Lipid nanoparticles with nucleic acid cargo

Abstract

Lipid nanoparticles (LNPs) are disclosed that encapsulate nucleic acid cargo, preferably comprising messenger ribonucleic acid (mRNA). The LNPs comprise at least a cationic lipid portion and a stabilizer portion. The stabilizer portion preferably comprises at least one polyethylene glycol (PEG) lipid. Furthermore, the LNPs comprise at least one glycerol dialkylglycerol tetraether (GDGT) lipid, e.g., from archaea of ​​the genus Sulfolobus, optionally present among other ether lipids. Pharmaceutical compositions comprising the LNPs, such as mRNA vaccines, are also disclosed.

Description

[Technical Field]

[0001] The field of the invention relates to lipid nanoparticles (LNPs) with nucleic acid cargo, particularly for use in messenger ribonucleic acid (mRNA) vaccines. [Background technology]

[0002] LNPs have recently attracted attention as LNP-based mRNA vaccines against SARS-CoV-2 (mainly elasomeran, marketed by Moderna under the trademark Spikebax®, and tosinameran, marketed by BioNTech / Pfizer under the trademark Comirnaty®) have been administered to hundreds of millions of individuals. LNPs as delivery systems for RNA-based vaccines in general are reviewed in Aldosari et al., 2021. LNPs for mRNA delivery are also discussed in detail in Hou et al., 2021.

[0003] Generally, LNPs carrying nucleic acid cargo (or payload) comprise a lipid layer and microdomains consisting of lipids and encapsulated nucleic acids. LNPs have a median diameter of 10 nm to 1000 nm (e.g., as determined by dynamic light scattering (DLS)) and can be, for example, spherical or polyhedral in shape. Depending on their specific lipid composition, LNPs can be multilayered. LNPs contain cationic lipids (particularly lipids that are protonated at low pH, i.e., in endosomes, also known as ionizable lipids). Typically, LNPs further contain stabilizers, such as polyethylene glycol (PEG) lipids, which reduce LNP aggregation. In addition, LNPs usually contain other types of lipids, such as phosphatidylcholine or phosphatidylethanolamine (often referred to as "helper lipids"), to improve properties such as delivery efficacy, tolerability, or biodistribution. Finally, LNPs can contain cholesterol or other sterols to modulate membrane integrity and rigidity.

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

[0005] WO 2017 / 099823 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 also relates to LNPs with PEG lipids.

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

[0008] WO 2021 / 123332 relates to cationic lipids and LNPs comprising said cationic lipids that are useful for the delivery of nucleic acids to living cells.

[0009] Despite recent advances in this field, there remains a need for improved LNPs, particularly with regard to storage stability and / or transfection efficiency. For example, the LNP-based SARS-CoV-2 vaccine Comirnaty® generally requires storage at -90°C to -60°C (Summary of Product Characteristics, September 13, 2022, EMEA / H / C / 005735-II / 0143, European Medicines Agency). Furthermore, improved transfection efficiency allows for lower doses, thereby reducing potential side effects. Summary of the Invention [Problem to be solved by the invention]

[0010] It is therefore an object of the present invention to provide improved LNPs carrying nucleic acid cargo (such as mRNA), particularly LNPs that have improved transformation efficiency and / or improved storage stability, especially at non-refrigerated temperatures (e.g., room temperature). [Means for solving the problem]

[0011] The present invention provides LNPs encapsulating a nucleic acid cargo, the LNPs comprising at least a cationic lipid portion (preferably comprising at least one ionizable lipid) and a stabilizer portion (preferably comprising a PEG lipid), the LNPs comprising at least one glycerol dialkyl glycerol tetraether (GDGT) lipid.

[0012] The present invention also provides pharmaceutical compositions (particularly vaccines) comprising the LNPs. The pharmaceutical compositions typically include further excipients. The pharmaceutical compositions are preferably for use in the prevention or treatment of a disease or condition in a (human) patient, particularly as a vaccine to prevent (or ameliorate) a disease, such as an infectious disease, or as a cancer vaccine.

[0013] During the course of this invention, experiments were conducted to improve LNPs known in the prior art (such as the LNPs used in the Comirnaty® mRNA vaccine). Surprisingly, it was found that the addition of GDGT lipids to LNP formulations improves both the transformation efficiency in target cells and the storage stability of the LNPs.

[0014] Importantly, LNPs (described in more detail above) are distinct from other lipid-containing delivery vehicles such as liposomes, lipo(poly)plexes, or archaeosomes, particularly in the context of mRNA packaging, and offer unique advantages but also challenges. For example, Midoux and Pichon (2014) provide an overview of various lipid-based mRNA vaccine delivery systems, distinguishing between lipoplexes, lipopolyplexes, LNPs, and cationic nanoemulsions. More broadly, drug delivery of RNA therapeutics (such as small interfering RNA and mRNA) is enabled by lipid-based vehicles such as micelles, liposomes, and LNPs (Paunovska et al., 2022).

[0015] In contrast to LNPs, liposomes are spherical lipid bilayer vesicles (lipid esters) surrounding an aqueous space. Liposomes are carriers for the administration of drugs, vaccines, genes, proteins, small molecules, antibiotics, and nutrients. Liposomes are made from phospholipids, primarily phosphatidylcholine and cholesterol, but may also contain other lipids such as phosphatidylethanolamine. Liposomes can be generated by dispersing phospholipids in an aqueous medium using a number of different methods (e.g., as reviewed by van Hoogevest, 2017, and Szoka et al., 1980), such as mechanical treatment (e.g., in a homogenizer, preferably by high-pressure homogenization) or ultrasonication. Liposomes vary in diameter, ranging from 0.02 to 10 μm.

[0016] Archaeosomes represent a special class of membrane lipid-based liposomes isolated from archaea. Archaeosomes are made of lipid ethers, namely diether structures (e.g., archaeoles) and tetraether structures (e.g., GDGTs such as cardoarchaeoles). See, for example, Kaur et al., 2016 and Patel et al., 1999. Diether structures typically consist of a glycerol moiety with two phytanyl chains (20–40 carbons in length) at the sn-2 and sn-3 positions. Tetraether structures typically have two di-phytanyl chains attached to two glycerol residues in either an antiparallel fashion (cardoarchaeoles) or a parallel fashion (iso-cardoarchaeoles). Additionally, one or several cyclopentane rings may be present.

[0017] As used herein, "cardoarchaeols" refers to the entire group of isoprenoid GDGT lipids containing zero to eight cyclopentane moieties. In particular, the nomenclature "GDGT-x" proposed by Schouten et al. (2013) (where x represents the number of cyclopentane moieties) is used herein, namely, GDGT-0, GDGT-1, GDGT-2, GDGT-3, GDGT-4, GDGT-5, GDGT-6, GDGT-7, and GDGT-8, all of which belong to the cardoarchaeols group.

[0018] Depending on the composition (eg, amount of archaeoles vs. cardoarchaeoles), the lipid layer of the archaeosomes may be monolayered or bilayered or a mixture thereof.

[0019] WO 2020 / 187526 discloses archaeosomes containing archaeal lipids from Sulfolobus cell cultures, primarily intended for oral acute or oral delayed delivery.

[0020] WO 2014 / 143806 relates to acid-stable liposome compositions that are claimed to have superior stability to conventional liposomes, thereby avoiding the need for intravenous delivery of bioactive agents.

[0021] Zavec et al., 2014, disclose that archaeosomes can efficiently deliver various types of cargo to epithelial cells in vitro.

[0022] Vishakarma et al. (2019) provide an overview of various lipid-based carriers for lymphatic transport, including liposomes and archaeosomes.

[0023] Furthermore, regarding archaeosomes, Daswani et al. (2021) disclosed that the use of polar lipid moiety E from Sulfolobus acidocaldarius as a liposomal drug stabilizer can reduce leakage of the antivascular drug combretastatin a4 disodium phosphate from tetraether / diester hybrid archaeosomes.

[0024] In contrast to archaeosomes, LNPs contain cationic lipids (especially ionizable lipids). [Brief explanation of the drawings]

[0025] [Figure 1a] GDGT increased the cellular uptake of LNPs by 30-fold compared to the reference LNP formulation used in the EMA-approved mRNA vaccine for SARS-CoV-2. (a) Partial replacement of the helper lipid DSPC in the LNP formulation with GDGT (tetraether lipid (“TEL”)) increases the rhodamine-lipid fluorescence signal in cells upon incubation with LNPs. The reference had DSPC at a concentration of 9.6 mol% of total lipids. [Figure 1b](b) Partial replacement of cholesterol (chol) in the LNP formulation with GDGT (TEL) increases the rhodamine-lipid fluorescence signal in cells upon incubation with LNPs. The reference had cholesterol at a concentration of 42.6 mol% of total lipids. [Figure 1c] (c) Repeated experiments using two references ("A" and "B") confirmed that partial replacement of the helper lipid DSPC in the LNP formulation with GDGT ("TEL") increased the rhodamine-lipid fluorescence signal in cells upon incubation with LNPs, with optimal results at approximately 8 mol% GDGT ("TEL"). [Figure 2] GDGT increased the transfection efficiency of LNPs by 90-fold compared to the reference LNP formulation used in the EMA-approved mRNA vaccine for SARS-CoV-2. Partial replacement of the helper lipid DSPC with GDGT ("TEL") in the LNP formulation increased enhanced green fluorescent protein (EGFP) mRNA expression in cells upon incubation with LNPs (encapsulating EGFP mRNA), as measured by fluorescence (arbitrary units, y-axis). The reference had DSPC at a concentration of 9.6 mol% total lipid. [Figure 3a] GDGT-LNP was stable at room temperature (RT, 25°C). For the three test formulations A, C, and D, (a) the encapsulation efficiency (EE) remained relatively constant over time, indicating excellent storage stability. [Figure 3b] GDGT-LNP was stable at room temperature (RT, 25°C). For the three test formulations A, C, and D, (b) particle diameter remained relatively constant over time, indicating excellent storage stability. [Figure 3c]GDGT-LNP was stable at room temperature (RT, 25°C). For the three test formulations A, C, and D, (c) particle concentration remained relatively constant over time, indicating excellent storage stability. DETAILED DESCRIPTION OF THE INVENTION

[0026] In a preferred embodiment of the invention, the cationic lipid is an ionizable lipid (i.e., a lipid that has an overall positive charge at endosomal pH, e.g., pH 5.0 to 6.5, e.g., pH 5.0, 5.5, 6, or 6.5, but is neutral at higher pHs, e.g., pH 7.0).

[0027] In another preferred embodiment, the cationic lipid moiety is 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-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]N1,N4 ... Sil-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl- 4-(2-Dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8[(3β)-cholest-5-en-3-yloxy]octyl}oxy)N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl

[0023] Alternatively, or in addition, the cationic lipid preferably comprises at least one cationic lipid (e.g., an ionizable lipid) selected from the group consisting of (20Z,23Z)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-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)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), and mixtures thereof.N-Dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-Dimemylhexacosa-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-Dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-Dimethyltricosa-14,17-dien- (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-9-amine, (18Z,21 Z)-N,N-dimethylheptacosa-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-dimethylhentriaconta-22,25-dien-10-amine, (21 (Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacos-18-en-10-amine, (17Z)-N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosane-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20, 23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicosa-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-Dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylhentriacontriacont-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclohexyl] N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-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]henicosan-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1-[(1 S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-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]hexadecan-8-amine Propyl]pentadecan-8-amine, RN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien -1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-i [(11Z,14Z)-Icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docosa-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docosa-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-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)propan-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine, and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-trien-10-amine, and pharmaceutically acceptable salts and stereoisomers thereof, and mixtures thereof.

[0028] Further suitable cationic lipids (especially ionizable lipids) are disclosed, for example, in Hou et al., 2021, U.S. Patent Nos. 7,404,969, 8,058,069, 9,364,435 and 9,404,127, WO 2017 / 099823, WO 2020 / 061284, WO 2020 / 219941 and WO 2021 / 123332, which are incorporated herein by reference in their entireties.

[0029] Further suitable cationic lipids are described, for example, in WO 2017 / 049245, WO 2017 / 112865, WO 2012 / 040184, WO 2011 / 153120, WO 2011 / 149733, WO 2011 / 090965, WO 2011 / 043913, WO 2011 / 022460, WO 2012 / 061259, WO 2012 / 054365, WO 2012 / 044638, WO 2010 / 080724, WO 2010 / 21865, WO 2008 / 103276, WO 2013 / 080724 ... Nos. 086373 and 2013 / 086354, U.S. Patent Nos. 7,893,302, 7,404,969, 8,283,333, and 8,466,122, and U.S. Patent Publication Nos. 20100036115, 20120202871, 20130064894, 20130129785, 20130150625, 20130178541, and 20130225836, all of which are incorporated herein by reference in their entireties.

[0030] The stabilizer moieties of the LNPs of the present invention are typically suitable for achieving one or more of: reducing LNP aggregation, increasing the mean particle size or hydrodynamic radius, extending the half-life of the LNPs in vivo (e.g., in humans, particularly in the blood circulation), and modifying the zeta potential.

[0031] According to a particularly preferred embodiment, the stabilizer moiety comprises at least one PEG-lipid. Suitable PEG-lipids include, for example, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), PEGylated diacylglycerol lipid (PEG-DAG), PEGylated ceramide lipid (PEG-Cer), PEGylated phosphatidylethanolamine lipid (PEG-PE), PEGylated succinic acid diacylglycerol lipid (PEG-S-DAG), PEGylated dialkoxypropylcarbamate lipid, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol ("PEG-DMG" or "DMG-PEG"), in particular PEG2000-DMG, 1,2-di capryl-rac-glycero-3-methylpolyoxyethylene glycol (CiO-diacylglycerol PEG), N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]} (comprising N-octanoyl-D-erythro-sphingosine (d18:1 / 8:0), also referred to as PEG-ceramide 8), or the PEG lipids disclosed in WO 2018 / 126084, WO 2020 / 093061, or WO 2020 / 219941 (all three references are incorporated by reference in their entirety), and any combination thereof.

[0032] Additional suitable PEG lipids are disclosed, for example, in Hou et al., 2021, WO 2017 / 099823, WO 2020 / 061284, WO 2020 / 219941, and WO 2021 / 123332, all of which are incorporated herein by reference in their entireties.

[0033] Alternatively, or in addition, the stabilizer moiety may include at least one non-PEG moiety, such as an XTEN peptide, which may or may not be bound to a lipid. Due to its hydrophilic nature, the XTEN peptide can form a hydration shell around the LNP. The XTEN peptide further serves to extend the half-life of the LNP compared to an LNP lacking (or not including) the stabilizer moiety. XTEN amino acid sequences are known in the art, including, for example, those reported in U.S. Pat. No. 9,062,299, which is incorporated herein by reference in its entirety. Alternatively, or in addition, in some embodiments, the stabilizer moiety may include a non-PEG moiety, such as a PAS peptide (which may or may not be bound to a lipid). PAS peptides are peptides that contain primarily, if not exclusively, proline, alanine, and serine. Like PEG and XTEN peptides, the PAS peptide can form a hydration shell around the LNP. The PAS peptide also serves to extend the half-life of the LNP compared to an LNP lacking (or not including) the stabilizer moiety. PAS amino acid sequences are known in the art, for example those reported in WO 2008 / 155134, which is incorporated herein by reference in its entirety.

[0034] GDGT lipids (also referred to herein as "GDGT") were discovered as membrane lipids in extremophilic archaea, but have more recently been identified as membrane components of some bacteria (see Schouten et al., 2013). Many lineages within archaea synthesize GDGTs. They form a monolayer, rather than a bilayer, in the cytoplasmic membrane. During the course of this invention, it was discovered that GDGT lipids are highly suitable for improving the properties (such as storage stability and transformation efficiency) of LNPs carrying nucleic acid cargo.

[0035] Thus, as noted above, the LNPs of the present invention comprise at least one GDGT lipid. This GDGT lipid may 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 a crenarcheol (unsubstituted, see Schouten et al., 2013, Figure 1, or substituted), or a branched GDGT such as GDGT-I, GDGT-II, or GDGT-III, or any mixture thereof. In particular, the GDGT lipid may include any of the GDGTs disclosed in Schouten et al., 2013 (particularly Figure 1), Kaur et al., 2016 (particularly Figures 2, 3, and 4), and WO 2020 / 187526 (all of which are incorporated by reference in their entireties). The GDGT may be substituted (eg, with a hexose moiety or a phosphatidylinositol moiety) or unsubstituted.

[0036] It has been found that cardoarchaeols (e.g., those obtained from Sulfolobus) are particularly suitable for the present invention and improve both the stability and transformation efficiency of the LNP (see also the Examples and Figures). Thus, according to a preferred embodiment, at least one GDGT lipid (of the ether lipid portion of the LNP) comprises at least one cardoarchaeols preferably selected from the group consisting of unsubstituted cardoarchaeols, phosphatidylinositol (PI)-cardoarchaeols, dihexose (2Hex)-cardoarchaeols and 2Hex-PI-cardoarchaeols (especially those disclosed in WO 2020 / 187526, FIG. 4), hexose (Hex)-cardoarchaeols and sulfono-trihexose (3Hex)-cardoarchaeols, sulfono-3Hex-PI-cardoarchaeols, and any mixtures thereof.

[0037] Particularly preferred Hex-cardoarchaeols are: [ka]

[0038] Particularly preferred sulfono-3Hex-PI-cardoarchaeols are: [ka]

[0039] The LNPs of the present invention are particularly suited for RNA cargo. Thus, in a preferred embodiment, the nucleic acid cargo comprises at least one (therapeutic) RNA. Examples of suitable RNA payloads are disclosed, for example, in Paunovska et al., 2022 (particularly Figure 1). In embodiments, the cargo may be a small interfering RNA (siRNA), an antisense oligonucleotide, an adenosine deaminase acting on RNA (ADAR) oligonucleotide, or an mRNA.

[0040] RNA may be chemically modified, for example, to improve its chemical stability. For example, RNA may include nucleoside analogs, such as analogs with chemically modified bases or sugars, and backbone modifications. In some embodiments, RNA may be modified with nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-deazaguanosine, 9-deazaguanosine, 10-deazaguanosine, 11-deazaguanosine, 12-deazaguanosine, 13-deazaguanosine, 14-deazaguanosine, 15-deazaguanosine, 16-deazaguanosine, 17-deazaguanosine, 18-deazaguanosine, 19-deazaguanosine, 20-deazaguanosine, 21-deazaguanosine, 22-deazaguanosine, 23-deazaguanosine, 24-deazaguanosine, 25-deazaguanosine, 26-deazaguanosine, 27-deazaguanosine, 28-deazaguanosine, 29-deazaguanosine, 30-deazaguanosine, 31-deazaguanosine, 32-deazaguanosine, 33 The bases may include, for example, 8-oxoadenosine, 8-oxoguanosine, 0(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 linkages). Further modifications are known to those skilled in the art. Suitable modifications are disclosed, for example, in International Publication Nos. 2017 / 099823 and 2020 / 061284 (each of which is incorporated by reference in its entirety).

[0041] During the course of the present invention, it was discovered that GDPD lipids improve the properties of LNPs, particularly for mRNA payloads. Thus, according to a particularly preferred embodiment, the nucleic acid cargo of the LNP comprises mRNA. The mRNA may be, for example, a therapeutic mRNA or an mRNA encoding a vaccine antigen. The mRNA may be codon-optimized. Examples of suitable mRNAs are provided, for example, in WO 2017 / 099823 and WO 2020 / 061284 (each incorporated by reference in its entirety), particularly paragraphs

[0178] to

[0180] of the latter.

[0042] According to a further preferred embodiment, the LNP comprises at least one further ether lipid, preferably a diether lipid (especially archaeol). In particular, the diether lipid may comprise any of the diether lipids disclosed in Kaur et al., 2016 and International Publication No. WO 2020 / 187526 (all of which are incorporated herein by reference in their entirety). The diether lipid (especially archaeol) may be substituted or unsubstituted (e.g., by a hexose moiety or phosphatidylinositol moiety). A particularly preferred substituted archaeol is phosphatidyl inositol-archaeol (PI-Arc).

[0043] According to another preferred embodiment, the LNP comprises an ether lipid moiety comprising at least one GDGT lipid (especially the "at least one GDGT lipid" disclosed above, especially caldarchaeol), and preferably at least one further ether lipid (especially the diether lipids disclosed above).

[0044] It is particularly preferred that the ether lipid moiety comprises ether lipids obtained by extraction from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acidocaldarius culture. Suitable growth conditions and extraction methods are disclosed, for example, in International Publication No. WO 2020 / 187526 (which is incorporated herein by reference in its entirety). In particular, the entire ether lipid moiety is obtained by extraction from said culture.

[0045] In embodiments, the archaeal culture may be, for example, 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. stadtmannae ... The culture medium may be a culture of H. manae, H. halobium, H. morrhuae, M. jannaschii, S. islandicus, S. solfataricus, S. shibatae, S. tokodaii, S. metallicus, M. sedula, H. hispanica, or H. volcanii, or a mixture (co-culture) thereof.

[0046] As an example, total archaeal lipids may be extracted from freeze-dried or spray-dried biomass by organic solvent extraction, e.g., using chloroform / methanol / water. Polar and neutral lipids may then be separated by precipitation with acetone. The resulting lipid extract may be used directly to prepare the ether lipid portion used in LNP production, or the resulting lipid extract may be further purified by chromatography to isolate specific classes of ether lipids for use in LNP production.

[0047] Methods for preparing lipids from or culturing archaea are also disclosed in, for example, U.S. Patent Application Publication No. 2017 / 0152533, U.S. Patent No. 6,316,260, EP 1 999 137, EP 0 883 624, EP 2 109 459, Siliakus et al., 2017, WO 2020 / 187526, Jain et al., 2014, each of which is incorporated herein by reference in its entirety.

[0048] The LNPs themselves may be produced by microfluidic mixing of LNP components (portions) containing GDGT lipids (e.g., present in isolated form or in ether lipid portions containing several ether lipids). Suitable LNP production methods are disclosed, for example, in International Publication Nos. 2017 / 099823, 2020 / 061284, and 2021 / 123332, each of which is incorporated herein by reference in its entirety. Additional LNP production methods are available to those skilled in the art.

[0049] According to another preferred embodiment, the LNP further comprises a sterol lipid moiety. Incorporation of the sterol lipid into the LNP reduces aggregation of other lipids in the LNP. The sterol lipid is preferably selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, phytosterols, and mixtures thereof. According to a preferred definition, "sterol lipid" includes the entire subgroup of steroids consisting of steroid alcohols.

[0050] In particular, the sterol lipid moiety comprises cholesterol.

[0051] According to yet another preferred embodiment, the LNP further comprises a helper lipid moiety. Helper lipids useful in the present invention comprise (and preferably consist of) non-cationic lipids. In particular, the helper lipid may be a phospholipid.

[0052] Preferably, the helper lipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DOPC), phosphatidylcholine (PC), and mixtures thereof. Further 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 (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dilinoleo ... -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-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-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 (1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt: DOPG), sphingomyelin, and mixtures thereof.

[0053] Further suitable helper lipids and sterol lipids are disclosed, for example, in WO 2017 / 099823 and WO 2020 / 061284, which are incorporated herein by reference.

[0054] It has been found that certain (molar) ratios in the LNP composition are particularly suitable for achieving improved LNPs, especially with regard to storage stability and / or transformation efficiency.

[0055] Therefore, it is preferred that the ether lipid portion (as defined above) accounts for 1 mol% to 20 mol% of the total lipids, preferably 2 mol% to 15 mol% of the total lipids, 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 of the LNP.

[0056] Alternatively, or in addition, the molar ratio of the ether lipid moiety to the helper lipid moiety is preferably 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, and especially preferably 7:1 to 5:1.

[0057] Furthermore, alternatively, or in addition to the above, the molar ratio of the ether lipid portion to the sterol lipid portion is preferably 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, and particularly preferably 1:3 to 1:5.

[0058] Particularly good results have been achieved, and therefore constitute another preferred embodiment of the invention, in LNPs (total lipids in the LNP, i.e. 100 mol %): (a) 40 mol% to 70 mol% of a cationic lipid moiety; (b) 5 mol% to 20 mol% of a helper lipid portion; (c) 20 mol% to 40 mol% of a sterol lipid portion; (d) 0.1 mol% to 4 mol% of a stabilizer moiety, and (e) 1 mol% to 20 mol% of an ether lipid portion (particularly 2 mol% to 15 mol%, more preferably 4 mol% to 12 mol%, particularly 6 mol% to 10 mol%, or even 7 mol% to 9 mol%) is.

[0059] As will be apparent to one of skill in the art upon reading this application, these ratios and mol% shown above may be ascertained as average values ​​across the entire population of LNPs (e.g., all LNPs present in a pharmaceutical composition).

[0060] Pharmaceutical compositions of the invention (comprising a plurality of LNPs of the invention) are preferably provided with at least one excipient. Suitable excipients for pharmaceutical compositions of the invention will be known to those skilled in the art upon reading this specification, and include, for example, water (especially water for injection), saline, Ringer's solution, dextrose solution, buffered saline solution, Hank's solution, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers, and preservatives. The pharmaceutical composition can be administered (as a medicament) to a patient or individual in need thereof (i.e., a patient or individual suffering from or at risk of developing a disease or condition referred to herein) by suitable procedures known to those skilled in the art upon reading this specification. The preferred route of administration of the pharmaceutical composition is parenteral administration, particularly via intraperitoneal, subcutaneous, intramuscular, and / or intravenous administration. The dosage and method of administration depend on the individual patient or individual being treated. The pharmaceutical composition can be administered at any suitable dosage known from other biological administration regimens or can be specifically assessed and optimized for a given individual. For example, in the pharmaceutical composition, the nucleic acid cargo may be present in an amount of 1 mg to 10 g, preferably 50 mg to 2 g, in particular 100 mg to 1 g. Usual dosages can also be determined based on the patient's kg body weight; for example, preferred dosages are in the range of 0.1 mg to 100 mg / kg body weight, in particular 1 to 10 mg / kg body weight (per administration session). Administration may be, for example, once daily, once every other day, once weekly, 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 ready to be dissolved in a liquid such as sterile, deionized, or distilled water, or sterile isotonic phosphate-buffered saline (PBS).Preferably, 1000 μg (dry weight) of such a composition contains 0.1-990 μg, preferably 1-900 μg, more preferably 10-200 μg of the compound, and optionally 1-500 μg, preferably 1-100 μg, more preferably 5-15 μg of a (buffer) salt (preferably to provide an isotonic buffer in the final volume), and optionally 0.1-999.9 μg, preferably 100-999.9 μg, more preferably 200-999 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 a final volume of 0.1-100 ml, preferably 0.5-20 ml, more preferably 1-10 ml.

[0061] According to particularly preferred embodiments, the LNPs of the present invention have a z-average diameter, determined by DLS, in the range of 10 nm to 900 nm, preferably 20 nm to 750 nm, more preferably 30 nm to 500 nm, especially 40 nm to 250 nm, or even 50 nm to 150 nm, in particular according to ISO 22412:2017. The z-average diameter as defined in ISO 22412-2017 is determined by the cumulant method, which yields the scattered light intensity-weighted harmonic mean particle diameter. For example, Markova et al., 2022, discloses in detail a method for measuring the z-average diameter of LNPs.

[0062] The present invention further relates to the following embodiments:

[0063] Embodiment 1 1. A LNP encapsulating a nucleic acid cargo, the LNP comprising at least a cationic lipid moiety, preferably comprising at least one ionizable lipid, and -Stabilizer part Including, The LNP comprises at least one GDGT lipid.

[0064] Embodiment 2 2. The LNP of embodiment 1, wherein said nucleic acid cargo comprises mRNA.

[0065] Embodiment 3 The LNP of embodiment 1 or embodiment 2, wherein the stabilizer moiety comprises at least one PEG lipid.

[0066] Embodiment 4 The LNP of any one of embodiments 1 to 3, wherein the LNP further comprises a sterol lipid moiety, preferably cholesterol.

[0067] Embodiment 5 The LNP of any one of embodiments 1 to 4, wherein the LNP further comprises a helper lipid moiety.

[0068] Embodiment 6 The LNP of any one of embodiments 1 to 5, wherein the at least one GDGT lipid comprises at least one cardoarchaeoI preferably selected from the group consisting of unsubstituted cardoarchaeoI, phosphatidylinositol (PI)-cardoarchaeoI, dihexose (2Hex)-cardoarchaeoI and 2Hex-PI-cardoarchaeoI, hexose (Hex)-cardoarchaeoI, sulfono-trihexose (3Hex)-cardoarchaeoI, sulfono-3Hex-PI-cardoarchaeoI, and any mixture thereof.

[0069] Embodiment 7 The LNP according to any one of embodiments 1 to 6, wherein the LNP comprises at least one further ether lipid, preferably a diether lipid, in particular an archaeal.

[0070] Embodiment 8 10. The LNP of any one of embodiments 1 to 7, wherein the LNP comprises an ether lipid portion 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 further ether lipid, in particular comprising archaeal and cardoarchaeal, preferably having the composition shown in Table 1 below.

[0071] Embodiment 9 9. The LNP of embodiment 8, wherein the ether lipid portion comprises an ether lipid obtained by extraction from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acidocaldarius culture.

[0072] Embodiment 10 The LNP of embodiment 9, wherein all of said ether lipid moieties are obtained from said culture by extraction.

[0073] Embodiment 11 The LNP according to any one of embodiments 1 to 10, wherein the ether lipid portion accounts for 1 mol% to 20 mol% of the total lipids, preferably 2 mol% to 15 mol% of the total lipids, more preferably 4 mol% to 12 mol% of the total lipids, particularly 6 mol% to 10 mol% of the total lipids, or even 7 mol% to 9 mol% of the total lipids.

[0074] Embodiment 12 12. The LNP of any one of embodiments 1 to 11, wherein the molar ratio of ether lipid moiety to helper lipid moiety (particularly 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, and especially 7:1 to 5:1.

[0075] Embodiment 13 The LNP of any one of embodiments 1 to 12, wherein the molar ratio of ether lipid moieties to sterol lipid moieties (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, and especially 1:3 to 1:5.

[0076] Embodiment 14 The total lipid content of the LNP is (a) 40 mol% to 70 mol% of a cationic lipid moiety; (b) 5 mol% to 20 mol% of a helper lipid portion; (c) 20 mol% to 40 mol% of a sterol lipid portion; (d) 0.1 mol% to 4 mol% of a stabilizer moiety, and / or (e) 1 mol% to 20 mol% of an ether lipid moiety The LNP according to any one of embodiments 1 to 13, comprising (preferably consisting of):

[0077] Embodiment 15 The cationic lipid moiety may be 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-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]N1,N4,N4-tridodecyl-1,4-piperazinediethaneamine (KL10), and N1-[2-(didodecylamino)ethyl]N1,N4,N4-tridodecyl-1,4-piperazinediethaneamine (KL10). amine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8[(3β)-cholest-5-en-3-yloxy]octyl}oxy)N,N-dimethyl-3-[(9Z,12Z)-octadeca-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)-octadeca-9,

[0039] The LNP of any one of embodiments 1-14, comprising at least one cationic lipid (e.g., an ionizable lipid) selected from the group consisting of (20Z, 12Z)-octadeca-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)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), and mixtures thereof. Alternatively, or additionally, 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-Dimemylhexacosa-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-Dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-Dimethyltricosa-14,17 -dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-9-amine, (18Z,21 Z)-N,N-dimethylheptacosa-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-dimethylhentriaconta-22,25-dien-10-amine, (21 (Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacos-18-en-10-amine, (17Z)-N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosane-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20, 23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicosa-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-Dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylhentriacontriacont-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclohexyl] N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-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]henicosan-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1-[(1 S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-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]hexadecan-8-amine Propyl]pentadecan-8-amine, RN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien -1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-i [(11Z,14Z)-Icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docosa-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docosa-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-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)propan-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine, and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-trien-10-amine, and pharmaceutically acceptable salts and stereoisomers thereof, and mixtures thereof.

[0078] Embodiment 16 The helper lipid moiety may be 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-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-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.

[0079] Embodiment 17 A pharmaceutical composition comprising an LNP according to any one of embodiments 1 to 16, and preferably at least one excipient.

[0080] Embodiment 18 18. The pharmaceutical composition of embodiment 17, wherein the pharmaceutical composition is a vaccine, preferably an mRNA vaccine.

[0081] Embodiment 19 19. The pharmaceutical composition of embodiment 17 or embodiment 18 for use in the prevention or treatment of a disease or condition in a patient.

[0082] Embodiment 20 20. The LNP or pharmaceutical composition of any one of embodiments 1 to 19, wherein the LNP has a z-average diameter (as determined by DLS, in particular according to ISO 22412:2017) of 10 nm to 900 nm, preferably 20 nm to 750 nm, more preferably 30 nm to 500 nm, in particular 40 nm to 250 nm, or even 50 nm to 150 nm.

[0083] The present invention is further illustrated by the following figures and examples, but is not limited thereto.

[0084] Figure 1: GDGT increased the cellular uptake of LNPs by 30-fold compared to the reference LNP formulation used in the EMA-approved mRNA vaccine for SARS-CoV-2. (a) Partial replacement of the helper lipid DSPC in the LNP formulation with GDGT (tetraether lipid (“TEL”)) increases the rhodamine-lipid fluorescence signal in cells incubated with LNPs. The reference had DSPC at a concentration of 9.6 mol% of total lipids. (b) Partial replacement of cholesterol (“Chol”) in the LNP formulation with GDGT (“TEL”) increases the rhodamine-lipid fluorescence signal in cells incubated with LNPs. The reference had cholesterol at a concentration of 42.6 mol% of total lipids. (c) Repeated experiments using two references (“A” and “B”) confirmed that partial replacement of the helper lipid DSPC in the LNP formulation with GDGT (“TEL”) increases the rhodamine-lipid fluorescence signal in cells incubated with LNPs, with optimal results achieved at approximately 8 mol% GDGT (“TEL”).

[0085] Figure 2: GDGT increased the transfection efficiency of LNPs by 90-fold compared to the reference LNP formulation used in the EMA-approved mRNA vaccine for SARS-CoV-2. Partial replacement of the helper lipid DSPC with GDGT ("TEL") in the LNP formulation increases enhanced green fluorescent protein (EGFP) mRNA expression in cells upon incubation with LNPs (encapsulating EGFP mRNA), as measured by fluorescence (arbitrary units, y-axis). The reference had DSPC at a concentration of 9.6 mol% total lipid.

[0086] Figure 3: GDGT-LNP was stable at room temperature (RT, 25°C). For the three test 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.

[0087] Example

[0088] LNP formulation Lipid stock solutions (archaeal membrane lipids obtained from Sulfolobus acidocaldarius, including GDGT (see Table 1 below and WO 2020 / 187526), ​​DSPC, ALC-0315, ALC-0159, cholesterol, and rhodamine) were filtered through a 0.2 μm polytetrafluoroethylene (PTFE) filter and mixed according to the molar ratios used in each experiment (see Tables 2 and 3 below). The organic solvent for the reference formulation was ethanol, whereas for formulations containing GDGT, a 2:3 mixture of dimethyl sulfoxide (DMSO) and 2-propanol was used as the organic solvent. Poly(A) (Carl Roth) or mRNA encoding EGFP (CleanCap, 5 moU) was dissolved in 10 mM citrate buffer (pH 4.0) to prepare the aqueous phase. LNPs were formulated using a NanoAssemble® Ignite (Precision Nanosystems) at a total flow rate of 12 mL / min and a flow ratio of 3:1 (aqueous phase:organic phase). Immediately after formulation, the LNPs were diluted 1:2 with 10 mM PBS (pH = 7.4). The organic solvent was removed by dialysis (Spectrapore, 6-8 kDa) against 10 mM phosphate-buffered saline (PBS) buffer (pH = 7.4). Physicochemical characterization of the LNPs was performed by Ribogreen assay (mRNA content) and Zetasizer analysis (size, polydispersity index (PDI), zeta potential). See Table 4 below. The Zetasizer is available from Malvern Panalytical, UK.

[0089] A representative lipid composition of a stock solution of GDGT-containing archaeal membrane lipids (i.e., the ether lipid portion) is shown in Table 1. [Table 1]

[0090] Table 2 shows the test variables for the helper lipid composition. [Table 2]

[0091] Table 3 shows the test variables for cholesterol lipid composition. [Table 3]

[0092] The properties (EE: encapsulation efficiency) of the tested LNPs are shown in Table 4. The z-average diameter was determined by DLS. [Table 4]

[0093] High encapsulation efficiency and low PDI (i.e., monodisperse distribution) were observed for the LNP samples.

[0094] LNP uptake Caco-2 cells were cultured twice and then grown in 1 mL of medium (Dulbecco's Modified Eagle Medium (DMEM) + L-glutamine, 10% fetal bovine serum (FBS), 1% antibiotic mixture) at a cell density of 10 5 Cells were seeded at 1000 μg / well. After 24 hours of incubation (37°C, 5% CO), LNPs were added to the wells at a cargo concentration of 5.2 μg poly(A) / well. After a further 24 hours of incubation, the cells were washed twice with 500 μL of 10 mM PBS buffer to remove any remaining LNPs in the supernatant. This was followed by the addition of Hoechst solution and an additional 5-minute incubation step. After two washing steps with 500 μL of 10 mM PBS buffer, LNP cellular uptake (excitation / emission = 570 nm / 590 nm) and the number of cell nuclei (excitation / emission = 460 nm / 490 nm) were analyzed using confocal microscopy (Olympus IX83).

[0095] GDGT increased the cellular uptake of LNPs compared to the reference LNP formulation used in the EMA-approved mRNA vaccine for SARS-CoV-2 (see Figure 1).

[0096] Transfection using LNPs After two passages, Caco-2 cells were cultured in 950 μL of medium (DMEM + L-glutamine, 10% FBS, 1% antibiotic mixture) in a 48-well plate at a cell density of 10 5 Cells were seeded at 1000 μL / well. After 24 hours of incubation (37°C, 5% CO2), 500 μL of depleted medium was replaced with fresh medium. LNPs encoding EGFP were then added to the wells at a concentration of 6.4 μg / well. Cells were incubated for 60 hours, and fluorescence intensity (excitation / emission = 488 nm / 509 nm) was measured using a confocal microscope (Olympus IX83) at 17, 24, 36, 48, and 60 hours.

[0097] GDGT increased the transfection efficiency of LNPs compared to the reference LNP formulation used in the EMA-approved mRNA vaccine for SARS-CoV-2 (see Figure 2).

[0098] LNP storage After formulation, the LNPs were stored in 2 mL Eppendorf tubes at 4° C. and 25° C. (room temperature), respectively. Physicochemical characterization by Ribogreen assay and Zetasizer analysis was performed at specific time intervals over a period of 6 weeks.

[0099] GDGT-LNP was found to be stable at room temperature (see Figure 3).

[0100] Non-patent literature Aldosari, Basmah N. et al., "Lipid nanoparticles as delivery systems for RNA-based vaccines." Pharmaceutics 13.2(2021):206 Daswani, Varsha P. et al., “The Polar Lipid Fraction E from Sulfolobus acidocaldarius Can Be Used as Liposomal Drug Stabilizing Agents to Reduce the Leakage of the Antivascular Drug Combretastatin A4 Disodium Phosphate from Tetraether / Diester Hybrid Archaeosomes.”, Biophysica 1.4(2021):474~486 Hou, Xucheng et al., "Lipid nanoparticles for mRNA delivery." Nature Reviews Materials 6.12(2021):1078-1094 Jain, Samta et al., "Biosynthesis of archaeal membrane ether lipids." Frontiers in microbiology 5 (2014): 641 Kaur, Gurmeet et al., "Archaeosomes: an excellent carrier for drug and cell delivery." Drug delivery 23.7(2016):2497-2512 "Biophysical Characterization of Viral and Lipid-Based Vectors for Vaccines and Therapeutics with Light Scattering and Calorimetric Techniques." by Markova, Natalia et al., Vaccines 10.1(2021):49 Midoux, Patrick et al., "Lipid-based mRNA vaccine delivery systems." Expert review of vaccines 14.2(2015):221-234 Patel, Girishchandra B. et al., "Archaeobacterial ether lipid liposomes (archaeosomes) as novel vaccine and drug delivery systems." Critical reviews in biotechnology 19.4 (1999): 317~357 Paunovska, Kalina et al., "Drug delivery systems for RNA therapeutics." Nature Reviews Genetics 23.5 (2022): 265-280 Schouten, Stefan et al., "The organic geochemistry of glycerol dialkyl glycerol tetraether lipids: A review." Organic geochemistry 54(2013):19-61 Siliakus, Melvin F. et al., "Adaptations of archaeal and bacterial membranes to variations in temperature, pH, and pressure." Extremophiles 21.4(2017):651-670 Szoka Jr., Frank et al., "Comparative properties and methods of preparation of lipid vesicles (liposomes)." Annual review of biophysics and bioengineering 9.1 (1980): 467-508 van Hoogevest,Peter「Review-an update on the use of oral phospholipid excipients.」、European Journal of Pharmaceutical Sciences 108(2017):1~12. Vishwakarma, Nikhar, “Lipid-based nanocarriers for lymphatic transport.” AAPS PharmSciTech 20.2(2019):1~13 Zavec,Apolonija Bedinaらによる「Archaeosomes can efficiently deliver different types of cargo into epithelial cells grown in vitro.」、Journal of Biotechnology 192(2014):130~135

Claims

1. A lipid nanoparticle (LNP) encapsulating a nucleic acid cargo, the LNP comprising at least a cationic lipid moiety, and Stabilizer part Including, The LNP is a lipid nanoparticle (LNP) encapsulating a nucleic acid cargo, the LNP comprising at least one glycerol dialkyl glycerol tetraether (GDGT) lipid.

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

3. 3. The LNP of claim 1 or claim 2, wherein the stabilizer moiety 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 moiety, preferably cholesterol.

5. The LNP further comprises a helper lipid moiety, and preferably, the helper lipid moiety is selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DOPE), and phosphatidylcholine (DOPE). 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DL ... phosphocholine (1,2-dimyristoyl-sn-glycero-phosphocholine: DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dipalmitoyl-sn-glycero-3-phosphocholine: DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (1,2-diundecanoyl-sn 5. The LNP of claim 1, further comprising a helper lipid selected from the group consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and mixtures thereof.

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

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

8. The LNPs of any one of claims 1 to 7, wherein the LNPs comprise an ether lipid portion comprising at least one GDGT lipid and at least one further ether lipid, preferably wherein the ether lipid portion comprises archaeal and cardoarchaeal.

9. 9. The LNP of claim 8, wherein the ether lipid portion comprises an ether lipid obtained by extraction from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acidocaldarius culture.

10. 10. The LNP of claim 9, wherein all of the ether lipid moieties are obtained from the culture by extraction.

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

12. The LNP according to any one of claims 1 to 11, wherein the molar ratio of the ether lipid moiety to the helper lipid moiety 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, and especially 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 moiety to the sterol lipid moiety 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, and especially 1:3 to 1:

5.

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

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

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