2 g, in particular 100 mg to 1 g. Common dosage can also be determined based on the patient's kg body weight, for example, preferred dosage is in the range of 0.1 mg / kg body weight to 100 mg / kg body weight, especially 1 to 10 mg / kg body weight (each administration cycle). Administration can be, for example, once a day, once every other day, once a week, or once every two weeks. Since the preferred mode of administration of the pharmaceutical composition of the present invention is parenteral administration, the pharmaceutical composition according to the present invention is preferably liquid or prepared to be dissolved in a liquid such as sterile, deionized water or distilled water or sterile isotonic phosphate buffered saline (PBS). Preferably, 1000 μg (dry weight) of such a composition comprises 0.1 to 990 μg, preferably 1 to 900 μg, more preferably 10 to 200 μg of the compound, and optionally 1 to 500 μg, preferably 1 to 100 μg, more preferably 5 to 15 μg (buffer) salt (preferably to produce an isotonic buffer in the final volume), and optionally 0.1 to 999.9 μg, preferably 100 to 999.9 μg, more preferably 200 to 999 μg of other excipients. Preferably, 100 mg of such a dry composition is dissolved in sterile deionized / distilled water or sterile isotonic phosphate buffered saline (PBS) to produce a final volume of 0.1 to 100 mL, preferably 0.5 to 20 mL, more preferably 1 to 10 mL.

[0067] Particularly preferably, the z-average diameter of the LNPs of the present invention measured by DLS, in particular according to ISO 22412:2017, is between 10 nm and 900 nm, preferably between 20 nm and 750 nm, more preferably between 30 nm and 500 nm, especially between 40 nm and 250 nm or even between 50 nm and 150 nm. The z-average diameter defined in ISO 22412-2017 is determined by cumulative analysis and produces a harmonic mean particle diameter weighted by the intensity of the scattered light. For example, Markova et al. (2022) discloses in detail how to measure the z-average diameter of LNPs.

[0068] The present invention also relates to the following embodiments:

[0069] Embodiment 1. A LNP encapsulating a nucleic acid load, wherein the LNP comprises at least:

[0070] a cationic lipid fraction, said cationic lipid fraction preferably comprising at least one ionizable lipid, and

[0071] - Stabilizer fraction;

[0072] Wherein the LNP comprises at least one GDGT lipid.

[0073] Embodiment 2. The LNP of embodiment 1, wherein the nucleic acid cargo comprises mRNA.

[0074] Embodiment 3. The LNP of embodiment 1 or 2, wherein the stabilizer fraction comprises at least one PEG lipid.

[0075] Embodiment 4. The LNP according to any one of embodiments 1 to 3, wherein the LNP further comprises a sterol lipid fraction, preferably cholesterol.

[0076] Embodiment 5. The LNP according to any one of embodiments 1 to 4, wherein the LNP further comprises a helper lipid fraction.

[0077] Embodiment 6. An LNP according to any one of embodiments 1 to 5, wherein the at least one GDGT lipid comprises at least one bisphytane diglycerol tetraether, and the at least one bisphytane diglycerol tetraether is preferably selected from the group consisting of: unsubstituted bisphytane diglycerol tetraether, phosphatidylinositol (PI)-bisphytane diglycerol tetraether, dihexose (2Hex)-bisphytane diglycerol tetraether and 2Hex-PI-bisphytane diglycerol tetraether, hexose (Hex)-bisphytane diglycerol tetraether, sulfo-trihexose (3Hex)-bisphytane diglycerol tetraether, sulfo-3Hex-PI-bisphytane diglycerol tetraether and any mixture thereof.

[0078] Embodiment 7. The LNP according to any one of embodiments 1 to 6, wherein the LNP comprises at least one additional ether lipid, preferably a diether lipid, in particular bisphytane glycerol diether.

[0079] Embodiment 8. The LNP according to any one of embodiments 1 to 7, wherein the LNP comprises an ether lipid fraction, the ether lipid fraction comprising at least one GDGT lipid (preferably at least two different GDGT lipids, more preferably at least three different GDGT lipids, especially at least four different GDGD lipids) and preferably at least one additional ether lipid; in particular comprising bisphytane diglycerol ether and bisphytane diglycerol tetraether, preferably having the composition given in Table 1 below.

[0080] Embodiment 9. The LNP according to embodiment 8, wherein the ether lipid fraction comprises ether lipids obtainable by extraction from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acidothermophilus culture.

[0081] Embodiment 10. The LNP according to embodiment 9, wherein the entire ether lipid fraction is obtainable by extraction from the culture.

[0082] Embodiment 11. The LNP according to any one of embodiments 1 to 10, wherein the ether lipid fraction accounts for 1 mol% to 20 mol% of the total lipids, preferably 2 mol% to 15 mol% of the total lipids, even more preferably 4 mol% to 12 mol% of the total lipids, especially 6 mol% to 10 mol% of the total lipids, or even 7 mol% to 9 mol% of the total lipids.

[0083] Embodiment 12. The LNP according to any one of embodiments 1 to 11, wherein the molar ratio of the ether lipid fraction to the auxiliary lipid fraction (especially to DSPC, if present) is 20:1 to 1:20, preferably 15:1 to 1:10, more preferably 12:1 to 1:3, even more preferably 8:1 to 4:1, especially 7:1 to 5:1.

[0084] Embodiment 13. The LNP according to any one of embodiments 1 to 12, wherein the molar ratio of the ether lipid fraction to the sterol lipid fraction (especially to cholesterol, if present) is 0.25:1 to 1:30, preferably 0.5:1 to 1:20, more preferably 1:1 to 1:10, even more preferably 1:2 to 1:6, especially 1:3 to 1:5.

[0085] Embodiment 14. The LNP of any one of embodiments 1 to 13, wherein the total lipids of the LNP comprise (preferably consist of):

[0086] (a) 40 mol% to 70 mol% cationic lipid fraction,

[0087] (b) 5 mol% to 20 mol% of a helper lipid fraction,

[0088] (c) 20 mol% to 40 mol% of a sterol lipid fraction,

[0089] (d) 0.1 mol% to 4 mol% of a stabilizer fraction, and / or

[0090] (e) 1 mol% to 20 mol% ether lipid fraction.

[0091] Embodiment 15. The LNP according to any one of embodiments 1 to 14, wherein the cationic lipid fraction comprises a cationic lipid (e.g., an ionizable lipid) selected from the group consisting of [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), 3-(didodecanylamino)-N1,N1,4-tridodecane 1-piperazineethylamine (KL10), N1-[2-(didodecylamino)ethyl]N1,N4,N4-triadecyl-1,4-piperazinediethylamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-triadecane (KL25), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleic acid-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptriacontane-6,9,28,31-tetraene- 19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyl-N,N-dimethylaminopropane (DODMA), 2-({8[(3β)-cholest-5-en-3-yloxy]octyl}oxy)N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R )-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), (2S)2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)) and mixtures thereof. Alternatively or in addition, the cationic lipid is preferably selected from the group consisting of: (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhexicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosac-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosac-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosac-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacosac-18,21-dien-8-amine, (17Z,20Z)-N , N-dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethyltriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacosa-18-en-10-amine, (17Z)-N,N-dimethylhexacosa-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosa-10-amine, (20Z,23Z)- N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonyleicos-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-10-amine, (15Z)-N,N-dimethylheptacos-15-en-10-amine, (14Z)-N,N-dimethylnonacosa-14-en-10-amine, (17Z)-N,N-dimethylnonacosa-17-en-10-amine, (24Z)-N,N-dimethyltricaracon-24-en-10-amine, (20Z)-N,N-dimethylnonacosa-20-en-10-amine, (22Z)-N,N-dimethyl 1-[(1S,2R)-2-octylcyclopropyl]-N,N-dimethylnonadecan-10-amine, (16Z)-N,N-dimethylpentacosan-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylheicosan-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonylheicosan-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]-heptadecan-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]heneicosane-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N,N-dimethyl-1-[(1R,2S)-2-undecylcyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N- Dimethylpentadecan-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, R—N,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)propane-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)propane-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy] -3-[(5Z)-octadec-5-en-1-yloxy]propane-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propane-2-amine, (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadec-6,9,12-trien-1-yloxy]-3-(octyloxy)propane-2-amine, (2S)-1-[(11Z,14Z)-eicosyl-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propane-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-eicosyl-11,14-dien-1-yloxy]-N,N-dimethylpropane-2-amine, 1-[(11Z,14Z)-eicosyl-11,14-dien-1-yloxy]1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propane-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propane-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropane-2-amine, (2S)-1-[(13Z)-docosa-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropane-2-amine, 1-[(13Z)-docosa-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propane-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propane-2-amine, (2R)-N ,N-dimethyl-H(1-formyloctyl)oxy]-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S, 2S)-2-[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propane-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propane-2-amine and (11E,20Z,23Z)-N,N-dimethylnonacoustic-11,20,23-trien-10-amine, and pharmaceutically acceptable salts and stereoisomers thereof, and mixtures thereof. ,

[0092] Embodiment 16. The LNP according to any one of embodiments 1 to 15, wherein the auxiliary lipid fraction comprises an auxiliary lipid selected from the group consisting of: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DOPC), phosphatidylcholine (PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dipalmitoyl-sn- Glycerol-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0DietherPC), 1-oleoyl-2-cholestylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diacetatetraenoyl-sn-glycero-3-phosphocholine, 1,2-didocotetraenoyl-sn-glycero-3-phosphocholine, 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocotetraenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof.

[0093] Embodiment 17. A pharmaceutical composition comprising the LNP according to any one of embodiments 1 to 16 and preferably at least one excipient.

[0094] Embodiment 18. A pharmaceutical composition according to embodiment 17, wherein the pharmaceutical composition is a vaccine, preferably an mRNA vaccine.

[0095] Embodiment 19. Use of the pharmaceutical composition according to embodiment 17 or 18 for preventing or treating a disease or condition in a patient.

[0096] Embodiment 20. The LNP or pharmaceutical composition according to any one of embodiments 1 to 19, wherein the z-average diameter of the LNP is between 10 nm and 900 nm, preferably between 20 nm and 750 nm, more preferably between 30 nm and 500 nm, especially between 40 nm and 250 nm, or even between 50 nm and 150 nm (determined by DLS, in particular according to ISO 22412:2017). BRIEF DESCRIPTION OF THE DRAWINGS

[0097] The present invention is further illustrated by the following figures and examples, but is not limited thereto.

[0098] Figure 1 : GDGT increases cellular uptake of LNPs by 30-fold compared to the reference LNP formulation used in the EMA-approved SARS-CoV-2 mRNA vaccine. (a) Partial replacement of the auxiliary lipid DSPC in the LNP formulation with GDGT ("TEL") results in an increase in the rhodamine-lipid fluorescence signal in cells after incubation with LNPs. The reference had a DSPC concentration of 9.6 mol% total lipids. (b) Partial replacement of cholesterol ("Chol") in the LNP formulation with GDGT ("TEL") results in an increase in the rhodamine-lipid fluorescence signal in cells after incubation with LNPs. The reference had a cholesterol concentration of 42.6 mol% total lipids. (c) Repeated experiments using two references ("A" and "B") confirmed that partial replacement of the auxiliary lipid DSPC in the LNP formulation with GDGT ("TEL") results in an increase in the rhodamine-lipid fluorescence signal in cells after incubation with LNPs, with optimal results obtained at approximately 8 mol% GDGT ("TEL").

[0099] Figure 2 : GDGT increases the transfection efficiency of LNP by 90-fold compared to the reference LNP formulation used in the EMA-approved SARS-CoV-2 mRNA vaccine. Partial replacement of the auxiliary lipid DSPC in the LNP formulation with GDGT ("TEL") results in increased expression of EGFP mRNA in cells after incubation with LNP (which encapsulates EGFP mRNA), as measured by fluorescence (arbitrary units, y-axis). The reference has a DSPC concentration of 9.6 mol% total lipids.

[0100] Figure 3 : GDGT-LNPs are stable at room temperature (RT, 25°C). For the three tested formulations A, C and D, (a) encapsulation efficiency (EE), (b) particle diameter and (c) particle concentration remained relatively constant over time, indicating excellent storage stability. DETAILED DESCRIPTION

[0101] Example

[0102] Preparation of LNP

[0103] Lipid stock solutions (archaeal membrane lipids including GDGT obtained from Sulfolobus acidothermophilus (see Table 1 below; see also WO 2020 / 187526 A1), DSPC, ALC-0315, ALC-0159, cholesterol, rhodamine) were filtered through a 0.2 μm polytetrafluoroethylene (PTFE) filter and mixed according to the molar ratios used in the corresponding experiments (see also Tables 2 and 3 below). The organic solvent of the reference preparation was ethanol, while in the preparation containing GDGT, a mixture of dimethyl sulfoxide (DMSO) and 2-propanol (2:3) was used as the organic solvent. The aqueous phase was prepared by dissolving Poly(A) (Carl Roth) or mRNA encoding EGFP (CleanCap, 5moU) in 10 mM citrate buffer (pH = 4.0), respectively. NanoAssembleR Ignite (Precision Nanosystems) was used to prepare LNPs at a total flow rate of 12 mL / min and a flow ratio of 3: 1 (aqueous phase: organic phase). After preparation, LNPs were immediately diluted 1: 2 in 10 mM PBS (pH = 7.4). The removal of organic solvents was achieved by dialysis (SpectraPor, 6-8 kDa) of 10 mM phosphate buffered saline (PBS) buffer (pH = 7.4). The physicochemical characterization of LNPs was performed by Ribogreen determination (mRNA content) and Zetasizer analysis (size, polydispersity index (PDI), zeta potential), see Table 4 below. Zetasizer was purchased from Malvern Panalytical Ltd, UK.

[0104] Typical lipid compositions of GDGT-containing archaeal membrane lipid stock solutions (i.e., ether lipid fractions) are given in Table 1:

[0105]

[0106] Table 2 gives the variations in the helper lipid composition tested:

[0107]

[0108] Table 3 gives the cholesterol lipid composition changes tested:

[0109]

[0110] Table 4 shows the characteristics of the tested LNPs (EE: Encapsulation Efficiency), the z-average diameter was determined by DLS:

[0111]

[0112] High encapsulation efficiency and low PDI (ie, monodisperse distribution) were observed for the LNP samples.

[0113] Uptake of LNPs

[0114] After two passages, Caco-2 cells were plated at 10 5 The cells were seeded at a density of 10 cells / well in 1 ml of culture medium (DMEM+L-glutamine, 10% fetal bovine serum (FBS), 1% antibiotic mixture). Incubate (37°C, 5% CO 2 ) 24 hours later, LNP was added to the wells with a loading concentration of 5.2 μg poly (A) / well. After incubation for another 24 hours, the cells were washed twice using 500 μL of 10mM PBS buffer to remove the residual LNP in the supernatant. This was followed by the step of adding Hoechst solution and incubating for another 5 minutes. After the step of washing twice using 500 μL of 10mM PBS buffer, confocal microscopy (Olympus IX83) was used to analyze the cellular uptake (excitation / emission=570nm / 590nm) and the number of nuclei (excitation / emission=460nm / 490nm) of LNP.

[0115] GDGT increased the cellular uptake of LNPs compared to the reference LNP formulation used in the EMA-approved SARS-CoV-2 mRNA vaccine (see Figure 1 ).

[0116] Transfection with LNPs

[0117] After two passages, Caco-2 cells were plated at 10 5 The cells were seeded at a density of 10 cells / well in 950 μL of culture medium (DMEM+L-glutamine, 10% FBS, 1% antibiotic mixture). Incubated (37°C, 5% CO 2 ) After 24 hours, 500 μL of consumed culture medium was replaced with new culture medium. Subsequently, LNP was added to the wells at a concentration of 6.4 μg of mRNA / well encoding EGFP. The cells were incubated for 60 hours, and the fluorescence intensity (excitation / emission=488nm / 509nm) was measured using a confocal microscope (Olympus IX83) after 17, 24, 36, 48, and 60 hours.

[0118] GDGT improves LNP transfection efficiency compared to the reference LNP formulation used in the EMA-approved SARS-CoV-2 mRNA vaccine (see Figure 2 ).

[0119] Storage of LNP

[0120] After formulation, LNPs were stored in 2 mL Eppendorf tubes at 4°C and 25°C (room temperature), respectively. Physicochemical characterization was performed at 6-week intervals by Ribogreen assay and ZetaSizer analysis.

[0121] GDGT-LNP is stable even at room temperature (see Figure 3 ).

[0122] Non-patent citations

[0123] Aldosari, Basmah N., et al. "Lipid nanoparticles as delivery systems for RNA-based vaccines." Pharmaceutics 13.2(2021):206.

[0124] Daswani,Varsha P.,et al."The Polar Lipid Fraction E from Sulfolobusacidocaldarius Can Be Used as Liposomal Drug Stabilizing Agents to Reduce theLeakage of the Antivascular Drug Combretastatin A4 Disodium Phosphate fromTetraether / Diester Hybrid Archaeosomes." Biophysica 1.4(2021):474-486.

[0125] Hou, Xucheng, et al. "Lipid nanoparticles for mRNA delivery." NatureReviews Materials 6.12(2021):1078-1094.

[0126] Jain, Samta, et al. "Biosynthesis of archaeal membrane ether lipids." Frontiers in microbiology 5(2014):641.

[0127] Kaur,Gurmeet,et al."Archaeosomes:an excellent carrier for drug andcell delivery."Drug delivery 23.7(2016):2497-2512.

[0128] Markova,Natalia,et al."Biophysical Characterization of Viral andLipid-Based Vectors for Vaccines and Therapeutics with Light Scattering andCalorimetric Techniques."Vaccines 10.1(2021):49.

[0129] Midoux,Patrick,et al."Lipid-based mRNA vaccine delivery systems."Expert review of vaccines 14.2(2015):221-234.

[0130] Patel,Girishchandra B.,et al."Archaeobacterial ether lipid liposomes(archaeosomes)as novel vaccine and drug delivery systems."Critical reviews inbiotechnology 19.4(1999):317-357.

[0131] Paunovska,Kalina,et al."Drug delivery systems for RNA therapeutics."NatureReviews Genetics 23.5(2022):265-280.

[0132] Schouten,Stefan,et al."The organic geochemistry of glycerol dialkylglyceroltetraether lipids:A review."Organic geochemistry 54(2013):19-61.

[0133] Siliakus,Melvin F.,et al."Adaptations of archaeal and bacterialmembranes tovariations in temperature,pH and pressure."Extremophiles 21.4(2017):651-670.

[0134] Szoka Jr,Frank,et al."Comparative properties and methods ofpreparation of lipidvesicles(liposomes)."Annual review of biophysics andbioengineering 9.1(1980):467-508.

[0135] van Hoogevest,Peter."Review–an update on the use of oralphospholipidexcipients."European journal of pharmaceutical sciences 108(2017):1-12.

[0136] Vishwakarma,Nikhar,et al."Lipid-based nanocarriers for lymphatictransportation."AAPS PharmSciTech 20.2(2019):1-13.

[0137] Zavec,Apolonija Bedina,et al."Archaeosomes can efficiently deliverdifferent typesof cargo into epithelial cells grown in vitro."Journal ofBiotechnology 192(2014):130-135.

Claims

1. A lipid nanoparticle (LNP) encapsulating a nucleic acid load, wherein the LNP comprises at least: - a cationic lipid fraction, and - Stabilizer fraction; wherein the LNP comprises at least one glycerol dialkyl glycerol tetraether (GDGT) lipid.

2. The LNP of claim 1, wherein the nucleic acid cargo comprises messenger ribonucleic acid (mRNA).

3. The LNP of claim 1 or 2, wherein the stabilizer fraction comprises at least one polyethylene glycol (PEG) lipid.

4. The LNP according to any one of claims 1 to 3, wherein the LNP further comprises a sterol lipid fraction, preferably cholesterol.

5. The LNP according to any one of claims 1 to 4, wherein the LNP further comprises an auxiliary lipid fraction; preferably, wherein the auxiliary lipid fraction comprises an auxiliary lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DOPC), phosphatidylcholine (PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and mixtures thereof.

6. The LNP according to any one of claims 1 to 5, wherein the at least one GDGT lipid comprises at least one bisphytane diglycerol tetraether, and the at least one bisphytane diglycerol tetraether is preferably selected from the group consisting of unsubstituted bisphytane diglycerol tetraether, phosphatidylinositol (PI)-bisphytane diglycerol tetraether, dihexose (2Hex)-bisphytane diglycerol tetraether and 2Hex-PI-bisphytane diglycerol tetraether, hexose (Hex)-bisphytane diglycerol tetraether, sulfo-trihexose (3Hex)-bisphytane diglycerol tetraether, sulfo-3Hex-PI-bisphytane diglycerol tetraether and any mixture thereof.

7. The LNP according to any one of claims 1 to 6, wherein the LNP comprises at least one additional ether lipid, preferably a diether lipid, in particular bisphytane glycerol diether.

8. The LNP according to any one of claims 1 to 7, wherein the LNP comprises an ether lipid fraction comprising at least one GDGT lipid and at least one additional ether lipid; preferably, wherein the ether lipid fraction comprises bisphytane glycerol diether and bisphytane diglycerol tetraether.

9. The LNP according to claim 8, wherein the ether lipid fraction comprises ether lipids obtainable by extraction from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acidothermophilus culture.

10. The LNP according to claim 9, wherein the entire ether lipid fraction can be obtained by extraction from the culture.

11. The LNP according to any one of claims 1 to 10, wherein the ether lipid fraction accounts for 1 to 20 mol% of the total lipids, preferably 2 to 15 mol% of the total lipids, even more preferably 4 to 12 mol% of the total lipids, especially 6 to 10 mol% of the total lipids, or even 7 to 9 mol% of the total lipids.

12. The LNP according to any one of claims 1 to 11, wherein the molar ratio of the ether lipid fraction to the helper lipid fraction is 20:1 to 1:20, preferably 15:1 to 1:10, more preferably 12:1 to 1:3, even more preferably 8:1 to 4:1, in particular 7:1 to 5:

1.

13. The LNP according to any one of claims 1 to 12, wherein the molar ratio of the ether lipid fraction to the sterol lipid fraction is 0.25:1 to 1:30, preferably 0.5:1 to 1:20, more preferably 1:1 to 1:10, even more preferably 1:2 to 1:6, in particular 1:3 to 1:

5.

14. The LNP according to any one of claims 1 to 13, wherein the total lipids of the LNP comprise: (a) 40 mol% to 70 mol% cationic lipid fraction, (b) 5 mol% to 20 mol% of a helper lipid fraction, (c) 20 mol% to 40 mol% of a sterol lipid fraction, (d) 0.1 mol% to 4 mol% of a stabilizer fraction, and (e) 1 mol% to 20 mol% ether lipid fraction.

15. A pharmaceutical composition comprising the LNP according to any one of claims 1 to 14, preferably wherein the pharmaceutical composition is a vaccine.

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