Lipid nanoparticle composition

Abstract

The present invention relates to the field of lipid nanoparticles (LNPs). In particular, the present invention relates to an LNP composition comprising a cationic or cationizable lipid or lipoid material, a helper lipid, a lipid polymer and a monoacylglycerol (MMG) analogue. The LNP composition is particularly suitable for use as a vaccine composition.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of lipid nanoparticles (LNPs). In particular, the present invention relates to a LNP composition comprising a cationic or cationizable lipid or lipidoid material, a helper lipid, a lipid polymer, and a monomycolyl glycerol (MMG) analog. The LNP composition is particularly suitable for use as a vaccine composition. BACKGROUND

[0002] RNA therapeutics, including mRNA vaccines, are susceptible to nuclease degradation and require a delivery system due to their large size and negative charge, which prevents them from penetrating the cell membrane. Lipid nanoparticles (LNPs) are a promising delivery system in this regard. Preventive vaccines based on mRNA-loaded LNPs (mRNA-LNPs) have proven to be very effective against infectious diseases, as evidenced by the mRNA-LNP vaccines developed during the worldwide coronavirus disease 2019 (COVID-19) pandemic. Despite the success of the COVID-19 mRNA vaccines, there are still several shortcomings of the first generation of mRNA vaccines. For example, LNP design is based on the LNP technology that was originally developed for the systemic liver targeting of small interfering RNA (siRNA) for the drug Onpattro® ® which was approved in 2018 for the treatment of polyneuropathy caused by hereditary transthyretin amyloidosis. Therefore, there is a need for 1) engineering RNA delivery systems to more precisely target the immune system and 2) optimizing the delivery system to specifically function as an antigen-encoding RNA carrier and adjuvant.

[0003] WO 2021 / 148511 A1 relates to the field of LNPs, more specifically, the technical solution comprises ionizable lipids, phospholipids, sterols, PEG lipids, and one or more nucleic acids. The prior art document discloses the use of LNPs for the immunogenic delivery of nucleic acid molecules, in particular mRNA, thereby making them particularly suitable for use in vaccines, such as for the treatment of cancer or infectious diseases. Although the LNPs described in WO 2021 / 148511 A1 were developed for mRNA delivery, they do not contain elements that specifically target the immune system, such as ligands for pattern recognition receptors (PRRs) such as integrated pathogen-associated molecular patterns (PAMPs). In view of this, an improved LNP composition would be advantageous, in particular an LNP composition with improved immunogenicity would be even more advantageous. SUMMARY

[0004] In view of the above problems, one object of the present invention relates to a lipid nanoparticle (LNP) composition with improved immunogenicity. In particular, one object of the present invention is to provide an LNP composition that solves the above-mentioned problems of the prior art, is colloidally stable, and demonstrates efficient intracellular delivery of nucleic acids.

[0005] Therefore, one aspect of the present invention relates to a lipid nanoparticle (LNP) composition comprising a cationic or cationic lipid or lipid-like material, an auxiliary lipid, a lipid polymer, and a monoacylglycerol (MMG) analog, wherein the cationic or cationic lipid or lipid-like material is selected from 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecyl-2-ol) (C12-200), N1,N16-bisdodecyl-4,7 ,13-Tris[3-(dodecylamino)-3-oxopropyl]-4,7,10,13-tetraazahexadecanediamide (98N12-5), tetra(8-methylnonyl)3,3',3'',3'''-(((methylazadiyl)bis(propane-3,1-diyl))bis(azatriyl))tetrapropionate (306Oi10), 3,3',3'',3'''-(ethane-1,2-diylbis(azatriyl))tetra(N-(2-((2-hydroxytetradecyl)amino)ethyl)propionamide) (G0-C14), 9-heptadecyl-8-{(2-hydroxy [6-oxo-6-(undecyloxy)hexyl]amino octanoate (SM-102), [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), N1,N3,N5-tris(3-(bisdodecylamino)propyl)phenyl-1,3,5-tricarboxamide (TT3), dilinoleoylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) The group consisting of hexa(octane-3-yl)-9,9',9'',9''',9'''',9''''-((((benzene-1,3,5-tricarbonyl)tri(azadiyl))tri(propane-3,1-diyl))tri(azatriyl))hexanoate (FTT5), dimethyl bis(octadecyl)ammonium bromide (DDAB), 1,2-dioleoyl-3-dimethylammonium chloride (DODAC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or any mixture thereof.

[0006] Among them, the auxiliary lipids are selected from 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphatidyl-(1'-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1,2-distearatel-sn-glycerol-3-phosphatidylcholine (DSPC), and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine. The group consisting of glycerol phosphate (DPPG), 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC), 1,2-diacyl-3-O-β-D-galactosyl-sn-glycerol (MGDG), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (SOPC), 1,2-diacyl-3-O-(α-D-galactosyl1-6)-α-D-galactosyl-sn-glycerol (DGDG) and sulfosucrose diacylglycerol (SQDG), or any mixture thereof, and

[0007] The lipid polymer is polyethylene glycol (PEG) or polysarcosine-lipid conjugate or PEG- or polysarcosine-lipid conjugate or any mixture thereof.

[0008] Another aspect relates to a vaccine composition comprising a lipid nanoparticle (LNP) composition according to the invention and at least one nucleic acid encoding an antigen. Yet another aspect relates to the said vaccine composition for the prevention and / or treatment of infectious diseases.

[0009] One aspect relates to a method for obtaining a lipid nanoparticle (LNP) composition according to the invention, the method comprising the steps of:

[0010] a) Provides cationic or cationic lipid or lipid-like materials, auxiliary lipids, lipid polymers, monoacylglycerol (MMG) analogs, and at least one nucleic acid.

[0011] b) Dissolve the cationic or cationic lipid or lipid-like material, auxiliary lipid, lipid polymer, or MMG analog from step a) in an organic solvent containing ethanol, preferably anhydrous ethanol with a purity close to 100%, thereby providing an organic phase.

[0012] c) Dilute at least one nucleic acid from step a) in an aqueous solvent containing a buffer solution with a pH in the range of 3 to 7.8, thereby providing an aqueous phase.

[0013] d) The organic phase from step b) is mixed with the aqueous phase from step c), and lipid nanoparticles (LNPs) are obtained by nanoprecipitation.

[0014] e) Filter the LNPs from step d), preferably by tangential flow filtration or dialysis, to obtain the LNP composition.

[0015] Among them, cationic or cationic lipids or lipid-like materials are selected from 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200), N1,N16-bisdodecyl-4,7,13-tris[3-(dodecylamino)-3-oxopropyl]-4,7,10,13-tetraazahexadecanediamide (98N12-5), tetra(8 -Methylnonyl)3,3',3'',3'''-(((methylazadiyl)bis(propane-3,1-diyl))bis(azatriyl))tetrapropionate (306Oi10), 3,3',3'',3'''-(ethane-1,2-diylbis(azatriyl))tetra(N-(2-((2-hydroxytetradecyl)amino)ethyl)propionamide) (G0-C14), 9-heptadecyl-8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), [ [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), N1,N3,N5-tris(3-(bisdodecylamino)propyl)phenyl-1,3,5-tricarboxamide (TT3), dilinoleoylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), hexa(octane-3-yl)-9,9',9'' The group consisting of ,9''',9'''',9''''-((((benzene-1,3,5-tricarbonyl)tri(azadiyl))tri(propane-3,1-diyl))tri(azatriyl))hexanoate (FTT5), dimethyl bis(octadecyl)ammonium bromide (DDAB), 1,2-dioleoyl-3-dimethylammonium chloride (DODAC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or any mixture thereof,

[0016] Among them, the auxiliary lipids are selected from 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphatidyl-(1'-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1,2-distearatel-sn-glycerol-3-phosphatidylcholine (DSPC), and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine. The group consisting of glycerol phosphate (DPPG), 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC), 1,2-diacyl-3-O-β-D-galactosyl-sn-glycerol (MGDG), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (SOPC), 1,2-diacyl-3-O-(α-D-galactosyl-1-6)-α-D-galactosyl-sn-glycerol (DGDG) and sulfosucrose diacylglycerol (SQDG), or any mixture thereof.

[0017] Wherein, the lipid polymer is a polyethylene glycol (PEG)- or polysarcosine-lipid conjugate or a PEG- or polysarcosine-lipid conjugate or any mixture thereof, and

[0018] The monoacylglycerol (MMG) analogues are selected from the group consisting of MMG-1, MMG-2, MMG-3, MMG-4, MMG-5, MMG-6 and MMG-7 or any mixture thereof.

[0019] Another aspect relates to lipid nanoparticle (LNP) compositions obtained using the methods of the present invention. Attached Figure Description

[0020] Figure 1A The encapsulation efficiency (%) of FLuc mRNA in different LNP formulations is shown, including C12-200, MMG-1, DOPE, cholesterol, and DMPE-PEG. 2000 Dissolve in an organic phase composed of anhydrous ethanol (gray column), or in an organic phase composed of 90% ethanol and citrate buffer (10 mM, pH 3) (black column).

[0021] Figure 1B shows a cryo-transmission electron microscopy (cryoTEM) image of C12-200 LNPs loaded with FLuc mRNA.

[0022] Figure 1C shows cryo-transmission electron microscopy (cryoTEM) images of C12-200 MMG 23 LNPs loaded with FLuc mRNA, with black arrows indicating the edge surface of C12-200 MMG 23.

[0023] Figure 1D shows cryo-transmission electron microscopy (cryoTEM) images of C12-200 MMG 47 LNPs loaded with FLuc mRNA. The black arrows indicate the polyhedral morphology of C12-200 MMG 47.

[0024] Figure 1E shows the normalized fluorescence intensity of 6-(p-toluidine)-2-naphthalenesulfonyl chloride (TNS) as a function of buffer pH. pKa is defined as the pH at which the normalized fluorescence intensity is 50% (dashed line). Data points represent mean ± sd (n = 3 technical replicates).

[0025] Figure 2 The diagram shows the enhanced eGFP expression in the HEK-Dectin 1 overexpressing immune cell line (right panel) compared to HEK-null cells (left panel) when C12-200 MMG 47 LNPs were transfected with eGFP mRNA-loaded. Data points represent mean ± SD (n=3, independent experiments with three pooled techniques replicated).

[0026] Figure 3 Representative whole-body images of female BALB / c mice injected subcutaneously with C12-200 LNP (1–4) and C12-200 MMG47 (5–8) formulations loaded with FLuc mRNA at the base of the tail are shown. Representative whole-body images were taken in prone and supine positions 6 hours after administration. Scale indicates brightness values ​​at the injection site and liver (range 0.1–2.0 × 10⁻⁶). 8 ).

[0027] Figure 4 The data show the total bioluminescent signal (total flux, supine position) quantitatively detected at the injection site (SOI) at different time points after subcutaneous injection of LNP. The dashed line represents the background, i.e., the total flux of mice treated with PBS and D-fluorescein alone. Data points represent mean ± SD (n=4–5).

[0028] Figure 5 The bioluminescent signal (total throughput, supine position) quantitatively detected at the injection site (SOI) is shown. The dashed line represents the background, i.e., the total throughput of mice treated with PBS and D-fluorescein. Data points represent mean ± SD (n=4–5). ***p<0.001 and ****p<0.0001 are based on one-way ANOVA combined with Dunnett's multiple comparison test.

[0029] Figure 6The ratio of bioluminescent signal (mean brightness value) at the injection site (SOI) and liver quantitatively detected 6 hours after subcutaneous injection of C12-200 LNPs and C12-200 MMG 47 LNPs is shown. Data points are expressed as mean ± SD (n=5).

[0030] Figure 7 The bioluminescent signal (total flux, supine position) was quantitatively detected at the injection site (SOI) 6 hours after subcutaneous injection of MMG-1 modified LNPs formulated with different cationic lipids or lipid-like materials, auxiliary lipids, and lipid polymers. Dashed lines represent background, i.e., the total flux of mice treated with PBS and fluorescein. Data points represent mean ± SD (n = 1–3).

[0031] Figure 8 Representative whole-body images of female BALB / c mice intramuscularly injected with C12-200 LNPs (1–3), C12-200 MMG 47 LNPs (4–6), and SM-102 MMG 38 LNPs (7–9) loaded with Fluc mRNA are shown. Mice injected with PBS (10). Representative whole-body images were taken in supine and lateral positions 6 hours after administration. Scale indicates luminance values ​​at the injection site (range 0.1–1.0 × 10⁻⁶). 8 ).

[0032] Figure 9 The bioluminescent signal (total flux, supine position) was quantitatively detected at the injection site (SOI) at different time points after intramuscular injection of LNP. The dashed line represents the background, i.e., the total flux of mice treated with PBS and D-fluorescein alone. Data points represent mean ± SD (n=3).

[0033] Figure 10 This demonstrates that in mice inoculated with C12-200 LNPs or MMG-1-modified C12-200 LNPs, the effect of OVA... 257-264 MHC-I Tet produces cytokines (IFN-γ, IL-2, IL-4, TNF-α, and IL-17A) after peptide restimulation of lymph node (LN) cells. + CD8 + CD44 + Percentage of T cells (mean ± SD, n=6). One-way ANOVA combined with Tukey's multiple comparison test. p-values ​​were *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0034] Figure 11 This demonstrates that in mice inoculated with C12-200 LNPs or MMG-1-modified C12-200 LNPs, the effect of OVA...323-339 MHC-II Tet peptides, upon restimulation of lymph node cells, produce cytokines (IFN-γ, IL-2, IL-4, TNF-α, and IL-17A). + CD4 + CD44 + Percentage of T cells (n=6, mean ± SD). One-way ANOVA using Tukey's multiple comparison test. p-values ​​were *p<0.05.

[0035] Figure 12A shows the OVA protein-specific T in mice inoculated with LNPs. FH Cell count (n=6, mean ± SD). One-way ANOVA combined with Tukey's multiple comparison test.

[0036] Figure 12B shows the effect of OVA on mice inoculated with LNPs. 257-264 Peptide-specific T FH Cell count (n=6, mean ± SD). One-way ANOVA combined with Tukey's multiple comparison test.

[0037] Figure 12C shows the effect of OVA on mice inoculated with LNPs. 323-339 Peptide-specific T FH Cell count (n=6, mean ± SD). One-way ANOVA combined with Tukey's multiple comparison test.

[0038] Figure 12D shows the number of OVA protein-specific GC B cells in mice inoculated with LNPs (n=6, mean ± SD). One-way ANOVA was performed using Tukey's multiple comparison test. p-values ​​were *p<0.05.

[0039] Figure 12E shows the effect of OVA on mice inoculated with LNPs. 257-264 Number of peptide-specific GC B cells (n=6, mean ± SD). One-way ANOVA combined with Tukey's multiple comparison test.

[0040] Figure 12F shows the effect of OVA on mice inoculated with LNPs. 323-339 Number of peptide-specific GC B cells (n=6, mean ± SD). One-way ANOVA combined with Tukey's multiple comparison test.

[0041] Figure 12G shows the midpoint titer (log EC50) of OVA-specific IgG measured by endpoint dilution ELISA using serum from immunized mice collected 6 weeks after primary immunization. 50Values). Bars represent the mean ± SD, n=6. One-way ANOVA combined with Tukey's multiple comparison test. p-values ​​are *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0042] Figure 12H shows the midpoint titer (log EC50) of OVA-specific IgG1 measured by endpoint dilution ELISA using serum from immunized mice collected 6 weeks after primary immunization. 50 Values). Bars represent the mean ± SD, n=6. One-way ANOVA combined with Tukey's multiple comparison test. p-values ​​are *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0043] Figure 12I shows the midpoint titer (log EC50) of OVA-specific IgG2c as determined by endpoint dilution ELISA using serum from immunized mice collected 6 weeks after primary immunization. 50 Values). Bars represent the mean ± SD, n=6. One-way ANOVA combined with Tukey's multiple comparison test. p-values ​​are *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0044] Figure 13 OVA shows that mice inoculated with LNPs produce cytokines (IFN-γ, IL-2, IL-4, TNF-α, and IL-17A). 257-264 Peptide-specific MHC-I Tet + CD8 + CD44 + Percentage of T cells (mean ± SD, n=6). One-way ANOVA combined with Tukey's multiple comparison test. p-values ​​were *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0045] Figure 14 OVA shows the production of cytokines (IFN-γ, IL-2, IL-4, TNF-α, and IL-17A) in the spleen of mice inoculated with LNPs. 323-339 Peptide-specific MHC-II Tet + CD4 + CD44 + Percentage of T cells (mean ± SD, n=6). One-way ANOVA combined with Tukey's multiple comparison test. p-values ​​are ***p<0.001.

[0046] Figure 15 shows that C12-200, C12-200 MMG 47, and SM-102 MMG 37 LNPs loaded with S mRNA induce S protein-specific CD8 in mouse spleen. + T cell response. (A) Spike protein tetramer (VNFNFNGL) positive CD8 in the spleen. + T cells. (BD)IFNγ + and TNF-α + CD8 + Percentage of T cells. Bars represent mean ± sd (uninoculated mice n=3, inoculated mice n=5-6), and data were analyzed using one-way ANOVA with Dunnett correction for multiple comparisons.

[0047] Figure 16 shows the S protein-specific IgG and virus-neutralizing antibodies in mouse serum induced by C12-200, C12-200 MMG 47, and SM-102 MMG 37 LNPs loaded with S mRNA. (A) Absorbance of mRNA-LNP treated mouse serum at 450 nm versus serial dilutions. (B) Spike protein-specific IgG titer. (C) Serial dilutions of serum incubated with B.1.351 SARS-CoV-2 in 96-well plates. (D) TCID of mouse serum calculated based on plaque reduction neutralizing titer assay. 50 Values. Bars represent the mean ± sd (uninoculated mice n=3, inoculated mice n=5-6), and the data were analyzed using one-way ANOVA with Dunnett correction.

[0048] Figure 17 shows the S protein-specific CD8 in the spleen of mice injected intramuscularly with (A) C12-200 and C12-200 MMG 47 LNPs and (B) SM-102 and SM-102 MMG 38 LNPs loaded with S mRNA. + T cell response. Bars represent mean ± sd (uninoculated mice n=3, inoculated mice n=4-6).

[0049] Figure 18 shows the anti-spike protein IgG titers in mouse serum after intramuscular immunization with (A) C12-200 and C12-200 MMG 47 LNPs loaded with S mRNA and (B) SM-102 and SM-102 MMG 38 LNPs. Bars represent mean ± sd (n=6).

[0050] Figure 19 shows the ability of uninoculated mice and mice inoculated with LNPs loaded with S mRNA to neutralize SARS-CoV-2. (A) Serial dilutions of serum incubated with B.1.351 SARS-CoV-2 in 96-well plates. (B) TCID of mouse serum calculated based on plaque reduction neutralizing titer assay. 50 Values. Data are expressed as mean ± sd (uninoculated mice n=3, inoculated mice n=5-6), and the data were analyzed using one-way ANOVA with Dunnett correction.

[0051] Figure 20 The bioluminescent signal (total throughput) in the supine position was quantitatively detected at different time points after subcutaneous injection of MMG-1 modified LNPs loaded with FLuc RNA. The dashed line represents the background, i.e., the total throughput of mice treated with PBS and D-luciferin alone. Data points represent mean ± SD (n=3).

[0052] Figure 21 This shows the total bioluminescent signal (total flux) quantified at the injection site (SOI) in the supine position 6 hours after intramuscular injection of an MMG analog-modified LNP loaded with FLuc mRNA. Corresponding to 10 5 The intersection of the Y and X axes in photons / second represents the background signal, i.e., the total flux of mice treated with PBS and D-fluorescein only. Data points are represented as mean ± SD (n=3) and mean (n=2).

[0053] Figure 22 This study demonstrates a dose-dependent decrease in the TNF-α gene gene in mouse macrophages when transfected with LNPs containing small interfering RNA (siRNA) targeting TNF-α. Data points represent mean ± SD (n = 3 technical replicates).

[0054] Figure 23 The bioluminescent signal (total throughput) in the supine position, quantified at the injection site (SOI), is shown 6 hours after subcutaneous injection of MMG-1-modified LNPs loaded with FLuc mRNA. The dashed line represents the background, i.e., the total throughput of mice treated only with PBS and D-luciferin. LNPs were added to the cryoprotectant sucrose before rapid freezing in liquid nitrogen and then stored at -80°C or -20°C for one week, thawed, and used for injection. Data points represent the mean (n=1–2).

[0055] The present invention will be described in more detail below. Detailed Implementation

[0056] definition

[0057] Before discussing the invention in further detail, the following terms and conventions are first defined.

[0058] Lipid nanoparticle (LNP)

[0059] In this article, the term "lipid nanoparticles" is abbreviated as LNP, referring to nanoparticles composed of lipids. Lipid nanoparticles are typically spherical, with an average diameter between 10 and 1000 nanometers.

[0060] LNPs typically comprise four components: cationic or cationizable lipids or lipid-like materials, accessory lipids, cholesterol, and lipid polymers. Cationic or cationizable lipids or lipid-like materials play a crucial role in protecting mRNA from nucleases and their intracellular release. Cationable lipids are cationic under acidic pH conditions, where they form electrostatic complexes with mRNA, but are neutral under physiological pH conditions to minimize cytotoxicity. Cationic lipids or lipid-like materials are unaffected by environmental factors and consistently maintain their cationic charge. Structural accessory lipids, such as phospholipid distearate phosphatidylcholine (DSPC) and dioleoylphosphatidylethanolamine (DOPE), maintain bilayer stability during storage and cycling and improve mRNA encapsulation performance. Cholesterol plays multiple roles, including filling intergranular gaps, limiting LNP-protein interactions, maintaining membrane integrity, and potentially promoting membrane fusion. Lipid polymers, which can be polyethylene glycol-modified lipids or polysarcosine-lipid conjugates, achieve colloidal stability of LNPs by forming a hydrophilic spatial barrier on the LNP surface, thereby preventing aggregation in formulations.

[0061] Cationic or cationizable lipid or lipidoid material

[0062] In this article, the term "cationic or cationizable lipid or lipid-like material" refers to lipids or lipid-like materials that are permanently positively charged (cationic) or become positively charged (cationizable) upon pH change. Specifically, "ionizable lipids or lipid-like materials" are lipids that are positively charged under acidic pH conditions and neutral under physiological pH conditions (~7.4). Ionizable lipids or lipid-like materials are positively charged under acidic pH conditions to concentrate RNAs and load them into LNPs, while remaining neutral under physiological pH conditions to minimize toxicity. After being taken up by cells, these lipids are protonated in acidic endosomes and interact with anionic endosome phospholipids to form pyramidal ion pairs incompatible with the lipid bilayer. These cation-anionic lipid pairs drive the bilayer structure toward an inverted hexagonal H+. II The phase transition is presumably to promote membrane fusion / disruption, endosome escape, and load release into the cytoplasm.

[0063] In this document, the terms "lipid" and "lipid-like materials" are broadly defined, referring to molecules comprising one or more hydrophobic moieties or groups and optionally one or more hydrophilic moieties or groups. Molecules comprising both hydrophobic and hydrophilic moieties are also often referred to as amphiphilic substances. Lipids typically have low solubility in water. In aqueous environments, their amphiphilic properties allow these molecules to self-assemble into ordered structures and different phases. One of these phases consists of lipid bilayers, present in vesicles, monolayer / multilayer liposomes, or membrane structures in aqueous environments. Hydrophobicity can be achieved by introducing nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups may include polar and / or charged groups, including carbohydrate, phosphate, carboxyl, sulfate, amino, thiol, nitro, hydroxyl, and other similar groups.

[0064] Lipidoid material or lipidoid conjugate

[0065] The terms “lipid-like material,” “lipid-like compound,” “lipid-like conjugate,” “lipid-like molecule,” or “lipid” refer to substances that are structurally and / or functionally related to lipids but are not strictly considered lipids. For example, the term encompasses compounds capable of forming amphiphilic layer structures when present in an aqueous environment in vesicles, monolayers / multilayers of liposomes, or membranes, and includes surfactants or synthetic compounds having both hydrophilic and hydrophobic portions. Generally, the term refers to molecules containing hydrophilic and hydrophobic portions with different structural organization, whose structures may be similar to or dissimilar to lipids. When used herein, unless otherwise stated or clearly contradicted by the context, the term “lipid” should be interpreted to encompass both lipids and lipid-like materials.

[0066] Helper lipid

[0067] Accessory lipids are a class of lipid molecules that increase the stability and mobility of LNPs (lipid-dependent protein particles). Several types of molecules can be used as accessory lipids, such as phospholipids represented by DSPC and DOPE, and sterols represented by cholesterol.

[0068] Lipopolymer

[0069] In this article, the term "lipid polymer" refers to any polymer covalently linked to a lipid (fatty acid or steroid) moiety, such as a polyethylene glycol-modified lipid or a polysarcosine lipid.

[0070] Monoacylglycerol (MMG) and MMG analogs

[0071] Mycobacterial cell wall lipids, specifically mono-branched acylglycerol (MMG) or its analogues, are glycerol lipids.

[0072] Glycerol lipids such as MMG have immunomodulatory properties and can enhance immune responses. However, MMG is too toxic for human use. Therefore, well-tolerated synthetic analogs of MMG have been developed.

[0073] This synthetic analog, designated MMG-1, consists of a hydrophilic glycerol head and a lipid acid, with two hydrophobic saturated C14 / C15 alkyl tails linked by ester bonds. Furthermore, a series of MMG analogs with varying alkyl chain lengths (MMG-2; C16 / C17, MMG-3; C10 / C11 and MMG-4; C6 / C7) or stereochemistry targeting the head (MMG-5; 2S) and lipid tails (MMG-6, MMG-7) have been designed.

[0074] MMG is preferably synthesized from the glycerol lipid MMG-1. Monoacylglycerol-1 (MMG-1) is a short-chain synthetic analog of monoacylglycerol (MMG), a lipid of the mycobacterial cell wall. MMG-1 binds to a pattern recognition receptor (PRR) C-type lectin receptor (CLR) called a mincle. After incorporation into liposomes, MMG-1 exerts immunomodulatory effects by activating dendritic cells (DCs), thereby promoting the secretion of the Th1 cytokine interferon-γ (IFN-γ) and the Th17 cytokine interleukin-17 (IL-17). The preferred MMG analog has the chemical structure of 3-hydroxy-2-tetradecyl-octadecanoate-2,3-dihydroxypropyl ester, preferably the diastereomer of (2R)-2,3-dihydroxypropyl-3-hydroxy-2-tetradecyl-octadecanoate. Both MMG-6 and MMG-7 exhibit an inverted hexagonal phase (H). II This gives it a stronger ability to escape endosomes, thus enabling more efficient release of nucleic acid payloads compared to other MMG variants. Therefore, MMG-6 and MMG-7 are preferred analogues used in this invention.

[0075] Cholesterol

[0076] Cholesterol is a sterol with the IUPAC name (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptane-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecano-1H-cyclopenta[a]phenanthrene-3-ol. Cholesterol is biosynthesized by all animal cells and constitutes a fundamental structural component of animal cell membranes by providing stability. In LNPs (lower neutrophils), cholesterol plays multiple roles, including filling intergranular spaces, limiting LNP-protein interactions, maintaining membrane integrity, and potentially promoting membrane fusion.

[0077] Nucleic acid

[0078] In this document, the term "nucleic acid" refers to deoxyribonucleic acid (DNA) or, preferably, ribonucleic acid (RNA), more preferably mRNA. Nucleic acid may also be referred to as a "nucleic acid payload" because it is not part of an LNP vector but is loaded into the LNP. Nucleic acids include, but are not limited to, genomic DNA, plasmid DNA, cDNA, mRNA, and recombinant-generated or chemically synthesized molecules. Nucleic acids according to the invention can be in single-stranded or double-stranded molecular form and can be linearly or covalently closed to form a loop. Nucleic acids can be used for cell introduction, i.e., cell transfection; for example, the RNA form can be prepared by enzymatic reactions via in vitro transcription based on a DNA template. Nucleic acids can be chemically modified by containing one or more chemically modified nucleosides, such as pseudouridine, N1-methylpseudouridine, or nucleosides with 2'-O-methylation. Furthermore, nucleic acids can also contain phosphodiester bonds, thiophosphate bonds, or mixtures thereof. Additionally, RNA can be modified prior to application by stabilizing the sequence, capping, and / or polyadenylation.

[0079] RNA molecule

[0080] In this invention, the term "RNA molecule" refers to a molecule containing ribonucleotide residues, and preferably consists entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of the β-D-furanose group. This term includes isolated RNA such as double-stranded RNA, single-stranded RNA, partially purified RNA, substantially pure RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA through the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide materials, for example, modification of one or more nucleotides of RNA at the ends or interior of the RNA. Nucleotides in RNA molecules may also include non-standard nucleotides, such as non-naturally occurring nucleotides, chemically synthesized nucleotides, or deoxynucleotides.

[0081] Organic phase

[0082] The term "organic phase" refers to an organic solvent in which lipids, auxiliary lipids, lipid polymers, MMG analogs, and optionally cholesterol are dissolved, such as cationic or cationic lipids or lipid-like materials. The organic solvent is ethanol, preferably anhydrous ethanol with a purity close to 100%.

[0083] Aqueous phase

[0084] The term "aqueous phase" refers to an aqueous solvent in which at least one nucleic acid is dissolved. The aqueous solvent is a buffer solution, preferably with a pH of 3-7.8.

[0085] Buffer

[0086] A buffer solution (more precisely, a pH buffer or hydrogen ion buffer) is an acidic or alkaline aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. The pH value changes very little when a small amount of a strong acid or base is added. In various chemical applications, buffer solutions are used as a means of maintaining a near-constant pH value. In a preferred embodiment, the buffer solution is a citrate buffer. A citrate buffer is a buffer mixture of sodium citrate and citric acid. By acting as a chelating agent, a citrate buffer can be used for RNA isolation by preventing alkaline hydrolysis and magnesium ion-dependent RNA breakage. In a preferred embodiment of the invention, the pH of the citrate buffer is in the range of 3.0-4.0.

[0087] Nano-precipitation

[0088] Nanoprecipitation is a method for generating LNPs, which involves mixing an organic phase containing different lipids dissolved in an organic solvent with an aqueous phase containing at least one nucleic acid dissolved in an aqueous phase. Nanoprecipitation can be achieved in various ways, including but not limited to mixing the two phases using pipettes, T-mixers, microfluidic mixing, or impingement jet mixers.

[0089] Microfluidic mixing

[0090] Microfluidic mixing enables thorough and rapid mixing of multiple solvents in microscale devices. In this invention, LNPs are generated by mixing an organic phase containing different lipids dissolved in an organic solvent with an aqueous phase containing at least one nucleic acid dissolved in an aqueous phase. Each phase is then injected into a microfluidic chip containing channels with dimensions ranging from tens to hundreds of micrometers.

[0091] Flow ratio

[0092] The term "flow ratio," abbreviated as FRR, is used in this paper in the context of microfluidic mixing, where the flow ratio is the ratio of the flow rate of the aqueous phase to the flow rate of the organic phase. Therefore, a flow ratio of 3:1 means that the aqueous phase flows through the microfluidic mixing chip three times faster than the organic phase.

[0093] Total flow

[0094] In this article, the term "total flow rate" is used in microfluidic mixing-related scenarios, referring to the total flow rate of the aqueous and organic phases in a microfluidic mixing chip.

[0095] Antigen

[0096] In this article, the term "antigen" refers to molecules such as immunogenic peptides that can induce an immune response. Immune responses generated by antigens can be B cell-driven (antibody-mediated immune responses) and / or T cell-driven (cellular immune responses).

[0097] Dosing

[0098] In this invention, the term "administration" refers to administration by various means, including systemic administration such as intramuscular injection, subcutaneous injection, intradermal injection, or intraperitoneal injection, or administration by means of delivery of a formulation or device, such as local administration, intradermal administration, intranasal administration, sublingual administration, oral administration, or pulmonary administration.

[0099] The LNP and / or vaccine compositions according to the invention are typically administered via non-oral routes, more typically by subcutaneous or intramuscular injection, at a frequency ranging from once every two weeks to once or twice a month to once or twice a year.

[0100] Lipid nanoparticle composition

[0101] One object of the present invention relates to a lipid nanoparticle (LNP) composition having improved immunogenicity, which is colloidally stable and exhibits efficient intracellular delivery of nucleic acids. Therefore, one aspect of the present invention relates to a lipid nanoparticle (LNP) composition comprising a cationic or cationically cationizable lipid or lipid-like material, an auxiliary lipid, a lipid polymer, and a monoacylglycerol (MMG) analog.

[0102] Among them, cationic or cationic lipids or lipid-like materials are selected from 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200), N1,N16-bisdodecyl-4,7,13-tris[3-(dodecylamino)-3-oxopropyl]-4,7,10,13-tetraazahexadecanediamide (98N12-5), tetra(8 -Methylnonyl)3,3',3'',3'''-(((methylazadiyl)bis(propane-3,1-diyl))bis(azatriyl))tetrapropionate (306Oi10), 3,3',3'',3'''-(ethane-1,2-diylbis(azatriyl))tetra(N-(2-((2-hydroxytetradecyl)amino)ethyl)propionamide) (G0-C14), 9-heptadecyl-8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), [ [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), N1,N3,N5-tris(3-(bisdodecylamino)propyl)phenyl-1,3,5-tricarboxamide (TT3), dilinoleoylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), hexa(octane-3-yl)-9,9',9'' The group consisting of ,9''',9'''',9''''-((((benzene-1,3,5-tricarbonyl)tri(azadiyl))tri(propane-3,1-diyl))tri(azatriyl))hexanoate (FTT5), dimethyl bis(octadecyl)ammonium bromide (DDAB), 1,2-dioleoyl-3-dimethylammonium chloride (DODAC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or any mixture thereof,

[0103] Among them, the auxiliary lipids are selected from 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphatidyl-(1'-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1,2-distearatel-sn-glycerol-3-phosphatidylcholine (DSPC), and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine. The group consisting of glycerol phosphate (DPPG), 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC), 1,2-diacyl-3-O-β-D-galactosyl-sn-glycerol (MGDG), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (SOPC), 1,2-diacyl-3-O-(α-D-galactosyl1-6)-α-D-galactosyl-sn-glycerol (DGDG) and sulfosucrose diacylglycerol (SQDG), or any mixture thereof, and

[0104] The lipid polymer is polyethylene glycol (PEG) or polysarcosine-lipid conjugate or PEG- or polysarcosine-lipid conjugate or any mixture thereof.

[0105] Another aspect of the present invention relates to a lipid nanoparticle (LNP) composition comprising cationic or cationic lipid or lipid-like materials, auxiliary lipids, lipid polymers, and monoacylglycerol (MMG) analogs.

[0106] Among them, cationic or cationic lipids or lipid-like materials are selected from 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200), N1,N16-bisdodecyl-4,7,13-tris[3-(dodecylamino)-3-oxopropyl]-4,7,10,13-tetraazahexadecanediamide (98N12-5), tetra(8 -Methylnonyl)3,3',3'',3'''-(((methylazadiyl)bis(propane-3,1-diyl))bis(azatriyl))tetrapropionate (306Oi10), 3,3',3'',3'''-(ethane-1,2-diylbis(azatriyl))tetra(N-(2-((2-hydroxytetradecyl)amino)ethyl)propionamide) (G0-C14), 9-heptadecyl-8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), [ [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), N1,N3,N5-tris(3-(bisdodecylamino)propyl)phenyl-1,3,5-tricarboxamide (TT3), dilinoleoylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), hexa(octane-3-yl)-9,9',9'' The group consisting of ,9''',9'''',9''''-((((benzene-1,3,5-tricarbonyl)tri(azadiyl))tri(propane-3,1-diyl))tri(azatriyl))hexanoate (FTT5), dimethyl bis(octadecyl)ammonium bromide (DDAB), 1,2-dioleoyl-3-dimethylammonium chloride (DODAC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or any mixture thereof,

[0107] Among them, the auxiliary lipids are selected from 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphatidyl-(1'-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1,2-distearatel-sn-glycerol-3-phosphatidylcholine (DSPC), and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine. The group consisting of glycerol phosphate (DPPG), 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC), 1,2-diacyl-3-O-β-D-galactosyl-sn-glycerol (MGDG), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (SOPC), 1,2-diacyl-3-O-(α-D-galactosyl1-6)-α-D-galactosyl-sn-glycerol (DGDG) and sulfosucrose diacylglycerol (SQDG), or any mixture thereof, and

[0108] The lipid polymer is polyethylene glycol (PEG) or polysarcosine-lipid conjugate or PEG- or polysarcosine-lipid conjugate or any mixture thereof.

[0109] Another aspect of the present invention relates to a lipid nanoparticle (LNP) composition comprising a cationic or cationic lipid or lipid-like material, an auxiliary lipid, a lipid polymer, and monoacylglycerol (MMG).

[0110] Among them, cationic or cationic lipids or lipid-like materials are selected from 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200), N1,N16-bisdodecyl-4,7,13-tris[3-(dodecylamino)-3-oxopropyl]-4,7,10,13-tetraazahexadecanediamide (98N12-5), tetra(8 -Methylnonyl)3,3',3'',3'''-(((methylazadiyl)bis(propane-3,1-diyl))bis(azatriyl))tetrapropionate (306Oi10), 3,3',3'',3'''-(ethane-1,2-diylbis(azatriyl))tetra(N-(2-((2-hydroxytetradecyl)amino)ethyl)propionamide) (G0-C14), 9-heptadecyl-8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), [ [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), N1,N3,N5-tris(3-(bisdodecylamino)propyl)phenyl-1,3,5-tricarboxamide (TT3), dilinoleoylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), hexa(octane-3-yl)-9,9',9'' The group consisting of ,9''',9'''',9''''-((((benzene-1,3,5-tricarbonyl)tri(azadiyl))tri(propane-3,1-diyl))tri(azatriyl))hexanoate (FTT5), dimethyl bis(octadecyl)ammonium bromide (DDAB), 1,2-dioleoyl-3-dimethylammonium chloride (DODAC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or any mixture thereof,

[0111] Among them, the auxiliary lipids are selected from 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphatidyl-(1'-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1,2-distearatel-sn-glycerol-3-phosphatidylcholine (DSPC), and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine. The group consisting of glycerol phosphate (DPPG), 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC), 1,2-diacyl-3-O-β-D-galactosyl-sn-glycerol (MGDG), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (SOPC), 1,2-diacyl-3-O-(α-D-galactosyl1-6)-α-D-galactosyl-sn-glycerol (DGDG) and sulfosucrose diacylglycerol (SQDG), or any mixture thereof, and

[0112] The lipid polymer is polyethylene glycol (PEG) or polysarcosine-lipid conjugate or PEG- or polysarcosine-lipid conjugate or any mixture thereof.

[0113] The lipid polymer in the LNP composition contributes to the colloidal stability of the LNPs. Polyethylene glycol (PEG)-lipid conjugates or PEG-lipid-like conjugates are typically used as the lipid polymer in the LNP composition. Therefore, in one embodiment, the lipid polymer is a polyethylene glycol (PEG)-lipid conjugate or a PEG-lipid-like conjugate. However, polysarcosine, possessing PEG-like properties such as excellent water solubility, protein resistance, low cytotoxicity, and non-immunogenicity, and being based on endogenous materials, is considered a promising alternative to PEG-lipids or PEG-lipid-like conjugates. Furthermore, some individuals are allergic to PEG, making it particularly important to provide an alternative to PEG-lipids or PEG-lipid-like conjugates in such cases. Polysarcosine-functionalized LNPs have been reported in the literature, and the described polysarcosine-based LNPs can safely and efficiently deliver mRNA, laying a good foundation for the development of PEG-free RNA therapeutics. Therefore, another embodiment relates to an LNP composition, wherein the lipid polymer is selected from 1,2-dimyristoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG). 2000 ), 1,2-Dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG) 2000), 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) 2000 ), 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE-PEG), 1,2-distearyl-rac-glycerol-3-methoxy polyethylene glycol (DSG-PEG), ceramide-PEG, 1,2-dipalmitoyl-rac-glycerol-3-methylpolyoxyethylene (DPG-PEG), 1,2-dioleoyl-rac-glycerol, methoxy polyethylene glycol The group consisting of (DOG-PEG), 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine-N-methylpolyoxyethylene (DOPE-PEG), N-tetradecyl polysarcosine 25, N-hexadecyl polysarcosine 25, N-octadecyl polysarcosine 25, N-dodecyl polysarcosine 25, N,N-bistetradecylamine-N-succinoyl[methyl(polysarcosine)45], N,N-bistetradecylamine-N-succinoyl[methyl(polysarcosine)35] and N,N-bistetradecyl polysarcosine-25 or mixtures thereof.

[0114] In one embodiment, the cationic or cationic lipid or lipid-like material is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200), tetra(8-methylnonyl)3,3',3'',3'''-(((methylazadiyl)bis(propane-3,1-diyl))bis(azatriyl))tetrapropionate (306Oi10), 9-heptadecyl-8-{(2-hydroxy The preferred formulations are: [6-oxo-6-(undecyloxy)hexyl]amino octanoate (SM-102), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), dimethyl bis(octadecyl)ammonium bromide (DDAB), 1,2-dioleoyl-3-dimethylammonium chloride (DODAC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), or [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), preferably C12-200 or SM-102.

[0115] In another embodiment, the auxiliary lipid is 1,2-distearyl-sn-glycerol-3-phosphatidylcholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (SOPC) or 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), preferably DSPC or DOPE.

[0116] In yet another embodiment, the lipid polymer is 1,2-dimyristoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG). 2000 ), 1,2-distearyl-rac-glycerol-3-methoxy polyethylene glycol (DSG-PEG), N-tetradecyl polysarcosine 25, 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) 2000 ) or 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG) 2000 ), preferably DMPE-PEG 2000 or DMG-PEG 2000 .

[0117] In a preferred embodiment, the cationic or cationic lipid or lipid-like material is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200), the auxiliary lipid is 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), and the lipid polymer is 1,2-dimyristoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG). 2000 ).

[0118] Determining MMG

[0119] Cholesterol is one of the four components typically contained in LNPs. However, embodiments of the present invention demonstrate that cholesterol can be successfully replaced by monoacylglycerol (MMG) analogs. MMG analogs have immunomodulatory properties, enhancing immune responses and thus making the resulting LNPs more immunogenic. Therefore, in one embodiment, the monoacylglycerol (MMG) analog is selected from the group consisting of MMG-1, MMG-2, MMG-3, MMG-4, MMG-5, MMG-6, and MMG-7, or any mixture thereof. In a preferred embodiment, the monoacylglycerol (MMG) analog is MMG-1, MMG-6, and / or MMG-7, preferably MMG-1.

[0120] In one embodiment, the monoacylglycerol (MMG) analog is added in a molar content ranging from 5 mol% to 70 mol% relative to the total molar content of the LNP composition, such as from 8 mol% to 60 mol%, or from 10 mol% to 50 mol%. As shown in Table 1, MMG-1 is added in a molar content ranging from 0 mol% to 48 mol% relative to the total molar content of the LNP composition.

[0121] Determining LNP composition - cholesterol

[0122] One embodiment of the present invention relates to a lipid nanoparticle (LNP) composition, wherein the LNP further comprises cholesterol. In one embodiment, cholesterol is added in a molar content ranging from 5 mol% to 70 mol% relative to the total molar content of the LNP composition, such as from 8 mol% to 60 mol%, or from 10 mol% to 50 mol%.

[0123] As shown in Table 1, in C12-200-based LNPs, cholesterol is gradually replaced by MMG-1. Therefore, in one embodiment, the ratio of MMG analog to cholesterol is in the range of 1:100 to 100:1, such as 1:4, 1:1, or 4:1.

[0124] Determining content of cationic or cationizable lipid or lipidoid material, helper lipid, and lipopolymer

[0125] The content of cationic or cationic lipids or lipid-like materials, auxiliary lipids, and lipid polymers can vary considerably depending on the type of lipids or lipid-like materials or lipid polymers used. Therefore, in one embodiment, the cationic or cationic lipids or lipid-like materials are added in a molar content ranging from 10 mol% to 60 mol% relative to the total molar content of the LNP composition, such as 15 mol% to 55 mol%, 20 mol% to 50 mol%, 25 mol% to 45 mol%, preferably 30 mol% to 40 mol%. In another embodiment of the invention, the auxiliary lipids are added in a molar content ranging from 5 mol% to 30 mol% relative to the total molar content of the LNP composition, such as 8 mol% to 25 mol%, preferably 10 mol% to 20 mol%. In yet another embodiment, the lipid polymers are added in a molar content ranging from 0.5 mol% to 50 mol% relative to the total molar content of the LNP composition, such as 0.8 mol% to 40 mol%, 1 mol% to 30 mol%, 2 mol% to 20 mol%, 5 mol% to 10 mol%, preferably 1 mol% to 2 mol%.

[0126] Determining LNP composition - nucleic acid

[0127] The LNP composition of the present invention can be used as a delivery system for several payloads such as peptides or nucleic acids. Therefore, in one embodiment, the LNP further comprises at least one nucleic acid. This nucleic acid can be a mixture of different nucleic acids or the same nucleic acid. Therefore, in one embodiment, the at least one nucleic acid is a mixture of nucleic acids.

[0128] Depending on the therapeutic purpose, several different nucleic acids may be relevant payloads, such as mRNA for mRNA vaccines, siRNA for knocking out the target protein, or gRNA for CRISPR / Cas9 technology to knock out the target protein. Therefore, in one embodiment of the invention, at least one nucleic acid is selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA, circular RNA (circRNA), plasmid DNA (pDNA), small interfering RNA (siRNA), single guide RNA (sgRNA), guide RNA (gRNA), long non-coding RNA (lncRNA), small activating RNA (saRNA), and splice-conversion antisense oligonucleotides (ASO).

[0129] For example, siRNA can be used to downregulate the expression of tumor necrosis factor-α (TNF-α). Therefore, in one embodiment, at least one nucleic acid is siRNA, preferably targeting tumor necrosis factor-α (TNF-α) (SEQ ID NO.: 3, SEQ ID NO.: 4). In a preferred embodiment, at least one nucleic acid is an RNA molecule or a mixture of RNA molecules. The nucleic acid used in the examples is RNA, especially mRNA. Therefore, in a more preferred embodiment, at least one nucleic acid is mRNA or a mixture of mRNA.

[0130] The nucleic acids used as load cells in these LNPs can be modified by chemically modified nucleosides or by different bonds. For example, these modifications can help stabilize the nucleic acids or make them more resistant to nucleases. Thus, in one embodiment, at least one nucleic acid may contain at least one chemically modified nucleoside, such as pseudouridine, N1-methylpseudouridine, and 2'-O-methylated nucleosides. In yet another embodiment, at least one nucleic acid may contain a phosphodiester bond, a thiophosphoester bond, or a mixture thereof, preferably a phosphodiester bond.

[0131] Vaccine composition

[0132] The LNP composition of the present invention is particularly suitable for use as a vaccine composition, as shown in Example 6, which exhibits improved immunogenicity compared to LNP compositions without MMG analogs. Therefore, one aspect of the present invention relates to a vaccine composition comprising a lipid nanoparticle (LNP) composition according to the present invention and at least one nucleic acid encoding an antigen. In one embodiment, the antigen is an antigen derived from a pathogen causing an infectious disease. In yet another embodiment, the antigen is selected from the group consisting of coronavirus antigens such as the SARS-CoV2 spike protein (SEQ ID NO.: 5) or receptor-binding domain (RBD) of SARS-CoV and MERS-CoV antigens, Mycobacterium tuberculosis antigen (SEQ ID NO.: 8-9), Plasmodium falciparum antigen (SEQ ID NO.: 10), respiratory syncytial virus (RSV) antigen (SEQ ID NO.: 11), Ebola virus antigen, Marburg virus antigen, Lassa virus antigen, Nipah virus antigen, Zika virus antigen, Crimean-Congo hemorrhagic fever virus antigen, human papillomavirus (HPV) antigen, and influenza virus antigen. As illustrated in the examples, LNP compositions can effectively deliver mRNA payloads, making them particularly useful in mRNA vaccines. Therefore, in one embodiment, the antigen is encoded by mRNA.

[0133] Use of vaccine composition

[0134] One aspect relates to a vaccine composition according to the invention for the prevention and / or treatment of infectious diseases. In one embodiment, the infectious disease is selected from the group consisting of tuberculosis, COVID-19, MERS-CoV infection, SARS-CoV infection, malaria, RSV infection, Ebola, Marburg virus infection, Lassa fever, Nipah virus infection, Zika virus infection, Crimean-Congo hemorrhagic fever, HPV infection, and influenza.

[0135] Vaccine dosing

[0136] In Examples 3 and 4, the LNP composition was administered to mice via subcutaneous injection. However, as a vaccine composition, the LNP composition according to the invention and at least one nucleic acid encoding an antigen can be administered via other routes of administration. Therefore, in one embodiment, the vaccine composition is administered to the subject via intradermal, intraperitoneal, intravenous, intramuscular, or subcutaneous injection. Another embodiment relates to a vaccine composition used according to the invention, wherein the subject is a mammal, such as a human, non-human primate, calf, pig, horse, sheep, goat, mink, ferret, hamster, cat, bird, or dog. In a preferred embodiment, the subject is a human.

[0137] In one embodiment, the vaccine composition is administered in a single dose. However, vaccines are typically administered in multiple-dose regimens, such as the COVID-19 mRNA vaccine SpikeVax. ® and Comirnaty ® Therefore, in one embodiment, the vaccine composition is administered at at least two doses, such as at least three doses.

[0138] Method of obtaining a lipid nanoparticle (LNP) composition

[0139] like Figure 1A As shown, the inventors discovered that C12-200, MMG-1, DOPE, cholesterol, and DMPE-PEG... 2000 When dissolved in an organic phase consisting of anhydrous ethanol with a purity of at least 99.5%, preferably at least 99.8%, the mRNA encapsulation efficiency of LNPs loaded with FLuc mRNA (SEQ ID NO.: 1) increases compared to the case with 90% ethanol and citrate buffer (10 mM, pH 3). Therefore, one aspect relates to a method for obtaining a lipid nanoparticle (LNP) composition according to the invention, the method comprising the following steps:

[0140] a) Provides cationic or cationic lipid or lipid-like materials, auxiliary lipids, lipid polymers, monoacylglycerol (MMG) analogs, and at least one nucleic acid.

[0141] b) Dissolve the cationic or cationic lipid or lipid-like material, auxiliary lipid, lipid polymer, or MMG analog from step a) in an organic solvent containing ethanol, preferably anhydrous ethanol with a purity close to 100%, thereby providing an organic phase.

[0142] c) Dilute at least one nucleic acid from step a) in an aqueous solvent containing a buffer solution with a pH in the range of 3 to 7.8, thereby providing an aqueous phase.

[0143] d) The organic phase from step b) is mixed with the aqueous phase from step c), and lipid nanoparticles (LNPs) are obtained by nanoprecipitation.

[0144] e) Filter the LNPs from step d), preferably by tangential flow filtration or dialysis, to obtain the LNP composition.

[0145] Among them, cationic or cationic lipids or lipid-like materials are selected from 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200), N1,N16-bisdodecyl-4,7,13-tris[3-(dodecylamino)-3-oxopropyl]-4,7,10,13-tetraazahexadecanediamide (98N12-5), tetra(8 -Methylnonyl)3,3',3'',3'''-(((methylazadiyl)bis(propane-3,1-diyl))bis(azatriyl))tetrapropionate (306Oi10), 3,3',3'',3'''-(ethane-1,2-diylbis(azatriyl))tetra(N-(2-((2-hydroxytetradecyl)amino)ethyl)propionamide) (G0-C14), 9-heptadecyl-8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), [ [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), N1,N3,N5-tris(3-(bisdodecylamino)propyl)phenyl-1,3,5-tricarboxamide (TT3), dilinoleoylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), hexa(octane-3-yl)-9,9',9'' The group consisting of ,9''',9'''',9''''-((((benzene-1,3,5-tricarbonyl)tri(azadiyl))tri(propane-3,1-diyl))tri(azatriyl))hexanoate (FTT5), dimethyl bis(octadecyl)ammonium bromide (DDAB), 1,2-dioleoyl-3-dimethylammonium chloride (DODAC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or any mixture thereof,

[0146] Among them, the auxiliary lipids are selected from 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphatidyl-(1'-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1,2-distearatel-sn-glycerol-3-phosphatidylcholine (DSPC), and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine. The group consisting of glycerol phosphate (DPPG), 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC), 1,2-diacyl-3-O-β-D-galactosyl-sn-glycerol (MGDG), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (SOPC), 1,2-diacyl-3-O-(α-D-galactosyl-1-6)-α-D-galactosyl-sn-glycerol (DGDG) and sulfosucrose diacylglycerol (SQDG), or any mixture thereof.

[0147] Wherein, the lipid polymer is a polyethylene glycol (PEG)- or polysarcosine-lipid conjugate or a PEG- or polysarcosine-lipid conjugate or any mixture thereof, and

[0148] The monoacylglycerol (MMG) analogues are selected from the group consisting of MMG-1, MMG-2, MMG-3, MMG-4, MMG-5, MMG-6 and MMG-7 or any mixture thereof.

[0149] In one embodiment, the method further includes the following steps:

[0150] f) Concentrate the lipid nanoparticle (LNP) composition using one of the group consisting of filtration, centrifugation, vacuum-assisted centrifugation, or any combination thereof, preferably filtration.

[0151] Determining cationic or cationizable lipid or lipidoid material, helper lipid, lipopolymer, and monoacylglycerol (MMG) analog of step a) Determining nucleic acid of step a)

[0152] One embodiment relates to a method according to the invention, wherein the lipid polymer is selected from 1,2-dimyristoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG). 2000 ), 1,2-Dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG) 2000 ), 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) 2000), 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE-PEG), 1,2-distearyl-rac-glycerol-3-methoxy polyethylene glycol (DSG-PEG), ceramide-PEG, 1,2-dipalmitoyl-rac-glycerol-3-methylpolyoxyethylene (DPG-PEG), 1,2-dioleoyl-rac-glycerol, methoxy polyethylene glycol The group consisting of (DOG-PEG), 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine-N-methylpolyoxyethylene (DOPE-PEG), N-tetradecyl polysarcosine 25, N-hexadecyl polysarcosine 25, N-octadecyl polysarcosine 25, N-dodecyl polysarcosine 25, N,N-bistetradecylamine-N-succinoyl[methyl(polysarcosine)45], N,N-bistetradecylamine-N-succinoyl[methyl(polysarcosine)35] and N,N-bistetradecyl polysarcosine-25 or mixtures thereof. In another embodiment, the cationic or cationic lipid or lipid-like material is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200), the auxiliary lipid is 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), and the lipid polymer is 1,2-dimyristoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG). 2000 In yet another embodiment, the monoacylglycerol (MMG) analogue is MMG-1, MMG-6, and / or MMG-7, preferably MMG-1.

[0153] Determining buffer of step c)

[0154] One embodiment relates to a method according to the invention, wherein at least one nucleic acid is selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (sRNA), circular RNA (circRNA), plasmid DNA (pDNA), small interfering RNA (siRNA), single guide RNA (sgRNA), guide RNA (gRNA), long non-coding RNA (lncRNA), small activating RNA (saRNA), and splice-conversion antisense oligonucleotides (ASO), preferably at least one nucleic acid being mRNA.

[0155] Mixing by nano-precipitation

[0156] One embodiment relates to the method according to the invention, wherein the pH of the buffer in step c) is in the range of 3.3 to 6, preferably 3.5 to 5, and more preferably 4.0. The pH of the buffer may vary depending on the cationic or cationically cationizable lipid or lipid-like material used. In one embodiment, the buffer is selected from the group consisting of citrate buffer, acetate buffer, Tris buffer, or HEPES buffer.

[0157] Determining filtration of step e)

[0158] Nanoprecipitation is a simple method for encapsulating hydrophilic and hydrophobic drugs in nanoparticles. Several different methods can be used for nanoprecipitation. Therefore, in one embodiment, the mixing in step d) is performed using microfluidic mixing, pipette mixing, a T-mixer, or an impingement jet mixer. In a preferred embodiment, microfluidic mixing is used. In another embodiment, the flow ratio of the aqueous phase to the organic phase during microfluidic mixing is in the range of 1:1 to 10:1, preferably 3:1. In yet another embodiment, the total flow rate during microfluidic mixing is selected from the range of 1.0 mL / min to 20 mL / min, preferably 12 mL / min.

[0159] Product of the method

[0160] In one embodiment, the filtration of lipid nanoparticles (LNPs) in step e) is performed against phosphate-buffered saline or Tris buffer, preferably against Tris buffer at pH 7.4.

[0161] Materials

[0162] One aspect relates to lipid nanoparticle (LNP) compositions obtained using the methods of the present invention.

[0163] It should be noted that the embodiments and features described in one aspect of the invention are also applicable to other aspects of the invention. The entire contents of all patent and non-patent references cited in this application are incorporated herein by reference.

[0164] The invention will now be described in further detail by way of non-limiting embodiments.

[0165] Example

[0166] Example 1: Materials and Methods

[0167] Preparation of mRNA-loaded lipid nanoparticles

[0168] Cleancap was completely replaced with 5-methoxyuridine (1 mg / mL in 1 mM sodium citrate buffer, pH 6.4). ® Enhanced green fluorescent protein (eGFP), FLuc (SEQ ID NO.: 1), and OVA mRNA (SEQ ID NO.: 2) were obtained from TriLink Biotechnologies (San Diego, CA, USA). C12-200 was synthesized, purified, and characterized according to previously reported methods (Love, KT et al. 2010). MMG-1 was purchased from Clausson Kaas (Farum, Denmark). 1,2-Distearyl-sn-glycerol-3-phosphatidylcholine (DSPC) and 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE) were obtained from Avanti. ® Polar Lipids (Alabaster, AL, USA). Cholesterol, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG), and 1,2-dimyristoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG) were obtained from Sigma-Aldrich (St. Louis, Mo, USA). SM-102 was obtained from MedChemExpress (Copenhagen, Denmark). Various ionizable lipids and co-phospholipids were obtained from commercial suppliers. MMG-2, MMG-3, MMG-4, MMG-5, and MMG-6 were synthesized and purified according to previously published methods (Nordly, P. et al. (2011), Martin-Bertelsen, B. et al. (2013)). Endofit™ OVA was obtained from Invivogen (Toulouse, France). Triton™ X-100 purchased from Sigma-Aldrich. Quant-iT™ RiboGreen ® RNA reagents and Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0, TE buffer) were obtained from Molecular Probes, Invitrogen (Paisley, UK). RNase-free water was used throughout the study. All other chemicals and reagents were analytical grade and obtained from commercial suppliers.

[0169] Statistical analysis

[0170] C12-200 LNPs use NanoAssemblr ® The Ignite™ microfluidic mixing system (PrecisionNanosystems Inc., Vancouver, Canada) was prepared using a microfluidic mixing method employing a toroidal micromixer chip. The aqueous phase consisted of mRNA (FLuc) dissolved in citrate buffer (10 mM, pH=3). C12-200 or SM-102, MMG-1, DOPE, cholesterol, and DMPE-PEG were dissolved in an organic phase consisting of 99.99% anhydrous ethanol, and the molar ratio of MMG-1 to cholesterol was systematically adjusted (Table 1). For ease of naming, the MMG-1 modified C12-200 LNPs are designated as C12-200 MMG 11 (11.88 mol% MMG-1), C12-200 MMG 23 (23.25 mol% MMG-1), C12-200 MMG 35 (35.63 mol% MMG-1), and C12-200 MMG 47 (47.5 mol% MMG-1). As controls, two C12-200 LNP formulations without MMG-1 were prepared: one containing cholesterol (designated C12-200), and the other without cholesterol (designated C12-200 DOPE DMPE-PEG). 2000For comparison, LNPs containing 0 mol% or 37.5 mol% MMG-1 were also prepared, with SM-102 used as the ionizable lipid (Table 1). The SM-102 LNPs were prepared using DSPC as the co-lipid, while “SM-102 DOPE MMG 38” contained DOPE as the co-lipid. LNPs were prepared by injecting the organic phase into the central inlet of the micromixer chip and the aqueous phase into the right inlet. The mRNA and lipid solutions were mixed at a flow rate ratio (FRR) of 3:1 (aqueous to organic phase). A total flow rate (TFR) of 12 mL / min was used. The calculated formulation volume accounted for the initial waste (0.25 mL), the final waste (0.10 mL), and the dead volume of the syringe, as well as potential losses during syringe loading into the microfluidic mixing system. The final formulation volume was 1.25 mL. LNPs based on C12-200 and MMG-1 used for immunogenicity studies were prepared in a similar manner (FFR = 3:1, TFR = 12 mL / min, final waste liquid = 1.5 mL), and loaded with 80 μg OVA mRNA in a final formulation volume of 4.5 mL. LNPs used for the immunization control group (Ctrl) were loaded with 50 μg eGFP mRNA for primary immunization and FLluc mRNA for booster immunization, respectively. A 10,000 MWCO dialysis kit (Slide-A-Lyzer) was used. ® The formulation was purified by dialyzing against phosphate-buffered saline (PBS) for 2.5 hours with magnetic stirring to exchange the solvent and neutralize the pH. Subsequently, it was purified using a 100 kDa MWCO 4 mL centrifuge filter (Amicon). ® The formulation was concentrated in PBS to a final lipid content of approximately 1.5 mg / mL by centrifugation at 20 °C and 1500 × g for 4 minutes (Merck KGaA, Darmstadt, Germany).

[0171] Table 1: Theoretical molar composition of C12-200 / SM-102 lipid nanoparticles (LNPs) modified with C12-200 / SM-102- and MMG-1-

[0172]

[0173] Objectives

[0174] Graphical plotting and statistical analysis were performed using GraphPad Prism V9 (GraphPad Software Inc., La Jolla, CA, USA). In vivo imaging data were analyzed using one-way ANOVA, with a significance level set at 0.05, and paired comparisons were performed using Tukey's post-hoc test. Immune responses between groups were analyzed using one-way ANOVA (analysis of cytokine-producing T cells, T...). FH Comparisons were performed using either GC B cells (antibodies) or two-way ANOVA (pluripotent T cells), with a significance level set at 0.05. Paired comparisons were performed using Tukey or Dunnett post-hoc tests, respectively. A p-value < 0.05 was considered statistically significant.

[0175] Example 2: Cholesterol in C12-200 LNPs and SM-102 LNPs can be replaced by MMG-1.

[0176] Materials and methods

[0177] This study aims to verify whether the gradual replacement of cholesterol with MMG-1 affects the physicochemical properties, mRNA encapsulation, and morphology of the obtained LNPs.

[0178] Results

[0179] Physicochemical characterization

[0180] LNP formulations were characterized based on mean intensity-weighted hydrodynamic diameter (Z-mean) and polydispersity index (PDI). Z-mean and PDI were determined by dynamic light scattering at 25°C using a ZetaSizer Nano SZ (Malvern Instruments, Worcestershire, UK) equipped with a 633 nm laser and a detection angle of 173°. Data acquisition and analysis were performed using Zetasizer 7.11 software (Malvern Instruments). Before analysis, 60 μL of the formulation was diluted with 440 μL of PBS in a 1 mL polystyrene cuvette. Measurements were repeated three times, with an equilibration time of 60 seconds. mRNA encapsulation efficiency was measured using Quant-iT™. RiboGreen ® The experiment was conducted and quantified in a manner largely in accordance with previously reported methods (Lokras, A. et al. 2022).

[0181] Morphology of LNPs modified by MMG-1

[0182] Morphological analysis of the FLuc mRNA-loaded LNP formulations C12-200, C12-200 MMG 23, and C12-200 MMG 47 was performed using cryo-TEM on a Tecnai G2 20 TWIN transmission electron microscope (Field Electron and Ion Company, Hillsboro, Oregon, USA). Samples were prepared by vitrification (rapid cooling) and transferred onto a Pelco Lacey carbon membrane grid using a Vitrobot™ Mark IV (Field Electron and Ion Company). Excess liquid was removed with filter paper, forming a thin film of approximately 10–500 nm. The samples were then immediately immersed in liquid ethane at -180°C and transported within a cryostat connected to the electron microscope. Throughout the experiment, the samples were maintained below -180°C. Analysis was performed in bright-field mode with an accelerating voltage of 120 mV. Digital images were recorded using a Gatan Imaging Filter 100 CCD camera (Gatan, Pleasanton, CA, USA).

[0183] Figure 1A

[0184] C12-200 LNPs loaded with FLuc and OVA mRNA, and modified C12-200 LNPs with varying molar amounts of MMG-1, were prepared using a microfluidic mixing method with the NanoAssemblr® Ignite™ microfluidic mixing system. SM-102 LNPs loaded with FLuc mRNA were also prepared using the NanoAssemblr® Ignite™ microfluidic mixing system. For all LNP formulations, the FLuc mRNA-loaded LNPs used for imaging had an average hydrodynamic diameter of less than 200 nm and an average PDI value of less than 0.25 (Table 2), indicating that the formulation was monodisperse. SM-102 LNPs generally had a lower average hydrodynamic diameter than C12-200 LNPs, but the incorporation of MMG-1 did not appear to affect this difference. Regardless of whether MMG-1 was used in preparation, LNPs loaded with OVA mRNA for in vivo immunogenicity studies showed an average hydrodynamic diameter of less than 140 nm and an average PDI value of less than 0.16 (Table 2). LNPs loaded with eGFP or Fluc mRNA, used as controls in immunoassays, showed average hydrodynamic diameters of 106 and 115 nm, and PDI values ​​of 0.149 and 0.075, respectively. Without considering MMG-1 modification, the encapsulation efficiency (average 88%) of LNPs loaded with OVA mRNA (for immunogenicity studies) was slightly lower than that of LNPs loaded with FLuc mRNA (average 94%, for imaging) (Table 2). Regarding the mRNA encapsulation efficiency of control formulations used in in vivo immunogenicity studies, the figure was 93% for eGFP mRNA and 95% for FLuc mRNA. There was no significant difference in encapsulation efficiency between MMG-1 modified and unmodified C12-200 LNPs used for imaging or in vivo immunogenicity studies. Interestingly, the inventors discovered that C12-200, MMG-1, DOPE, cholesterol, and DMPE-PEG... 2000 When dissolved in an organic phase composed of anhydrous ethanol, the mRNA encapsulation efficiency of LNPs loaded with FLuc mRNA increased from approximately 80% to approximately 90-96% compared to the case with citrate buffer (10 mM, pH 3) containing 90% ethanol. Conclusions Therefore, LNPs prepared with anhydrous ethanol were used in subsequent experiments.

[0185] Table 2: LNP characteristics, data represent the average of two independent formulations.

[0186]

[0187] To perform morphological analysis of LNPs using cryo-TEM, C12-200, C12-200 MMG 23, and C12-200 MMG 47, loaded with Fluc mRNA, were selected (Figs. 1B-D). All LNPs had diameters of approximately 100-200 nm, consistent with the average hydrodynamic diameter of these LNPs. C12-200 LNPs exhibited a spherical morphology with uniform curvature (Fig. 1B), while C12-200 MMG 23 LNPs showed less sphericity and a sharper surface (Fig. 1C). On the other hand, C12-200 MMG 47 LNPs exhibited a typical polyhedral morphology (Fig. 1D). Apparent pK values ​​of C12-200 and C12-200 MMG 47 LNPs were also analyzed. a The values ​​were 7.22 and 7.14 respectively, while the PK values ​​for SM-102 and SM-102 MMG 38 LNPs were... a The values ​​were 6.69 and 6.67, respectively (Figure 1E). Therefore, replacing cholesterol with MMG-1 did not change the pK values ​​of C12-200 and SM-102 LNPs. a value.

[0188] Objectives

[0189] Compared to unmodified C12-200 LNPs, the average hydrodynamic diameter (Z-mean), polydispersity index (PDI), and mRNA encapsulation efficiency of MMG-1 modified C12-200 LNPs showed no significant differences. However, as more cholesterol was replaced by MMG-1, the morphology of the LNPs changed: C12-200 LNPs initially exhibited a uniformly curved spherical shape, gradually losing their spherical characteristics and becoming more angular in C12-200 MMG 23, and exhibiting a polyhedral morphology in C12-200 MMG 47. Furthermore, the MMG-1 modified LNPs could be formulated using SM-102, indicating the use of other cationic or cationic lipid or lipid-like materials. Additionally, there were no significant pK differences between unmodified and MMG-1 modified C12-200 LNPs or between unmodified and MMG-1 modified SM-102 LNPs. a Differences. Therefore, in both C12-200 LNPs and SM-102 LNPs, cholesterol can be replaced by MMG-1.

[0190] Example 3: Effects of replacing cholesterol with MMG-1 on eGFP mRNA-loaded C12-200 and C12-200 MMG 47 LNPs in HEK-null and HEK-overexpressing Dectin-1 receptor cell lines, and on the biodistribution of FLuc-loaded C12-200 LNPs and SM-102 LNPs.

[0191] Materials and methods

[0192] This study aimed to verify whether MMG-1 modified C12-200 LNPs could mediate the expression of eGFP protein in vitro in HEK-null and HEK-overexpressing Dectin-1 receptor cell lines. Another objective was to investigate whether replacing cholesterol with MMG-1 in C12-200 and SM-102 LNPs affected the biodistribution and duration of protein production mediated by FLuc mRNA-loaded C12-200 and SM-102 LNPs.

[0193] HEK-null and HEK-Dectin-1 cell assays

[0194] Results

[0195] HEK cells expressing Dectin-1

[0196] SEAP reporter 293 cells expressing the human Dectin-1A gene (HEK-Blue™ hDectin-1a) and the parental NF-κB-induced SEAP cell line (HEK-Blue™ Null1-v) were both derived from human embryonic kidney (HEK) 293 cells, obtained by InvivoGen (San Diego, CA, USA). Cells were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine, 1 μg / mL puromycin, and 1X HEK-Blue CLR selector (hDectin-1a) or 100 μg / mL nomoxantrone (Null1-v, Invivogen). Dectin-1 expression on the cell surface was confirmed by flow cytometry. For transfection experiments, cells were decelled with PBS after reaching 80% confluence and then transfected at 2.6 × 10⁶ cells / mL. 5L cells were seeded in antibiotic-free, complete DMEM. Cells were then transfected with LNP formulations C12-200 and C12-200 / MMG (100%) loaded with eGFP mRNA and incubated at 37°C and 5% CO2. eGFP expression was evaluated at specified time points using a Tecan Spark® (Tecan Group Ltd., Männedorf, Switzerland) plate reader via fluorescence measurements at excitation wavelengths of 485 nm and emission wavelengths of 535 nm.

[0197] Bioluminescence imaging

[0198] Female 8-10 week old BALB / cOlaHsd mice (ENVIGO, Horst, Netherlands) were obtained and acclimatized for one week prior to imaging. Food and water were provided freely. All experimental work was approved by the Danish National Laboratory Inspection Agency, license number 2022-15-0201-01221, and conducted in accordance with EU Directive 86 / 609 on the care and use of laboratory animals. 4 μg of FLuc mRNA, loaded into C12-200 and MMG-1 modified C12-200 LNPs or SM-102 and MMG-1 modified SM-102 LNPs, was subcutaneously injected (sc) into the base of the tail of mice using 100 μL of PBS (pH 7.4). Mice were intraperitoneally injected (ip) with 150 mg / kg body weight of XenoLight D-fluorescein potassium bioluminescent substrate (PerkinElmer, Waltham, MA, USA) at 6, 24, 48, 72, and 96 hours after administration, followed by 15 minutes of rest. Mice were then anesthetized with isoflurane, and imaging was performed using an in vivo imaging system (IVIS) Lumina XRMS (PerkinElmer). Bioluminescent emission was measured using Living Image. ® Software v4.7.4 (PerkinElmer) and brightness (photons / s / cm) 2 The intensity of the emitted light is quantified by sphericity and total flux (p / s).

[0199] Figure 2

[0200] To investigate the expression of eGFP in HEK-null and HEK-Dectin-1 cell lines, the inventors used the reported HEK-null and HEK-Dectin-1 cell lines to quantify eGFP expression mediated by C12-200 and C12-200 MMG 47 LNPs. Figure 2Interestingly, within the dose range of 10-160 ng of EGFP mRNA, C12-200 MMG 47 LNPs mediated approximately 1.5-2.0 times higher eGFP expression in the HEK-Dectin-1 cell line compared to the case with C12-200 LNPs. Figures 3-4 The results showed that Mycobacterium tuberculosis directly interacts with Dectin-1, thereby enhancing bacterial binding to splenic dendritic cells, while Dectin-1 blockers reduce this binding. This enhanced binding may lead to greater accumulation of C12-200 MMG 47 LNPs, resulting in endosome escape. Another benefit of enhanced binding to receptors on primary gated immune cells is that the selected antigens may be taken up by these cells more readily, resulting in a reduced dose of ionizable lipids required to achieve the same level of antigen expression and decreased dose-dependent reactivity of ionizable lipids. However, the 1.5–2.0-fold enhancement in vitro would significantly reduce the amount of lipids and mRNA required for global production of mRNA vaccines.

[0201] To examine the biodistribution and duration of protein production mediated by mRNA-loaded LNPs in vivo, unmodified and MMG-1 modified C12-200 and SM-102 LNPs loaded with FLuc mRNA were subcutaneously injected into the tail root of mice at a dose of 4 μg mRNA (Table 2). Spatiotemporal FLuc expression was imaged and quantified at 6, 24, 48, 72, and 96 hours. Figure 2 ). Figure 3 Only images taken 6 hours after LNP administration are shown. Whole-body images of mice in a supine position show strong bioluminescent signals at the injection site (SOI), i.e., the tail base, for all LNP formulations 6 hours post-administration. Figure 3 Bioluminescence images taken in the supine position show that, compared to unmodified C12-200 LNPs, some MMG-1-modified C12-200 LNPs in mice migrated to the liver, while most unmodified C12-200 LNPs remained in the SOI (somatic oxygenation site). Figure 4 No significant difference in bioluminescent signal was observed between C12-200 and C12-200 MMG 47 LNP at any time point, and the signal was at least 10 times higher than the background at 96 hours post-injection. Figure 5Quantitative analysis of FLuc expression at the SOI (supine position) showed that mice injected with C12-200 MMG11, 23, and 35 LNP formulations had significantly lower protein production at 6 hours post-administration compared to C12-200 and C12-200 MMG 47 LNP formulations (***p<0.001, **p<0.0001). Figure 5 Furthermore, high FLuc expression was observed in mice treated with SM-102 LNPs, with levels comparable to those measured in mice treated with the SM-102MMG 37 formulation. Interestingly, LNPs without cholesterol or MMG-1 did not mediate FLuc expression. Figure 6 When calculating the ratio of mean brightness values ​​at the injection site to those in the liver, FLuc expression mediated by C12-200 MMG 47 LNPs was significantly concentrated at the injection site compared to C12-200 LNPs. Conclusions This satisfies one of the important criteria for vaccine design: that it does not spread throughout the body.

[0202] Objectives

[0203] MMG-1-based LNPs mediated higher protein expression in cells modified to overexpress immune receptors, suggesting their potential use as an adjuvant. Strong bioluminescent signal intensity at the SOI at 6 hours post-drug administration in mice confirmed that MMG-1-modified C12-200 / SM-102 LNPs mediated in vivo transfection and intracellular delivery of mRNA as effectively as unmodified C12-200 LNPs.

[0204] Example 4: Effects of using various cationic or cationic lipid or lipid-like materials, auxiliary lipids, and lipid polymers to formulate sterol-free (MMG-1-based) LNPs on the physicochemical properties and biodistribution of FLuc-loaded MMG-1-based LNPs.

[0205] Materials and methods

[0206] This study investigated the effects of replacing components in MMG-1-modified LNPs with different cationic or cationic lipid or lipid-like materials, auxiliary lipids, and lipid polymers. The replacements were evaluated based on the physicochemical properties of the MMG-1-modified LNPs and their spatiotemporal expression in mice.

[0207] The following cationic lipid or lipid-like materials, auxiliary lipids, and lipid polymers were tested.

[0208] 1) Cationic or cationic lipids or lipid-like materials: cKK-E12, 306Oi10, 4A3-SC8, DLin-MC3 DMA, ALC-0315, SM-102, DLin-KC2-DMA and C12-200

[0209] 2) Assisting lipids: DOPE, DSPC, SOPC

[0210] 3) Lipid polymers: DMPE-PEG2k, DMG-PEG2k, ALC-0159, polysarcosine pSar25 and DSPE-PEG2k

[0211] Results

[0212] Bioluminescence imaging

[0213] Female 8-10 week old BALB / cOlaHsD mice (ENVIGO, Horst, Netherlands) were obtained and acclimatized for one week prior to imaging. Food and water were provided freely. All experimental work was approved by the Danish National Laboratory Inspection Agency, license number 2022-15-0201-01221, and conducted in accordance with EU Directive 86 / 609 on the care and use of laboratory animals. 4 μg of FLuc mRNA loaded with the formulation in Table 3 was injected subcutaneously (sc) at the base of the tail of the mice using 100 μL of PBS (pH 7.4). At 6, 24, 48, 72, and 96 hours post-administration, mice were intraperitoneally (ip) injected with 150 mg fluorescein / kg body weight of XenoLight D-fluorescein potassium bioluminescent substrate (PerkinElmer, Waltham, MA, USA) followed by a 15-minute rest. Mice were then anesthetized with isoflurane and imaged using an in vivo imaging system (IVIS) Lumina XRMS (PerkinElmer). Bioluminescence emission was measured using Living Image. ® Software v4.7.4 (PerkinElmer) and luminance values ​​(photons / second / cm) 2 The intensity of emitted light is quantified using sphericity and total flux (p / s).

[0214] Figure 7

[0215] LNPs with specific compositions were formulated (Table 3). All LNPs had hydrodynamic dimensions less than 200 nm, polydispersity index (PDI) of approximately or less than 0.2, and mRNA encapsulation efficiency greater than 90% (Table 4).

[0216] Table 3: Composition of LNPs prepared in Example 4

[0217]

[0218] Table 4: Physicochemical properties of LNPs loaded with FLuc mRNA prepared according to Example 4

[0219]

[0220] LNPs loaded with mRNA were subcutaneously injected into the base of the tail of mice at a dose of 4 μg mRNA, and spatiotemporal FLuc expression was quantitatively detected 6 hours later. Conclusions No significant differences in FLuc expression were observed among the groups (combined with Dunnett-corrected one-way ANOVA), indicating that MMG-1-based LNPs are compatible with various cationic lipid or lipid-like materials. When DSPE-PEG2k was used as the lipid polymer, FLuc expression levels were generally lower, consistent with the expectation that the longer C18 carbon tail would hinder LNP uptake. When using a composition of DLin-KC2-DMA and SOPC phospholipids, FLuc expression levels were also reduced by approximately one order of magnitude, as this composition is more suitable for delivering plasmid DNA than mRNA.

[0221] Objectives

[0222] Strong bioluminescent signal intensity was observed in the SOI region 6 hours after drug administration to mice, confirming that MMG-1 modified LNPs can mediate in vivo transfection and intracellular delivery of mRNA when using different components, especially different cationic lipids or lipid-like materials, auxiliary lipids and lipid polymers.

[0223] Example 5: Intramuscular administration of FLuc-loaded C12-200 LNPs and SM-102 LNPs to mice

[0224] Results

[0225] In previous embodiments, the LNP formulation of the present invention was administered subcutaneously. Therefore, this study aims to investigate the effects of intramuscular injection of MMG-1 modified LNPs.

[0226] Bioluminescence imaging

[0227] Female 8-10 week old BALB / cOlaHsD mice (ENVIGO, Horst, Netherlands) were obtained and acclimatized for one week prior to imaging. Food and water were provided freely. All experimental work was approved by the Danish National Laboratory Inspection Agency, license number 2022-15-0201-01221, and performed in accordance with EU Directive 86 / 609 on the care and use of laboratory animals. 2 μg of FLuc mRNA loaded with MMG-1 modified C12-200 and SM-102 LNPs was injected intramuscularly (im) into the thigh of the hind limb of mice using a dose volume of 50 μL PBS (pH 7.4). At 6, 24, 52, 75, and 120 hours post-administration, mice were injected intraperitoneally (ip) with 150 mg fluorescein / kg body weight of XenoLight D-fluorescein potassium bioluminescent substrate (PerkinElmer, Waltham, MA, USA) followed by a 15-minute rest period. Mice were then anesthetized with isoflurane and imaged using an in vivo imaging system (IVIS) Lumina XRMS (PerkinElmer). Bioluminescence emission was measured using LivingImage. ® Software v4.7.4 (PerkinElmer) and luminance values ​​(photons / second / cm) 2 The intensity of emitted light is quantified using sphericity and total flux (p / s).

[0228] Figure 8

[0229] A strong bioluminescent signal intensity was observed in the SOI region 6 hours after drug administration to mice, confirming that MMG-1 modified LNPs can mediate in vivo transfection and intracellular delivery of mRNA via intramuscular injection at a dose of 2 μg. Figure 9 Protein expression remained above baseline levels for at least 120 hours after administration. Conclusions ).

[0230] Objectives

[0231] MMG-1 modified LNPs can mediate in vivo transfection and intracellular delivery of mRNA via intramuscular administration.

[0232] Example 6: Effect of MMG on immunogenicity using model mRNA encoding ovalbumin (OVA) when cholesterol is replaced by MMG.

[0233] Materials and methods

[0234] This study aims to examine whether replacing cholesterol with MMG-1 in C12-200 LNPs affects the immunogenicity of the LNPs loaded with OVA mRNA.

[0235] Results

[0236] Immunization

[0237] Six-week-old female C57BL / 6 mice (ENVIGO) were obtained and acclimatized for one week prior to experimental procedures. Animals had free access to food and water. All experimental work was approved by the Danish National Laboratory Inspection Agency, license number 2022-15-0201-0122. The research procedures followed EU Directive 86 / 609 on the care and use of laboratory animals. Mice were divided into six groups of six. All mice underwent two immunizations, administered subcutaneously via tail root injection at two-week intervals using 200 μL of PBS (pH 7.4). Control group (Ctrl) mice were initially immunized with saline (n=3) and 10 µg of C12-200 LNPs loaded with eGFP mRNA (n=3), followed by booster immunizations with saline (n=3) and 10 µg of FLuc mRNA-C12-200 LNPs (n=3). Mice in the C12-200 group were immunized twice with 10 µg of C12-200 LNPs (C12-200, n=6) loaded with OVA mRNA. Mice in the last four groups were immunized twice with 10 µg of C12-200 LNPs loaded with OVA mRNA. The LNPs contained 11 (C12-200 MMG 11, n=6), 23 (C12-200 MMG 23, n=6), 35 (C12-200 MMG 35, n=6), and 47 (C12-200 MMG 47, n=6) mol% MMG-1, respectively. For the control group, C12-200 group, C12-200MMG 11 group, C12-200 MMG 23 group, C12-200 MMG 35 group and C12-200 MMG 47 group, the final doses of C12-200 / DOPE / cholesterol / MMG-1 / DMPE-PEG were 267 / 80 / 123 / 0 / 27, 267 / 80 / 123 / 0 / 27, 267 / 80 / 92 / 45 / 27, 267 / 80 / 61 / 91 / 27, 267 / 80 / 31 / 136 / 27 and 267 / 80 / 0 / 182 / 27µg / mouse / immunization.

[0238] Sample collection and cell preparation

[0239] In week 4 of the study, two weeks after booster immunization, animals were euthanized. Blood was collected via cardiac puncture, and serum was separated by spontaneous coagulation at room temperature (RT). Serum was extracted using a Heraeus Multifuge 3SR+ (Thermo Fischer Scientific) at 2000×g for 10 minutes. Serum was stored at -20°C until antibody detection. Spleens and inguinal lymph nodes from the subcutaneous administration site were aseptically collected from euthanized mice. Spleens and lymph nodes were homogenized using a 70 μm nylon mesh cell filter (Falcon, Durham, NC, USA) and washed twice with PBS to obtain a single-cell suspension. Subsequently, spleen and lymph node cells were seeded into 96-well microtiter plates (Nunc, Roskilde, Denmark), with 2×10⁶ cells per well. 5 10 cells per well for cytokine detection, or 1 × 10 cells per well. 6 Cells were used for flow cytometry analysis and incubated in 100 µL RPMI-1640 (Sigma-Aldrich) medium supplemented with 5 × 10⁶ ppm of 500 mg / L. -5 M 2-mercaptoethanol (Gibco Thermo Fisher), 1% sodium pyruvate (Sigma-Aldrich), 1% penicillin-streptomycin (Gibco Thermo Fisher), 1% HEPES (Gibco Thermo Fisher), and 10% FCS (Gibco Thermo Fisher).

[0240] Antibody testing

[0241] ELISA is used to detect antibodies in serum samples. Simply put, Maxisorp... TMPlates (Nunc) were coated with 1 μg / mLOVA carbonate-bicarbonate buffer (pH=9.6). Serum samples were started at a 1:5 dilution and serially diluted 11 times with bicarbonate buffer. OVA-specific IgG, IgG1, and IgG2c were detected using horseradish peroxidase-labeled secondary antibodies: rabbit anti-mouse IgG (ThermoFisher; dilution 1:2,500), goat anti-mouse IgG1 (Southern Biotech, Birmingham, AL, USA; dilution 1:20,000), and goat anti-mouse IgG2c (Southern Biotech; dilution 1:5,000). 3,3',5,5'-Tetramethylbenzidine Plus2 (Kem-En-Tec, Taastrup, Denmark) was used as the substrate. The enzymatic reaction was terminated by adding 0.2 M H₂SO₄, and the absorbance was read at 450 nm using a FLUOstar optima plate reader (BMG Labtech, Ortenberg, Germany). Serum OD values ​​were analyzed using nonlinear regression to calculate the midpoint titer (ECG) of the ELISA, following a previously described method (Thakur, A. et al. 2018). 50 .

[0242] Flow cytometry

[0243] Spleen cells and lymph node cells were treated with OVA (SEQ ID NO.: 2, 5 μg / mL), OVA, etc. 257-264 (SIINFEKL, SEQ ID NO.: 6) and OVA 323-339 The peptide (ISQAVHAAHAEINEAGR, SEQ ID NO.: 7) was used for stimulation (5 μg / mL, AnaSpec, Fremont, CA, USA), supplemented with anti-CD28 (37.51) and anti-CD49d (9C10) co-stimulatory antibodies (1 μg / mL, BD Biosciences). The culture was incubated at 37°C and 5% CO2 for 6 hours. Bleomycin A (10 μg / mL, Sigma-Aldrich) and monensin / Golgi inhibitor (0.7 µL / mL, BD Biosciences) were added during the last 5 hours of incubation. Separate culture media and concanavalin A (5 μg / mL, Sigma-Aldrich) were used as negative and positive controls, respectively. For multifunctional epitope-specific CD4... +T cell detection was performed using a previously described protocol combining MHC-II tetramer and intracellular cytokine staining (ICS) (Pastore, G. et al. 2019). Based on this protocol, the multifunctional epitope-specific CD8 staining was optimized by combining MHC-I pentamer staining with ICS. + T cell detection. Splenic and lymph node cells were stored overnight at 4°C and then labeled with fixable live cell staining agent (FVS) 510 (BD Biosciences, 1:1, 100 μL / well) for 20 min at 4°C in the dark, followed by washing twice with PBS. Cells were fixed with BD Cytofix / Cytoperm (BD Biosciences) at 4°C and permeabilized for 20 min. Subsequently, samples were blocked for 30 min at 4°C in Fc blocking solution (5 μg / mL CD16 / CD32 mAb, BD Biosciences) and then separately diluted with PE-labeled H-2Kb-SIINFEKL (OVA) with permeation / wash buffer at room temperature. 257-264 (MHC-I) pentamer (dilution ratio 1:8, ProImmune, Oxford, UK) and PE-labeled IA b -ISQAVHAHAEINEAGR (OVA) 323-339 MHC-II tetramer (dilution ratio 1:8, ProImmune) staining for 1 hour. During the last 20 minutes of tetramer incubation, the following fluorescent antibody mixture was added: anti-CD4-BUV395 (RM4-5; BD Biosciences), anti-CD8-BUV737 (53-6.7; BDBiosciences), anti-CD44-FITC (IM7; BD Biosciences), anti-CD62L-PE-CF594 (MEL-14; BDBiosciences), anti-IFN-γ-PE-Cy7 (XMG1.2; eBiosciences), anti-TNF-α-BV711 (MP6-XT22; BDBiosciences), anti-IL-2-APC (JES6-5H4; eBiosciences), anti-IL-4-BV421 (11B11; BDBiosciences), and anti-IL-17-PerCP-Cy5.5 (eBio17B7; eBiosciences). For T FHFor the detection of GC B cells, lymph node cells were stained with anti-CD3-BV711 (145-2C11; BD Biosciences), anti-CD4-BUV395 (RM4-5; BD Biosciences), anti-B220-BV480 (RA3-6B2; BD Biosciences), anti-CD95-AF647 (Jo2; BD Biosciences), anti-CXCR5-PE (2G8; BD Biosciences), anti-GL7-FITC (GL7; BD Biosciences), and anti-PD-1-BV421 (J43; BD Biosciences). Dead cells were excluded using the fixable viable dye FVS780 (BD Biosciences). All cells were washed twice, resuspended in FACS buffer, and analyzed using an LSR Tortessa flow cytometer (BD Biosciences). Surface label gating was based on a fluorescence minus one control. The gating strategy used to identify specific cell populations in the spleen and draining lymph nodes was based on previous reports (Vono, M. et al. (2019), Christensen, D. et al. (2017), Thakur, A. et al. (2018)). All flow cytometry data were analyzed using FlowJo software v10 (Tree Star, Ashland, OR, USA).

[0244] Figure 10

[0245] Unmodified and MMG-1 modified C12-200 LNPs loaded with OVA mRNA induced high levels of CD8 in draining lymph nodes at the subcutaneous injection site. + T cell response

[0246] To investigate the effect of replacing cholesterol with MMG-1 on immunogenicity, the inventors determined the immunogenicity of LNPs loaded with OVA mRNA in mice after primary and booster immunizations administered subcutaneously at 2-week intervals. Isolated lymph node cells were restimulated in vitro with the antigen, and the immune response was evaluated by combining intracellular cytokine staining (ICS) and tetramer staining with quantitative detection of phenotypic marker expression. Figure 10 Antigen-specific CD4 was determined by combined Boolean gating analysis. + CD44 + and CD8 + CD44 +The function of T cells was examined, focusing on the expression of IFN-γ, TNF-α, IL-2, IL-4, and IL-17 in these cells. Generally, immunization with unmodified and MMG-1-modified C12-200 LNPs loaded with OVA mRNA results in a very high percentage of IFN-γ-secreting OVA in draining lymph nodes. 257-264 Specific MHC-I (SIINFEKL, SEQ ID NO.: 6) Tet + CD8 + CD44 + T cells. Furthermore, compared to immunization with C12-200 LNPs loaded with OVA mRNA, immunization with MMG-1-modified C12-200 LNPs loaded with OVA mRNA resulted in increased secretion of TNF-α by OVA cells. 257-264 Specific MHC-I (SIINFEKL, SEQ ID NO.: 6) Tet + CD8 + CD44 + The percentage of T cells was significantly increased (C12-200 MMG group loaded with OVA mRNA 23*p<0.05, Figure 11 Compared with mice inoculated with C12-200 LNPs loaded with OVA mRNA, mice inoculated with C12-200 MMG 11 (*P<0.05) and C12-200 MMG 35 LNPs (****P<0.0001) loaded with OVA mRNA, respectively, co-secreted IFN-γ and TNF-α OVA. 257-264 Specific CD8 + CD44 + The percentage of T cells was statistically significantly increased. Regarding pluripotent CD8 cells... + CD44 + Regarding T-cell responses, no other significant differences were observed between LNP formulations (data not shown).

[0247] Replacing cholesterol with MMG-1 does not affect the CD4-induced activity of C12-200 LNPs loaded with OVA mRNA in draining lymph nodes. + T cell response

[0248] Subsequently, the inventors measured the OVA in the draining lymph nodes. 323-339 Specific CD4 + CD44 + T cells. Regarding OVA, which secretes cytokines. 323-339 Specific MHC-II (ISQAVHAAHAEINEAGR, SEQ ID NO.: 7) Tet+ CD4 + CD44 + Regarding the percentage of T cells, there was essentially no statistically significant difference between the different groups. Figure 13 OVA secretes IL-4. 323-339 Specific CD4 + CD44 + For T cells, only C12-200 MMG 11, which was loaded with OVA mRNA, showed a significant increase compared to C12-200 MMG 47 loaded with OVA mRNA.

[0249] Unmodified and MMG-1 modified C12-200 LNPs effectively promote T FH and GC differentiation and humoral response

[0250] It has been reported that nucleoside-modified mRNA-LNP vaccines induce strong T... FH Cell generation and GC formation. Therefore, the inventors measured T cells in the draining lymph nodes (ILNs) at the subcutaneous injection site of immunized mice. FH The number of GC B cells (Figure 12). Animals immunized with MMG-1-modified C12-200 LNPs loaded with OVA mRNA showed a higher number of T cells compared to unmodified LNPs and unimmunized animals. FH (Figure 12A-C) and GC B cells (Figure 12D-F), but the differences were not statistically significant. Among MMG-1 modified LNPs, C12-200 MMG 11 LNPs had the highest number of OVAs (Figure 12A), OVA 257-264 (Figure 12B) and OVA 323-339 Specific T FH (Fig. 12C), but the difference was not statistically significant. Almost the same trend was observed in GC B cells (Fig. 12D-F). To further determine the quality of the antibody response, the inventors performed ELISA and evaluated the midpoint titers of immunoglobulin G (IgG), IgG1, and IgG2c induced in serum two weeks after booster immunization with the mRNA-LNP vaccine. The total IgG (Fig. 12G), IgG1 (Fig. 12H), and IgG2c (Fig. 12I) titers induced by all OVA mRNA-loaded LNP vaccines were significantly higher than those in unimmunized animals. However, there was no difference in antibody titers between MMG-1-modified and unmodified C12-200 LNPs. These data suggest that OVA mRNA-loaded C12-200 LNPs can induce functionally diverse humoral responses, and these responses are not affected by the incorporation of MMG-1 into the LNPs.

[0251] Unmodified and MMG-1 modified C12-200 LNPs induce extremely high levels of CD8 in the spleen. + T cell response

[0252] Subsequently, the inventors evaluated the effect of LNPs loaded with OVA mRNA on inducing antigen-specific CD4 in the spleen. + and CD8 + The capacity of T cells. Similar to lymph node cells, the immune response in the spleen was assessed by restimulating spleen cells with ex vivo antigens, followed by ICS-based tetramer staining, as well as phenotypic marker expression and Boolean gating analysis. Figure 13 Immunization with MMG-1-modified C12-200 LNPs loaded with OVA mRNA induced the secretion of IFN-γ by OVA. 257-264 Specific MHC-I (SIINFEKL, SEQ ID NO.: 6) TET + CD8 + CD44 + In terms of the percentage of T cells, it was comparable to that of immunization with unmodified C12-200 LNPs loaded with OVA mRNA, and no difference in T cell production from other cytokines was observed. Figure 14 Typically, these cells have an extremely high average frequency, reaching 40-50%, almost twice the frequency induced in lymph nodes. Assessment of multifunctional T cell responses showed that, compared to mice inoculated with unmodified LNPs loaded with OVA mRNA, mice inoculated with MMG-1-modified C12-200 LNPs loaded with OVA mRNA co-secreted IFN-γ and TNF-α in the spleen. 257-264 Specific CD8 + CD44 + The percentage of T cells was significantly increased (*p<0.0001, data not shown).

[0253] Replacing cholesterol with MMG-1 does not affect CD4 in the spleen. + T cell response

[0254] Subsequently, the inventors measured the spleen OVA. 323-339 Specific MHC-II (ISQAVHAAHAEINEAGR, SEQ ID NO.: 7) Tet + CD4 + CD44 + T cells. No cytokine-producing OVAs were observed between the LNP groups. 323-339 Specific MHC-II (ISQAVHAAHAEINEAGR, SEQ ID NO.: 7) Tet +CD4 + CD44 + Differences in T cells ( Conclusions Furthermore, the average frequency of these cells is almost the same as the frequency induced in lymph nodes (LNs).

[0255] Objectives

[0256] CD8 + T cells are crucial for cytotoxic effector functions in infection, cancer, and autoimmunity; their effector functions and memory responses require CD4+. + T-cell assistance. Typically, after immunization with all LNP preparations, extremely high levels of MHC-I Tet are observed in lymph nodes and spleen. + CD8 + CD44 + T cell responses, characterized by highly defined cytokine responses polarized with IFN-γ, IL-2, and TNF-α. Compared to unmodified LNPs, MMG-1-modified LNPs induced the production of IFN-γ (12-15%) and TNF-α (7-10%) CD8+ in lymph nodes. + The frequency of T cells was slightly higher. However, in the spleen, all LNPs induced approximately 55-60% and 25-35% of IFN-γ, respectively. + and TNF-α + CD8 + T cells. Both IFN-γ and TNF-α have pro-inflammatory effects and are key regulators of cell-mediated immunity against intracellular pathogens and cancer. However, MMG-1 incorporation did not affect the T cells induced by C12-200 LNPs. FH It affects the differentiation of GC B cells and their ability to induce humoral responses.

[0257] Example 7: The effect of replacing cholesterol with MMG on the immunogenicity of using clinically relevant antigenic mRNA encoding the SARS-CoV-2 spike protein (S).

[0258] Materials and methods

[0259] This study aimed to test whether replacing cholesterol with MMG-1 in C12-200 and SM-102 LNPs would affect the immunogenicity of the LNPs loaded with S mRNA administered subcutaneously or intramuscularly. Another objective was to titrate the S mRNA dose to establish a dose-response relationship.

[0260] Results

[0261] Immunization

[0262] Six-week-old female C57BL / 6 mice (ENVIGO) were acclimatized for one week prior to the experimental procedures. Food and water were provided freely. All experimental work was approved by the Danish National Laboratory Inspection Agency, license numbers 2016-15-0201-01026 and 2022-15-0201-01203, respectively. The research procedures followed EU Directive 86 / 609 on the care and use of laboratory animals. For subcutaneous administration, six mice in each treatment group were immunized twice with 0.5 mg / kg of encapsulated S (SEQ ID NO.: 5) and eGFP mRNA (SEQ ID NO.: 12), respectively, two weeks apart, via subcutaneous administration at the base of the tail, with a dose volume of 100 μL. Control mice were initially immunized with saline (n=3) and C12-200 LNPs loaded with eGFP mRNA (n=3), and booster immunizations were performed with saline (n=3) and FLuc mRNA-C12-200 LNPs (n=3), respectively, to ensure unimmunized and irrelevant mRNA controls. For intramuscular administration, six mice in each treatment group were immunized twice with encapsulated SmRNA (SEQ ID NO.: 5) at doses of 0.01, 0.03, and 0.09 mg / kg, respectively, injected intramuscularly into the thigh muscle of the hind limb two weeks apart, with a dose volume of 50 μL. Control mice were initially immunized with PBS (n=3), and booster immunizations were also performed with PBS (n=3), thus serving as unimmunized controls.

[0263] Sample collection and cell preparation

[0264] At four weeks into the study, two weeks after booster immunization, mice were euthanized. Blood was collected via cardiac puncture, and serum was separated after natural coagulation at room temperature. Serum was then extracted using a microcentrifuge (Ole Dich, Hvidovre, Denmark) at 10,000×g for 10 minutes. Serum was stored at -20°C until antibody detection. Spleens and draining inguinal lymph nodes (iLNs) were aseptically collected from euthanized mice. Spleens and iLNs were homogenized using a Falcon 70μm nylon mesh cell filter (Corning, Durham, NC, USA), washed twice with PBS to obtain a single-cell suspension, and then resuspended in PBS supplemented with 5×10⁻⁶ cells / mL. -5 RPMI-1640 (hereinafter referred to as cRPMI+10% FBS) was prepared with β-mercaptoethanol, 1% sodium pyruvate, 1% penicillin-streptomycin, 1% HEPES, and 10% fetal bovine serum. Splenic and inguinal lymph node cells were transferred to 96-well round-bottom microtiter plates (Nunc), 100 µL per well containing 2 × 10⁻⁶ cells. 5Cells were collected for cytokine assay and transferred to 96-well V-bottom microtiter plates (Corning, New York, USA), 100 µL per well containing 1 × 10⁻⁶ cells. 6 Quantitative cells were selected for intracellular and tetrameric flow cytometry analysis, respectively.

[0265] ELISA

[0266] Immunoglobulin G

[0267] Enzyme-linked immunosorbent assay (ELISA) was used to detect S protein-specific IgG in serum samples. In short, S protein (Statens Serum Institut, Denmark) was dissolved at a concentration of 0.5 μg / mL in carbonate buffer (pH=9.6), and this solution was used to coat Maxisorp plates (Nunc) and incubated overnight at 4°C. The plates were washed three times with PBS + 0.2% Tween 20 and blocked with PBS + 2% BSA. Serum samples were serially diluted 11 times (5-fold) with PBS + 1% BSA, starting from a 1:10 dilution. S protein-specific IgG was detected using horseradish peroxidase-labeled rabbit anti-mouse IgG (1:2500, Invitrogen, Carslbad, CA, USA). TMB Plus2 was used as the substrate. The enzymatic reaction was terminated by adding 0.5 M H2SO4, and the absorbance was read at 450 nm using a Sunrise microplate reader (Tecan Group Ltd.), corrected for at 570 nm. The serum OD value was used to calculate the ELISA endpoint titer through nonlinear regression analysis, which yielded the dilution factor required for an OD value greater than 0.2.

[0268] Flow cytometry

[0269] Intracellular staining

[0270] For intracellular staining, spleen cells and lymph node cells were stimulated overnight at 37°C with recombinant S protein (1 μg / mL) and the smallest CD8 epitope of S protein (VNFNFNGL, SEQ ID NO.: 13, 1 μg / mL), along with anti-CD28 (37.51, BD Biosciences) and anti-CD49d (9C10, BD Biosciences), both at a concentration of 1 μg / mL. As negative and positive controls, cRPMI + 10% fetal bovine serum (FBS) and a mixture of phorbol 12-myristate 13-acetate (0.05 μg / mL, Sigma-Aldrich) and iomycin (1 μg / mL, Sigma-Aldrich) were used, respectively. Bleomycin A (10 μg / mL, Sigma-Aldrich) was added, followed by incubation for another 5 hours. To detect CD8+ producing cytokines... + T cells were stained using a combination of surface CD4, CD8, and CD44 staining and intracellular cytokine staining (as described below).

[0271] After stimulation, spleen and lymph node cells were washed with PBS + 1% fetal bovine serum (FBS). Each well was then stained with a mixture of anti-CD44-FITC (1:600, eBioscience, Thermo Fisher Scientific, Cat# 11-0441-85), anti-CD8-PerCP-Cy5.5 (1:600, eBioscience, Thermo Fisher Scientific, Cat# 45-0081-82), and anti-CD4-APC-eFluor780 (1:600, eBioscience, Thermo Fisher Scientific, Cat# 47-0042-82). Cells were incubated at 4°C in the dark for 20 minutes. Cells were then washed twice, fixed with 100 µL / well of Cytofix / Cytoperm (BD Biosciences), and permeabilized at 4°C in the dark for 20 minutes. Cells were washed twice with osmosis / wash buffer (BD Biosciences), and 50 µL of a mixture of anti-IFN-γ-PE-Cy7 (1:200, eBioscience, Thermo Fisher Scientific, Cat# 25-7311-82), anti-TNF-α-PE (1:200, BD Biosciences Cat# 554418), and anti-IL-2-APC (1:200, eBioscience, Thermo Fisher Scientific, Cat# 17-7021-82) was added to each well. Cells were incubated at 4°C in the dark for 20 min. Cells were then washed twice with PBS + 1% fetal bovine serum (FBS) and analyzed using LSR Tortessa (BD Biosciences).

[0272] Tetramer staining was performed to determine the spike protein-specific CD8. +Percentage of spleen cells. Spleen cells were stained at 37°C for 30 minutes in the dark with a mixture of 10 µL / well FC-Block (1:100) and PE-labeled VNFNFNGL tetramer (1:50, obtained from the NIH Tetramer Core Facility). Cells were washed twice with PBS + 1% fetal bovine serum (FBS), and then stained at 4°C in the dark for 20 minutes with a mixture of 50 µL / well of anti-CD62L-FITC (1:200, BD Biosciences Cat#553150), anti-CD8-PerCP-Cy5.5 (1:200, eBioscience Cat#45-0081-82), anti-CD19-PE-Cy7 (1:200, BD Biosciences Cat#552854), anti-CD44-APC (1:400, BD Biosciences Cat#559250), anti-CD4-APC-eFluor780 (1:200, eBioscience Cat#47-0042-82) and fixable live cell eFluor-506 (1:500, eBioscience Cat#65-0866-18). Cells were then washed twice with PBS + 1% fetal bovine serum (FBS), resuspended in 200 µL / well of PBS + 1% FBS, and analyzed using LSR Fretessa (BDBiosciences). All flow cytometry data were analyzed using FlowJo software v10 (Tree Star, Ashland, OR, USA).

[0273] Plaque Reduction Neutralization (PRNT) Test

[0274] SARS-CoV-2 variant β (B.1.351, GISAID accession number EPI_ISL_678615) was provided by Professor Alex Sigal of the African Institute of Health, South Africa. SARS-CoV-2 B.1.351 was amplified and cultured in A549 cells expressing human ACE-2 (provided by Benjamin Tenoever, New York University). At 72 hours post-infection, supernatant containing newly generated viral progeny was collected and centrifuged at 3000×g for 10 minutes to precipitate cell debris. The virus-containing supernatant was then filtered through a 0.45µm filter and concentrated using a MilliporeSigma centrifuge at 4000×g for 30 minutes. Viral titer was determined by the TCID50 assay and calculated using the Reed-Muench method.

[0275] Figures 16A-B

[0276] Administration of S mRNA-loaded LNPs via the subcutaneous route

[0277] S mRNA-loaded LNPs can be formulated into preparations with a particle size less than 130 nm, a polydispersity index (PDI) less than 0.2, and an mRNA encapsulation efficiency greater than 90% (Table 5).

[0278] Table 5: Physicochemical properties of S mRNA-loaded LNPs

[0279]

[0280] Approved COVID-19 mRNA vaccines induce high-intensity S protein-specific CD4 + and CD8 + T cell responses and antibodies that have been shown to effectively neutralize SARS-CoV-2. To investigate the effect of replacing cholesterol with MMG-1 in LNPs on the immune response upon subcutaneous administration, mRNA encoding the S protein (SEQ ID NO.: 5) was loaded into C12-200, C12-200MMG 23, C12-200MMG 47, C12-200MMG 47 (WR 10:1), and SM-102 MMG 37 LNPs, respectively (Table 1). When using the H-2K(b)-restricted SARS-CoV-2 spike protein 539-546 (VNFNFNGL) tetramer to evaluate CD8 + T cell responses, the inventors found that both C12-200 and C12-200 MMG LNPs induced a high frequency of tetramer-specific CD8 + T cells in the spleen (Figure 15A), and the frequency was significantly higher than that measured in the unvaccinated control group (0.0034 < p < 0.0001, combined with one-way ANOVA with Dunnett's correction for multiple comparisons). Intracellular cytokine staining confirmed this, and the results showed that restimulation of splenocytes with the VNFNFNGL CD8 minimal epitope of the S protein led to high levels of antigen-specific CD8 + T cells secreting IFN-γ (Figure 15B) and TNF-α (Figure 15C). Interestingly, compared with other groups, the C12-200MMG 47 (WR 10:1) LNP group showed a higher trend of CD8 + T cell responses. When comparing this preparation (C12-200 MMG 47 (WR10:1)) with C12-200 MMG 47 (WR 20:1), it was observed that CD8 +The percentage of T cells was significantly increased (P values ​​were 0.0473, 0.0009, and 0.0001, respectively; Figures 15B-C). Therefore, C12-200 MMG 47 (WR10:1) will be referred to as C12-200 MMG 47 below. Similar results were observed in previous reports, where OVA-specific CD8 increased with increasing ionizable lipid content, even when the weight ratio remained constant. + The percentage of T cells also decreases.

[0281] Anti-SARS-CoV-2 IgG effectively reduced the risk of death in humans and mice, highlighting the crucial role of antibodies in virus neutralization. Therefore, the inventors tested the ability of C12-200, C12-200 MMG, and SM-102 MMG-GLNPs loaded with S mRNA to mediate IgG production and virus neutralization (Figures 16A-D). The inventors found no significant difference in S protein-specific IgG endpoint titers between the C12-200 and C12-200 MMG groups. Compared to C12-200, SM-102 MMG immunized mice showed lower S protein-specific IgG endpoint titers (P=0.0531). Figures 17A-B This may be due to the high PEG-lipid content in SM-102 / MMG LNPs, which inhibits cellular uptake of LNPs, leading to lower protein expression. However, all vaccinated mice showed significantly higher IgG titers compared to unvaccinated mice. (Tissue culture infection dose 50 (TCID50)) 50 The TCID50 assay is a technique suitable for the quantitative detection of viruses that produce cytopathic effects in cells. It can be used to determine the virus-neutralizing activity of serum, which may be positively correlated with the protective effect against the virus. The inventors found that serum derived from inoculated mice could neutralize the virus, while serum derived from uninoculated mice could not (Figure 16C). No significant differences in TCID50 values ​​were observed between the groups (Figure 16D), indicating that replacing cholesterol with MMG-1 neither impaired the antibody response nor weakened the virus-neutralizing activity.

[0282] LNPs loaded with S mRNA administered via intramuscular route

[0283] To investigate the effect of replacing cholesterol with MMG-1 in LNPs on the immune response, mRNA encoding the S protein was loaded into C12-200, C12-200 MMG 47, SM-102, and SM-102 MMG 38 LNPs (Table 1). H-2K(b)-restricted SARS-CoV-2 spike protein was used. 539-546 (VNFNFNGL) Tetramer Evaluation CD8 +During T cell responses, the inventors discovered that all LNPs induce a high frequency of tetramer-specific CD8 in the spleen. + T cells ( Figures 18A-B The frequency of MMG-1 modified LNPs was significantly higher than that measured in the unvaccinated control (p<0.0001, combined with Dunnett's one-way ANOVA with multiple comparison correction), confirming that MMG-1 modified LNPs can induce an immune response similar to that of cholesterol-based LNPs.

[0284] The inventors also tested the IgG production mediated by C12-200, C12-200 MMG 47, SM-102, and SM-102 MMG38 LNPs loaded with S mRNA. Figures 19A-B ) and virus neutralization ( Conclusions The inventors found no significant difference in S protein-specific IgG endpoint titers between MMG-1 modified and cholesterol-based LNPs. The S protein-specific IgG endpoint titer depended on the dose of mRNA encapsulated in the LNPs, with C12-200 and C12-200 MMG 47 LNPs reaching saturation at a dose of 0.6 μg (Figure 18A), while SM-102 MMG 38 LNPs showed dose-dependent titers at all doses (Figure 18B). All vaccinated mice showed significantly higher IgG titers than unvaccinated mice (data not shown). Tissue culture infection dose 50 (TCID50) 50 The TCID assay is a technique suitable for the quantitative detection of viruses that produce cytopathic effects in cells. It can be used to determine the virus-neutralizing activity of serum, which may be positively correlated with protective effects against the virus. The inventors found that serum derived from inoculated mice could neutralize the virus, while serum derived from uninoculated mice could not (Figure 19A). No differences in TCID were observed between groups. 50 The values ​​showed significant differences (Figure 19B), indicating that replacing cholesterol with MMG-1 neither impaired the antibody response nor weakened viral neutralizing activity, and these results confirm that the cationic lipids C12-200 and SM-102 are interchangeable.

[0285] Objectives

[0286] When MMG-1 was used to replace cholesterol in C12-200 and SM-102 LNPs, the immunogenicity of the LNPs in terms of antibody response or viral neutralization activity was not affected regardless of the route of administration (subcutaneous or intramuscular). Notably, the CD8+ of the C12-200 MMG 47 LNPs group... + The T-cell response showed a trend of being higher than in other groups, indicating that replacing cholesterol with MMG enhanced immunogenicity.

[0287] Example 8: Testing the ability of MMG-1-modified LNPs to deliver circular RNA

[0288] Materials and methods

[0289] This study aims to verify whether MMG-1 modified LNPs can form nanoparticles with good physicochemical properties and mediate the expression of FLuc, which encodes circular RNA.

[0290] Results

[0291] The circular RNA encoding FLuc was purchased from Seattle Genova, USA. The method for measuring bioluminescence was the same as described in Examples 3-4.

[0292] Figure 20

[0293] MMG-1 modified LNPs loaded with circular FLuc RNA were successfully prepared. As their physicochemical properties indicate, they had a size of 133 nm, a particle size distribution index (PDI) of 0.133, and a circular FLuc RNA encapsulation efficiency of 96.5%. Six hours after subcutaneous injection, the bioluminescent signal (total flux) at the SOI in the supine position was approximately 10. 8 Photons / second ( Conclusions The signal originating from linear FLuc mRNA is almost 10 times that from circular RNA.

[0294] Objectives

[0295] Although the bioluminescent signal observed in MMG-1-modified LNPs loaded with circular FLucRNA was attenuated compared to the signal from MMG-1-modified LNPs derived from linear FLuc mRNA, this embodiment demonstrates that the MMG-1-modified LNPs of the present invention can contain different types of nucleic acids.

[0296] Example 9: Effects of replacing cholesterol with MMG analogs on physicochemical properties and in vivo protein expression

[0297] Materials and methods

[0298] This study aims to investigate whether LNPs can be formulated by replacing cholesterol components with different MMG analogs, specifically MMG-1, MMG-2, MMG-3, MMG-4, MMG-5, and MMG-6, and whether these LNPs can mediate FLuc expression in mice after intramuscular injection.

[0299] Results

[0300] Various MMG analogs were synthesized according to previously reported methods (Nordly, P. et al. 2011, Martin-Bertelsen, B. et al. (2013)). The bioluminescence determination method was the same as described in Example 5. The molar composition of the C12-200-based MMG-modified LNPs was 35 mol% C12-200: 16 mol% DOPE: 47.5 mol% MMG-X: 1.5 mol% DMPE-PEG. 2000 Where X ranges from 1 to 6. For ease of naming, this series of LNPs is designated as C12-200 MMG-1 47, C12-200 MMG-2 47, etc. The molar composition of SM-102 MMG-6 is 50 mol% SM-102: 10% DSPC: 38.5 mol% MMG-6: 1.5 mol% DMG-PEG. 2000 .

[0301] Figure 21

[0302] For LNPs modified with MMG using C12-200 as a cationic or cationic lipid or lipid-like material, their size is between 132.6 and 160.9 nm, the particle size dispersion index (PDI) is less than 0.1, and the mRNA encapsulation efficiency is greater than 90%, while the size of SM-102 MMG-6 is 87.2 nm (Table 6).

[0303] Table 6: Physicochemical properties of LNPs modified with various MMG analogs

[0304]

[0305] Mice injected with C12-200 MMG-1 47, C12-200 MMG-2 47, C12-200 MMG-647, and SM-102 MMG-1 38 LNPs loaded with FLuc mRNA showed very strong bioluminescent signals. Conclusions Other LNPs failed to mediate the same level of protein expression, presumably due to the shorter chain lengths of MMG-3 and MMG-4 and the different stereochemical structure of MMG-5 (2S rather than 2R). However, protein expression mediated by C12-200 MMG-347, C12-200 MMG-447, C12-200 MMG-547, and SM-102 MMG-638 remained above background levels, which in this example had a background value of 10. 5 That is, the point where the X and Y axes intersect.

[0306] Objectives

[0307] The MMG-modified LNPs of the present invention, using different MMG analogs, can induce protein expression in vivo.

[0308] Example 10: Effects of replacing cholesterol with MMG-1 on physicochemical properties and in vitro gene silencing

[0309] Materials and methods

[0310] This study aims to explore the physicochemical properties of LNPs by replacing cholesterol with MMG-1, and to verify whether the replaced LNPs can mediate in vitro gene silencing in mouse macrophages.

[0311] Results

[0312] In vitro gene silencing

[0313] The 2'-O-methyl-modified Dicer substrate asymmetric small interfering RNA (siRNA) duplexes targeting tumor necrosis factor-α (TNF-α) siRNA (17928.334 g / mol) were provided by GlaxoSmithKline (Stevenage, UK) as dried, purified, and desalted duplexes (SEQ ID NO.: 3 and SEQ ID NO.: 4). These siRNA duplexes were re-annealed according to the recommendations of Integrated DNA Technologies (IDT, Coralville, IA, USA).

[0314] The mouse macrophage cell line RAW 264.7 was purchased from the American Type Culture Collection (TIP71, Manassas, VA, USA). Cells were maintained in Dulbecco-modified Eagle medium with high (4.5 g / L) glucose (DMEM+, Fisher Scientific Biotech Line, Slangerup, Denmark), supplemented with 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM glutamine (all from Sigma-Aldrich), and 10% fetal bovine serum (FBS, Gibco, Life Technologies). Cells were cultured and grown in an incubator with 5% CO2, 95% atmospheric air, and 37°C. The growth medium was changed every other day, and cells were removed from the culture flask (75 cm²) using a cell scraper. 2 Cells were scraped off using a Sigma-Aldrich medium and passaged twice weekly. Cells were cultured at a rate of 1.0 × 10⁶ cells / week. 6Cells were seeded at a density of 10 cells / well in 6-well tissue culture plates (Sigma-Aldrich). Subsequently, a nanoparticle suspension was added to each well to achieve final siRNA concentrations of 2.8, 5.6, 11.3, 27.9, and 55.8 nM, with each concentration replicated in duplicate, followed by incubation for 21 hours. Lipopolysaccharide (LPS, Sigma-Aldrich) was added to each well to a final concentration of 5 ng / mL, and cells were incubated for another 3 hours. After 24 hours, cells were lysed with 350 μL of NucleoSpin cell lysis buffer (Macherey-Nagel, Düren, Germany), and total RNA was isolated and purified using the NucleoSpin RNA Plus kit (Macherey-Nagel). The purity of total RNA was determined and quantified using UV-Vis spectrophotometry (Nanodrop 2000, ThermoFisher Scientific). The purified RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). Real-time polymerase chain reaction (PCR) or quantitative PCR (qPCR) was performed using a LightCycler® 480 (Roche, Basel, Switzerland) and SYBR I Green® Master Mix (Roche). β-actin (ACTB) and β-glucuronidase (GUSB), serving as reference housekeeping genes, were analyzed twice, while TNF-α was analyzed three times. The concentrations of ACTB, GUSB, and TNF-α primers in the reaction mixture were 1.0, 0.5, and 1.0 μM, respectively. Cross-point (CP) analysis was performed using LightCycler® 480 software v.1.5.0 (Roche), followed by quantification relative to LPS-treated cells using the ΔΔCP comparison method.

[0315] Figure 22

[0316] LNPs loaded with siRNA targeting TNF-α (SEQ ID NO.: 3 and 4) and FLuc were successfully formulated, and these formulations exhibited good physicochemical properties. The size was less than 140 nm, the polydispersity index (PDI) was less than 0.15, and the siRNA encapsulation efficiency was greater than 90% (Table 7).

[0317] Table 7: Physicochemical properties of cholesterol and MMG-1 modified LNPs loaded with siRNA targeting TNF-α

[0318]

[0319] After incubating mouse macrophages with LNPs loaded with siRNA for 24 hours, TNF-α gene expression was downregulated by approximately 70%. Conclusions There was no significant difference in gene silencing mediated by C12-200 and C12-200 MMG 47 LNPs. As an unrelated siRNA control, LNPs loaded with FLuc siRNA did not mediate TNF-α gene silencing (data not shown), highlighting the specificity of the silencing effect.

[0320] Objectives

[0321] MMG-1 modified LNPs can also be formulated with small nucleic acid payloads such as siRNA, and the gene silencing mediated by these LNPs is at the same level as that mediated by cholesterol-based LNPs.

[0322] Example 11: Effects of Tris-HCl buffer and freeze-thaw cycles on FLuc expression in mice when MMG-1 is used to replace cholesterol.

[0323] Materials and methods

[0324] This study aimed to investigate the physicochemical properties of C12-200 MMG47 LNPs prepared using Tris-HCl as dispersion buffer (20 mM, pH 7.4) and the in vivo FLuc expression of these LNPs after one freeze-thaw cycle.

[0325] Results

[0326] The LNPs were prepared according to the method described in Example 1, except that the dialysis was performed in 20 mM Tris-HCl buffer (pH 7.4). After collecting the LNPs, 40% sucrose (Sigma Aldrich, molecular biology grade) was prepared in 20 mM Tris-HCl buffer (pH 7.4). A sufficient volume of this solution was added to the LNPs to achieve a final sucrose concentration of 8.5% by volume in the LNP dispersion. The LNPs were rapidly frozen in liquid nitrogen and then immediately transferred to a freezer at -80°C or -20°C. After one week of storage, the LNPs were thawed in a water bath at room temperature. Subsequently, FLuc mRNA-LNPs were subcutaneously injected into the tail base of mice at a dose of 0.2 mg FLuc mRNA / kg mouse body weight encapsulated in the LNPs, and the bioluminescent signal (total flux) was quantitatively detected at the injection site 6 hours after administration.

[0327] Formulation number

[0328] Compared to LNPs prepared with PBS (Table 2), LNPs prepared with 20 mM Tris-HCl buffer exhibited larger sizes (Table 8), possibly due to the low ionic strength of the buffer. However, their size was less than 180 nm, their polydispersity index was less than 0.15, and they showed an mRNA encapsulation efficiency greater than 90%.

[0329] Table 8: Physicochemical properties of MMG-1 modified LNPs loaded with FLuc mRNA in Tris-HCl buffer (20 mM, pH 7.4)

[0330] Z-average (nm) Polydispersity index (PDI) mRNA encapsulation efficiency (%) C12-200 MMG 47 Tris Figure 23 173.2 0.085 95.2

[0331] After thawing at room temperature and injecting the LNP formulation into mice, LNPs loaded with mRNA that had previously been frozen at -80°C or -20°C for one week showed a strong bioluminescent signal (>10). 8 (photons / second), this signal is comparable to that observed in fresh LNPs ( Conclusions This indicates that these formulations remain stable during freeze-thaw cycles and can effectively mediate FLuc protein expression.

[0332] ​

[0333] MMG-1 modified LNPs can be rapidly frozen and then stored at -80°C or -20°C for one week without affecting their ability to mediate protein expression.

[0334] References

[0335] WO 2021 / 148511 A1

[0336] Love, KT, Mahon, KP, Levins, CG, Whitehead, KA, Querbes, W., Dorkin, JR, Qin, J., Cantley, W., Qin, LL, Racie, T. et al. (2010) Lipid-like materials for low-dose, in vivo gene silencing. Proc Natl Acad Sci U SA, 107, 1864-1869

[0337] Lokras, A., Chakravarty, A., Rades, T., Christensen, D., Franzyk, H.,Thakur, A. and Foged, C. (2022) Simultaneous quantification of multiple RNAcargos co-loaded into nanoparticle-based delivery systems. Int J Pharm, 626,122171.

[0338] Thakur, A., Ingvarsson, P.T., Schmidt, S.T., Rose, F., Andersen, P.,Christensen, D. and Foged, C. (2018) Immunological and physical evaluation ofthe multistage tuberculosis subunit vaccine candidate H56 / CAF01 formulated asa spray-dried powder. Vaccine, 36, 3331-3339

[0339] Pastore, G., Carraro, M., Pettini, E., Nolfi, E., Medaglini, D. andCiabattini, A. (2019) Optimized Protocol for the Detection of MultifunctionalEpitope-Specific CD4(+) T Cells Combining MHC-II Tetramer and IntracellularCytokine Staining Technologies. Front Immunol, 10, 2304.

[0340] Vono, M., Eberhardt, C.S., Auderset, F., Mastelic-Gavillet, B.,Lemeille, S., Christensen, D., Andersen, P., Lambert, P.H. and Siegrist, C.A.(2019) Maternal Antibodies Inhibit Neonatal and Infant Responses toVaccination by Shaping the Early-Life B Cell Repertoire within GerminalCenters. Cell Rep, 28, 1773-1784 e1775.

[0341] Christensen, D., Mortensen, R., Rosenkrands, I., Dietrich, J. andAndersen, P. (2017) Vaccine-induced Th17 cells are established as residentmemory cells in the lung and promote local IgA responses. Mucosal Immunol,10, 260-270.

[0342] Thakur, A., Rodriguez-Rodriguez, C., Saatchi, K., Rose, F., Esposito,T., Nosrati, Z., Andersen, P., Christensen, D., Hafeli, U.O. and Foged, C.(2018) Dual-Isotope SPECT / CT Imaging of the Tuberculosis Subunit Vaccine H56 / CAF01: Induction of Strong Systemic and Mucosal IgA and T-Cell Responses inMice Upon Subcutaneous Prime and Intrapulmonary Boost Immunization. FrontImmunol, 9, 2825.

[0343] Nordly P, Korsholm KS, Pedersen EA, Khilji TS, Franzyk H, JorgensenL, Nielsen HM, Agger EM, Foged C. (2011) Incorporation of a syntheticmycobacterial monomycoloyl glycerol analogue stabilizesdimethyldioctadecylammonium liposomes and potentiates their adjuvant effect in vivo. Eur J Pharm Biopharm. 2011 Jan;77(1):89-98.

[0344] Martin-Bertelsen, B.; Korsholm, KS; Rose, F.; Nordly, P.; Franzyk, H.; Andersen, P.; Agger, EM; Christensen, D.; Yaghmur, A.; Foged, C. Thesupramolecular structure is decisive for the immunostimulatory properties ofsynthetic analogues of a mycobacterial lipid in vitro. RSC Adv. 2013, 3,20673-20683.

[0345] sequence list

[0346]

[0347] * Lowercase letters indicate deoxyribonucleotides, and p indicates phosphate residues.

Claims

1. A lipid nanoparticle (LNP) composition comprising cationic or cationic lipid or lipid-like materials, auxiliary lipids, lipid polymers, and monoacylglycerol (MMG) analogs. in, The cationic or cationic lipid or lipid-like material is selected from 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200), N1,N16-bisdodecyl-4,7,13-tris[3-(dodecylamino)-3-oxopropyl]-4,7,10,13-tetraazahexadecanediamide (98N12-5), tetra(8-methylnonyl)3,3',3'' 3'''-(((methylazadiyl)bis(propane-3,1-diyl))bis(azatriyl))tetrapropionate (306Oi10), 3,3',3'',3'''-(ethane-1,2-diylbis(azatriyl))tetra(N-(2-((2-hydroxytetradecyl)amino)ethyl)propionamide) (G0-C14), 9-heptadecyl-8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis( 2-Hexyldecanoate (ALC-0315), N1,N3,N5-Tris(3-(bis(dodecylamino)propyl)phenyl-1,3,5-tricarboxamide (TT3), Dilinoleoylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-Dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Hexa(octane-3-yl)-9,9',9'',9''',9''',9''''-((((benzene-1,3,5-tricarbonyl)tri(azadiyl) Tris(propane-3,1-diyl)tris(azatriyl)hexanoate (FTT5), dimethyl bis(octadecyl)ammonium bromide (DDAB), 1,2-dioleoyl-3-dimethylammonium chloride (DODAC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione (cKK-E12), 2-[3-[3-[3-[bis[3-[2-(2-methyl-3-octylthiopropionyl)oxyethoxy]-3-oxopropyl] The group consisting of [amino]propyl-methylamino]propyl-[3-[2-(2-methyl-3-octylthiopropionyl)oxyethoxy]-3-oxopropyl]amino]propionyloxy]ethyl-2-methyl-3-octylthiopropionate (4A3-SC8) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) or any mixture thereof. The auxiliary lipid is selected from 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphatidyl-(1'-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1,2-distearate-sn-glycerol-3-phosphatidylcholine (DSPC), and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine. The group consisting of glycerol phosphate (DPPG), 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC), 1,2-diacyl-3-O-β-D-galactosyl-sn-glycerol (MGDG), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (SOPC), 1,2-diacyl-3-O-(α-D-galactosyl-1-6)-α-D-galactosyl-sn-glycerol (DGDG) and sulfosucrose diacylglycerol (SQDG), or any mixture thereof, and The lipid polymer is polyethylene glycol (PEG) or polysarcosine-lipid conjugate or PEG- or polysarcosine-lipid conjugate or any mixture thereof.

2. The lipid nanoparticle (LNP) composition according to claim 1, wherein, The lipid polymer is selected from 1,2-dimyristoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG). 2000 ), 1,2-Dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG) 2000 ), 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) 2000 ), 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE-PEG), 1,2-distearyl-rac-glycerol-3-methoxy polyethylene glycol (DSG-PEG), ceramide-PEG, 1,2-dipalmitoyl-rac-glycerol-3-methylpolyoxyethylene (DPG-PEG), 1,2-dioleoyl-rac-glycerol, methoxy polyethylene glycol The group consisting of (DOG-PEG), 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine-N-methylpolyoxyethylene (DOPE-PEG), N-tetradecyl polysarcosine 25, N-hexadecyl polysarcosine 25, N-octadecyl polysarcosine 25, N-dodecyl polysarcosine 25, N,N-bistetradecylamine-N-succinoyl[methyl(polysarcosine)45], N,N-bistetradecylamine-N-succinoyl[methyl(polysarcosine)35] and N,N-bistetradecyl polysarcosine-25 or mixtures thereof.

3. The lipid nanoparticle (LNP) composition according to any one of claims 1 or 2, wherein, The cationic or cationic lipid or lipid-like material is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200), tetra(8-methylnonyl)3,3',3'',3'''-(((methylazadiyl)bis(propane-3,1-diyl))bis(azatriyl) 9-Heptadecanyl-8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione (cKK-E12), 2-[3-[3-[3-[bis[3-[2-(2-methyl-3-octylthiopropionyl)oxyethoxy]-3-oxopropyl] [Amino]propyl-methylamino]propyl-[3-[2-(2-methyl-3-octylthiopropionyl)oxyethoxy]-3-oxopropyl]amino]propionyloxy]ethyl-2-methyl-3-octylthiopropionate (4A3-SC8), dilinoleoylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1,2-dioleoyl -3-trimethylammonium propane (DOTAP), dimethyl bis(octadecyl)ammonium bromide (DDAB), 1,2-dioleoyl-3-dimethylammonium chloride (DODAC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) or [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), preferably C12-200, SM-102, ALC-0315, DLin-KC2-DMA, DLin-MC3-DMA, cKK-E12 or 4A3-SC8, more preferably C12-200 or SM-102.

4. The lipid nanoparticle (LNP) composition according to any one of the preceding claims, wherein, The auxiliary lipid is 1,2-distearyl-sn-glycerol-3-phosphatidylcholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (SOPC) or 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), preferably DSPC or DOPE.

5. The lipid nanoparticle (LNP) composition according to any one of the preceding claims, wherein, The lipid polymer is 1,2-dimyristoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG). 2000 ), 1,2-distearyl-rac-glycerol-3-methoxy polyethylene glycol (DSG-PEG), 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) 2000 ), 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159) or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG) 2000 ), preferably DMPE-PEG 2000 DMG-PEG 2000 ALC-0159, N-Tetradecyl Polysarcosine 25 or DSPE-PEG 2000 More preferably, DMPE-PEG 2000 or DMG-PEG 2000 .

6. The lipid nanoparticle (LNP) composition according to any one of the preceding claims, wherein, The monoacylglycerol (MMG) analogue is selected from the group consisting of MMG-1, MMG-2, MMG-3, MMG-4, MMG-5, MMG-6 and MMG-7 or any mixture thereof, preferably MMG-1, MMG-2, MMG-6 and / or MMG-7, more preferably MMG-1, MMG-6 and / or MMG-7, and most preferably MMG-1.

7. The lipid nanoparticle (LNP) composition according to any one of the preceding claims, wherein, The LNP also contains cholesterol.

8. The lipid nanoparticle (LNP) composition according to any one of the preceding claims, wherein, The LNP also contains at least one nucleic acid.

9. The lipid nanoparticle (LNP) composition according to claim 8, wherein, The at least one nucleic acid is selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA, circular RNA (circRNA), plasmid DNA (pDNA), small interfering RNA (siRNA), single guide RNA (sgRNA), guide RNA (gRNA), long non-coding RNA (lncRNA), small activating RNA (saRNA), and splice-conversion antisense oligonucleotides (ASO).

10. A vaccine composition comprising a lipid nanoparticle (LNP) composition according to any one of the preceding claims and at least one nucleic acid encoding an antigen.

11. The vaccine composition according to claim 10, wherein, The antigens are selected from the group consisting of coronavirus antigens such as SARS-CoV2 spike protein (SEQ ID NO.: 5) or receptor-binding domain (RBD) of SARS-CoV and MERS-CoV antigens, Mycobacterium tuberculosis antigen (SEQ ID NO.: 8-9), Plasmodium falciparum antigen (SEQ ID NO.: 10), respiratory syncytial virus (RSV) antigen (SEQ ID NO.: 11), Ebola virus antigen, Marburg virus antigen, Lassa virus antigen, Nipah virus antigen, Zika virus antigen, Crimean-Congo hemorrhagic fever virus antigen, human papillomavirus (HPV) antigen, and influenza virus antigen.

12. Use of the vaccine composition according to any one of claims 10 or 11 for the prevention and / or treatment of infectious diseases.

13. A method for obtaining a lipid nanoparticle (LNP) composition according to any one of claims 8 or 9, the method comprising the steps of: a) Provides cationic or cationic lipid or lipid-like materials, auxiliary lipids, lipid polymers, monoacylglycerol (MMG) analogs, and at least one nucleic acid. b) Dissolve the cationic or cationic lipid or lipid-like material from step a), the auxiliary lipid, the lipid polymer, and the MMG analog in an organic solvent containing ethanol, preferably anhydrous ethanol with a purity close to 100%, thereby providing an organic phase. c) Dilute the at least one nucleic acid from step a) in an aqueous solvent containing a buffer solution with a pH in the range of 3 to 7.8 to provide an aqueous phase. d) The organic phase from step b) is mixed with the aqueous phase from step c), and lipid nanoparticles (LNPs) are obtained by nanoprecipitation. e) Filter the LNPs obtained in step d), preferably by tangential flow filtration or dialysis, to obtain the LNP composition. The cationic or cationic lipid or lipid-like material is selected from 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200), N1,N16-bisdodecyl-4,7,13-tris[3-(dodecylamino)-3-oxopropyl]-4,7,10,13-tetraazahexadecanediamide (98N12-5), tetra(8-methylnonyl)3,3',3 '',3'''-(((methylazadiyl)bis(propane-3,1-diyl))bis(azatriyl))tetrapropionate (306Oi10), 3,3',3'',3'''-(ethane-1,2-diylbis(azatriyl))tetra(N-(2-(((2-hydroxytetradecyl)amino)ethyl)propionamide) (G0-C14), 9-heptadecyl-8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis (2-Hexyldecanoate) (ALC-0315), N1,N3,N5-Tris(3-(bis(dodecylamino)propyl)phenyl-1,3,5-tricarboxamide (TT3), Dilinoleoylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-Dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Hexa(octane-3-yl)-9,9',9'',9''',9''',9'''',9''''-((((benzene-1,3,5-tricarbonyl)tris(azadi) Tris(propane-3,1-diyl)tris(azatriyl)hexanoate (FTT5), dimethyl bis(octadecyl)ammonium bromide (DDAB), 1,2-dioleoyl-3-dimethylammonium chloride (DODAC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione (cKK-E12), 2-[3-[3-[3-[bis[3-[2-(2-methyl-3-octylthiopropionyl)oxyethoxy]-3-oxopropyl] The group consisting of [amino]propyl-methylamino]propyl-[3-[2-(2-methyl-3-octylthiopropionyl)oxyethoxy]-3-oxopropyl]amino]propionyloxy]ethyl-2-methyl-3-octylthiopropionate (4A3-SC8) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) or any mixture thereof. The auxiliary lipid is selected from 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphatidyl-(1'-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1,2-distearate-sn-glycerol-3-phosphatidylcholine (DSPC), 1,2-dipalmitoyl-sn-glycerol- The group consisting of glycerol 3-phosphate (DPPG), 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC), 1,2-diacyl-3-O-β-D-galactosyl-sn-glycerol (MGDG), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (SOPC), 1,2-diacyl-3-O-(α-D-galactosyl1-6)-α-D-galactosyl-sn-glycerol (DGDG) and sulfosucrose diacylglycerol (SQDG), or any mixture thereof. Wherein, the lipid polymer is a polyethylene glycol (PEG)- or polysarcosine-lipid conjugate or a PEG- or polysarcosine-lipid conjugate or any mixture thereof, and The monoacylglycerol (MMG) analogues are selected from the group consisting of MMG-1, MMG-2, MMG-3, MMG-4, MMG-5, MMG-6 and MMG-7 or any mixture thereof.

14. The method of claim 13, further comprising the following steps: f) Concentrate the lipid nanoparticle (LNP) composition using one of the group consisting of filtration, centrifugation, vacuum-assisted centrifugation, or any combination thereof, preferably filtration.

15. A lipid nanoparticle (LNP) composition obtained using the method of any one of claims 13 or 14.

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