Methods and compositions for vaccines targeting dendritic cells

Abstract

The present disclosure provides novel compounds, methods, and cell-targeting mRNA vaccine formulations for targeted delivery, such as delivery to dendritic cells. The compounds and formulations provided herein are designed to have a targeting moiety configured to provide a selective delivery feature with specificity for dendritic cells and a lipid tail for incorporation into a bilayer membrane of formed lipid nanoparticles.

Description

[0001] Cross-reference of related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 458,102, filed April 8, 2023; U.S. Provisional Patent Application No. 63 / 587,231, filed October 2, 2023; U.S. Provisional Patent Application No. 63 / 588,932, filed October 9, 2023; U.S. Provisional Patent Application No. 63 / 549,343, filed February 2, 2024; U.S. Provisional Patent Application No. 63 / 575,093, filed April 5, 2024; and PCT Patent Publication No. 63 / METHODS AND COMPOSITIONS FOR DENDRITIC CELL TARGETING NANO-DELIVERY, filed April 8, 2024. The entire contents of the foregoing applications are incorporated herein by reference.

[0003] sequence list This application contains a sequence list, which has been submitted electronically in ASCII XML format in accordance with Rule ST.26 and is incorporated herein by full reference. The ASCII copy created on April 5, 2024, is named A1000-01100PCT-3_20240405_SeqListing.xml and is 168 bytes in size. Technical Field

[0004] This disclosure relates to novel vaccine formulations for targeted nanoparticle delivery. Specifically, this disclosure relates to compositions and methods for novel bifunctional nanoparticle vaccine formulations capable of selectively delivering a payload to desired regions in tissues or specific cell types, including dendritic cells. Background Technology

[0005] Local delivery using nanotechnology has been widely used in scientific, industrial, and clinical applications. It has gradually emerged as a promising drug delivery method, offering advantages including improved solubility and permeability of drug molecules. In a recent example of mRNA vaccine development against the COVID-19 virus, considering the instability of mRNA molecules and the need for cryogenic storage (e.g., -70°C), lipid nanoparticles (LNPs) were developed to encapsulate and stabilize mRNA molecules after transport and injection into the human body. Lipid nanoparticles are typically composed of several types of lipids. The ratio of these lipids needs fine-tuning, and the manufacturing cost of lipid nanoparticles can be high. Most importantly, lipid nanoparticles generally cannot deliver mRNA molecules with high local selectivity. Therefore, the need for novel vaccine nanoparticle formulations with selective delivery capabilities remains unmet. Summary of the Invention

[0006] One aspect of this disclosure relates to a bifunctional compound for forming lipid nanoparticles suitable for pharmaceutical vaccine formulations. The components comprise the following formula:

[0007] Formula 1; or

[0008] Formula 2;

[0009] R1 contains substituted or unsubstituted glycosidic groups;

[0010] X1 and X2 are independently hydrogen and C, respectively. 1-30 Alkyl, C 1-30 alkenyl, C 1-30 Alkyne, aryl, aryloxy or their substituted forms, or

[0011] -(CH2)nX4, where n is 0 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic, or a substituted form thereof, provided that when X4 is a heterocyclic group, the heterocyclic group comprises 1 to 3 heteroatoms selected from the group consisting of O, S, and N, or combinations thereof; and

[0012] Where X3 is hydrogen and C 1-6 Alkyl or hydroxyl.

[0013] One aspect of this disclosure relates to a formulation for forming lipid nanoparticles comprising the compounds of this disclosure, wherein the compounds constitute 1 to 10 mol of the composition.

[0014] One aspect of this disclosure relates to a vaccine targeting dendritic cells, comprising lipid nanoparticles and / or lipid nanoparticle formulations. The lipid nanoparticles and / or lipid nanoparticle formulations comprise a membrane defining an internal space, wherein said membrane is formed of a plurality of lipid components comprising compounds of this disclosure.

[0015] One aspect of this disclosure relates to a bifunctional targeted nanocarrier vaccine composition / formulation comprising the lipid nanoparticles of this disclosure.

[0016] One aspect of this disclosure relates to a kit and / or reagent mixture for preparing lipid nanoparticles, comprising: a first reagent comprising a compound of the present disclosure; and a second reagent comprising an ionizable lipid, an auxiliary lipid, or a mixture thereof.

[0017] One aspect of this disclosure relates to a method for targeted delivery of an immunogenic payload to an individual in need, comprising administering to the individual an effective amount of the targeted lipid nanoparticles of this disclosure in the form of a pharmaceutically acceptable vaccine formulation, wherein the payload is a nucleic acid, compound, peptide, protein, polysaccharide, or combination thereof, and the payload is encapsulated by lipid nanoparticles.

[0018] One aspect of this disclosure relates to a method for preventing or treating a disease in an individual in need, comprising administering to the individual an effective amount of the targeted lipid nanoparticles of this disclosure in the form of a pharmaceutically acceptable vaccine formulation, wherein the effective load is a nucleic acid, compound, peptide, protein, polysaccharide, or combination thereof, and the effective load is encapsulated within the lipid nanoparticles; and wherein the effective load is an immunogenic agent / therapeutic agent or a derived immunogenic agent / therapeutic agent.

[0019] One aspect of this disclosure relates to a method for enhancing an adaptive immune response, comprising administering to an individual an effective amount of the targeted lipid nanoparticles of this disclosure in the form of a pharmaceutically acceptable vaccine formulation, wherein the payload is a nucleic acid, compound, peptide, protein, polysaccharide, or combination thereof, and the payload is encapsulated within the lipid nanoparticles; and wherein the payload is an immunogenic or a derivative of an immunogenic biomolecule.

[0020] In some embodiments of bifunctional molecules and their vaccine formulations, Formula 1 may be a structural and / or functional analog / mimicry with cell-targeting function, and Formula 2 may be a structural / functional analog / mimicry with lipid membrane insertion / anchoring function. Attached Figure Description

[0021] Figure 1 The accompanying illustrations of FACS analysis results demonstrate the efficacy of exemplary embodiments of the invention. Experiments show that the uptake of novel nanodelivery formulations targeting BDMCs according to embodiments of this disclosure is significantly improved compared to conventional LNPs. The FITC+ values ​​shown in the figures represent fluorescence intensity in arbitrary units (AU).

[0022] Figure 2The accompanying illustrations of FACS analysis results demonstrate the efficacy of the exemplary novel dendritic cell-targeting formulations of this disclosure. Experiments show the uptake of the exemplary novel dendritic cell-targeting formulations according to embodiments of this disclosure by targeting dendritic cells (DCs), B cells, and T cells compared to conventional LNPs. The FITC+ values ​​shown in the figures represent fluorescence intensity in arbitrary units (AU).

[0023] Figure 3 A graphical representation of FACS analysis results demonstrates the efficacy of the exemplary novel dendritic targeting formulations of this disclosure. Experiments show the uptake of the BDMC-targeting dendritic cell formulations according to embodiments of this disclosure compared to conventional LNPs. FITC+ values ​​shown in the figures represent fluorescence intensity in arbitrary units (AU). 22-LNP indicates a targeting formulation prepared using compound 22 of this disclosure, and the percentage in parentheses indicates the molar ratio of compound 22. Similarly, 23-LNP indicates a targeting formulation prepared using compound 23 of this disclosure, and the percentage in parentheses indicates the molar ratio of compound 23. The negative control is an LNP without the novel targeting compound / formulation of this disclosure (i.e., a “conventional LNP” as described herein).

[0024] Figure 4 A graphical representation of FACS analysis results demonstrates the efficacy of the exemplary novel dendritic cell-targeting formulations of this disclosure. Experiments show transfection of the BDMC-targeting formulations compared to LNPs without the compounds of this disclosure. FITC+ values ​​shown in the figures represent fluorescence intensity in arbitrary units (AU). 22-LNP represents a targeting formulation prepared using compound 22 of this disclosure, and the percentage in parentheses indicates the molar ratio of compound 22. Similarly, 23-LNP represents a targeting formulation prepared using compound 23 of this disclosure, and the percentage in parentheses indicates the molar ratio of compound 23. The negative control is an LNP formed without the novel targeting compounds / formulations of this disclosure (i.e., a “conventional LNP” as described herein).

[0025] Figure 5 This study provides illustrations demonstrating the targeting efficacy and specificity of exemplary formulations targeting LNP distribution in animal models. LNPs carry mRNA configured to encode luciferase in the targeted cells of the tested animal. If the LNP is successfully transfected into cells and the cells express luciferase, the assay will produce detectable cold light. The results clearly show tissue-specific targeting of the exemplary formulations to the spleen and lymphoid tissues, thereby providing supporting evidence for the specificity of immune cells (e.g., dendritic cells).

[0026] Figure 6 Compound 12 of this disclosure is shown 1H NMR spectrum.

[0027] Figure 7 Compound 12 of this disclosure is shown 13 C NMR spectrum.

[0028] Figure 8 The bar graph illustrates the in vivo induction of IFNγ (A) and IL-4 (B) by an exemplary LNP-targeting formulation according to one embodiment of the present disclosure. Serum was collected from experimental animals at 2 hours, 24 hours, and 48 hours after administration.

[0029] Figure 9 The illustrations demonstrate the neutralizing inhibitory effect of the exemplary LNP of this disclosure compared to the control LNP. The neutralizing inhibitory effect was assessed against different dilution factors to illustrate the differences between samples.

[0030] Figure 10 A bar graph is provided to demonstrate that an exemplary LNP carrying mRNA encoding a broad spike protein, according to this disclosure, can induce IgG production in vivo against wild-type virus and its delta and o strains.

[0031] Figure 11 The accompanying bar charts demonstrate the IgG titer (A) and neutralizing capacity (B) induced by commercially available LNPs and LNP formulations based on and / or constructed using the exemplary compounds according to embodiments of this disclosure. Both LNPs carry mRNA encoding a broad spike protein.

[0032] Figure 12 The LCMS spectrum of compound 21 disclosed herein is shown. Detailed Implementation

[0033] Nanoparticles have been widely used in various applications. Among them, lipid nanoparticles (such as liposomes) have become the most mature drug delivery system due to their biocompatibility and biodegradability. Typical liposomes possess a bilayer structure formed from phospholipids due to their amphiphilic properties. The bilayer structure (i.e., a double membrane) surrounds an internal space that can encapsulate hydrophilic molecules, while the bilayer itself can carry hydrophobic molecules. Lipid nanoparticles (LNPs) are also the most studied medium for delivering nucleic acids (such as RNA). Through encapsulation, LNPs protect nucleic acids from extracellular nucleases, thus allowing for safe delivery into cells.

[0034] Liposomes typically contain four lipid components in desired ratios: (i) helper lipids that encapsulate the delivery vehicle, (ii) ionizable lipids that enhance endosome escape and delivery, (iii) cholesterol that promotes stability, and (iv) lipid-anchored polyethylene glycol (PEG-lipids) that reduce immune system recognition and improve biodistribution. Cholesterol and PEG-lipids can also be classified as helper lipids. The properties of liposomes can be modulated by selecting desired lipid components or their ratios. Where it is not desirable to be bound by theory, the targeting portion can also be bound to the lipid components to provide selective delivery.

[0035] Targeted lipid compounds

[0036] One aspect of this disclosure provides a compound for forming formulations of lipid nanoparticles (LNPs) suitable for specific cell targeting. In some embodiments, the compound is a bifunctional compound containing a glycan-based cell-targeting portion and a lipid portion, the lipid portion being incorporated into a lipid bilayer (e.g., an LNP). In some embodiments, the bifunctional compound is designed to have a targeting portion and an exemplary lipid tail portion, the targeting portion being configured to provide selective delivery functionality as part of the bifunctional molecule, the exemplary lipid tail portion being capable of incorporating into a lipid bilayer membrane of an exemplary formulation (including lipid nanoparticles). The compounds of this disclosure may have a dual-tailed structure comprising two extended structures (e.g., hereinafter X1 and X2 groups). At least one of the two extended structures is configured to be incorporated into a bilayer membrane of the formed lipid nanoparticles. Hereinafter, the term "incorporated into a bilayer" describes at least a portion of the extended structure being incorporated into the bilayer. In some embodiments, the entire extended structure is incorporated into the bilayer, but the term is not limited to this context.

[0037] In one exemplary aspect, the compounds disclosed herein comprise:

[0038] Formula 1; or

[0039] Formula 2;

[0040] R1 comprises a substituted or unsubstituted glycosidic group; X1 and X2 are each independently hydrogen, alkyl, alkenyl, alkynyl, aryloxy, or a substituted form thereof, or -(CH2)nX4, where n is 0 to 50, and X4 is hydrogen, aryl, aryloxy, heterocyclic, or a substituted form thereof, provided that when X4 is a heterocyclic group, the heterocyclic group comprises 1 to 3 heteroatoms selected from the group consisting of O, S, and N; and X3 is hydrogen, C 1-6 Alkyl or hydroxyl.

[0041] R 1 Group

[0042] Targeting function In some embodiments, the R1 group is configured to provide selective or targeted delivery functionality to exemplary LNP formulations formed from the components of this disclosure. In some embodiments, the R1 group is configured to target antigen-presenting cells (e.g., dendritic cells). In some embodiments, the target cell may be other types of immune cells. In other embodiments, the target may be any biological cell designed to deliver the payload. In some embodiments, the R1 group is designed to have a targeting portion, which may be a ligand of a receptor on the target cell. For example, the R1 group may be configured to target dendritic cells (DC-SIGN).

[0043] Without being bound by theory, it is believed that mannosides and fucosides can specifically bind to dendritic cells (e.g., via DC-SIGN). Therefore, in some embodiments, the R1 group comprises mannoside, fucoside, or both as the targeting moiety. Mannoside and / or fucoside can be terminal mannose or terminal fucoside of the R1 group, which may provide a better chance of interaction with dendritic cells.

[0044] In some other embodiments, the R1 group is configured to target Siglec-1, and thus the glycoside may comprise 9-N-(4H-thieno[3,2-c]chromene-2-carbamoyl)-Neu5Ac-α2,3-Gal-GlcNAc. In some embodiments, the R1 group is configured to target Siglec-2, and the glycoside may comprise 9-biphenylNeu5Ac-α2,6-Gal-GlcNAc. In some embodiments, the R1 group is configured to target Siglec-5 / E, and the glycoside may comprise Neu5Ac-α2,3-Gal-GlcNAc.

[0045] In some embodiments, the R1 group includes the formula R2-R A - where R2 is a substituted or unsubstituted glycosidic group, and R A It is a linking group, and wherein the linking group is aryl, alkyl, amide, alkylamide, combinations thereof, or covalently bonded. In some embodiments, the aryl group comprises 0 to 3 substituents (e.g., 1 to 3 substituents), wherein the substituents of the aryl group are C. 1-6 Alkyl, halide or C 1-6 Alkyl halides. In some embodiments, the linking group is configured to provide structural flexibility and / or facilitate binding between the targeting moiety and the target. In some embodiments, R2 is linked to R at the carbon atom of the glycosidic group. A Covalent bonding leads to O-glycosylation.

[0046] Combining under acidic conditionsIn some embodiments, the binding between the glycosidic group of R1 and the target is Ca2+. 2+ Relatedly, calcium coordination may decrease at low pH, leading to lower binding affinity. Therefore, to provide better binding affinity under acidic conditions, the linking group may contain an aryl group. Without wishing to be bound by any theory, the aryl group can participate in CH-π and hydrophobic interactions, thereby enhancing binding under acidic conditions. The aryl group can be unsubstituted benzene or benzene substituted with a halide or alkyl halide (e.g., CF3). In some embodiments, the aryl group is coupled to the targeting moiety. For example, the R1 group may contain an O-arylmannoside.

[0047] spacer base In some embodiments, the linking group of R1 comprises a spacer group. The spacer group is configured to provide structural flexibility to R1. This flexibility allows the glycosidic group of R1 to move during the interaction between the targeting moiety and the target, thereby facilitating their binding, where theoretical constraints are not desired.

[0048] In some embodiments, the preferred spacer group is biocompatible. In some embodiments, the initiator spacer group comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof. For example, the spacer group can be a polyethylene glycol (PEG) moiety formed of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 24, 30, 36, 40, 48, 50, 55, 60, 65, or 72 (OCH2CH2) subunits, or any range defined by the aforementioned endpoints, such as 2 to 72, 2 to 60, 2 to 48, 2 to 36, 2 to 24, 2 to 18, 2 to 15, 2 to 10, 4 to 72, 4 to 60, 4 to 48, 4 to 36, 4 to 24, 4 to 18, 4 to 15, 4 to 10, 8 to 72, 8 to 60, 8 to 48, 8 to 36, 8 to 24, 8 to 18, 8 to 15, or 8 to 10 (OCH2CH2) subunits. In some embodiments, the PEG portion may be a straight chain, a branched chain, or a star-shaped structure.

[0049] Structural configuration In some embodiments, the glycoside can be a linear or branched structure. In some embodiments, the glycoside may have multiple targeting moieties, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 targeting moieties. The multiple targeting moieties can be arranged in a linear, branched, or star configuration. For example, the glycoside can comprise monomannoside, dimannoside, or trimannoside, and when the glycoside comprises trimannoside, the trimannoside can be in a linear form or a branched structure, such as α-1,3-α-1,6-trimannoside. In some embodiments, it has been noted that branched configurations (e.g., trimannoside glycan heads) exhibit superior binding affinity to their target receptors.

[0050] In some embodiments, the R1 group is a substituted glycosidic group. The glycosidic group may contain 1 to 6 substituents, and each substituent may be C1. 1-6 Alkyl, C 1-6 alkenyl, halogen, C 1-6 Alkyl halides, C 1-6 Alkoxy, amine, nitro, C 1-6 Alkylamines, amides, azides, aryls, cycloalkyls, heterocycloalkyls, sulfites, or their substituted forms, or combinations thereof. In some embodiments, the substituent is directly bonded to the carbon of the glycosidic group or bonded to the carbon via an O-group (e.g., by replacing a hydrogen atom of a hydroxyl group on the carbon).

[0051] In some embodiments, the substituents of the glycosidic group are selected from the group consisting of aryl, 5-membered cycloalkyl, 6-membered cycloalkyl, 5-membered heterocycloalkyl, and 6-membered heterocycloalkyl and their substituted forms, wherein the glycosidic group comprises 1 to 6 substituents selected from the group consisting of C 1-6 Alkyl, halogen, C 1-6 Alkyl halides, C 1-6 Alkoxy, amine, nitro, C 1-6 Alkylamine, azide, amide, carboxyl, hydroxyl, aryl, cycloalkyl, heterocycloalkyl, or their substituted forms, or combinations thereof. In some embodiments, the heterocycloalkyl group comprises one to three heteroatoms selected from the group consisting of O, S, and N.

[0052] In some embodiments, the substituent of the glycosidic group is a substituted or unsubstituted aryl group, such as a substituted or unsubstituted phenyl group. In some embodiments, the aryl group is substituted by 1 to 6 substituents, each substituent being independently selected from the group consisting of: C 1-6 Alkyl, halogen, C 1-6 Alkyl halides, C 1-6 Alkoxy, amine, nitro, C 1-6 Alkylamine, azide, amide, carboxyl, hydroxyl, aryl, cycloalkyl, heterocycloalkyl, or their substituted forms, or combinations thereof. In some embodiments, the substituent of the glycosidic group is a phenyl (benzene ring) substituted with OH, CH3, NH2, CF3, OCH3, F, Br, Cl, NO2, N3, or combinations thereof. For example, the substituted benzene ring may be a phenolic group.

[0053] In some embodiments, the R1 group is a monomannoside substituted with 1 to 6 substituents, and each substituent may be C16-C ... 1-6 Alkyl, C 1-6 alkenyl, halogen, C 1-6 Alkyl halides, amines, C 1-6Alkylamines, amides, aryl groups, cycloalkyl groups, heterocycloalkyl groups, sulfites, or their substituted forms, or combinations thereof. In some embodiments, the R1 group is a monomannoside substituted with a first substituent and a second substituent; each of the first and second substituents is independently selected from the group consisting of: C 1-6 Alkyl, C 1-6 alkenyl, halogen, C 1-6 Alkyl halides, amines, C 1-6 Alkylamines, amides, aryl groups, cycloalkyl groups, heterocycloalkyl groups, and sulfites.

[0054] In some embodiments, the R1 group comprises a first mannoside and a second mannoside. Each of the first mannoside and the second mannoside is independently substituted by 1 to 6 substituents, and each substituent may be C16-C ... 1-6 Alkyl, C 1-6 alkenyl, halogen, C 1-6 Alkyl halides, amines, C 1-6 Alkylamines, amides, aryl groups, cycloalkyl groups, heterocycloalkyl groups, sulfites, or their substituted forms, or combinations thereof.

[0055] Combining affinity In some embodiments, the binding affinity between the glycosidic group of R1 and the target can be determined by the dissociation constant (K). D (Definition). In some embodiments, K at pH 7.4 DIt can be 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750, or 8000. nM, or any range defined by the aforementioned endpoints, such as 5 to 8000, 5 to 7000, 5 to 6000, 5 to 5000, 5 to 4000, 5 to 3000, 5 to 2500, 5 to 2000, 5 to 1500, 5 to 1250, 5 to 1000, 5 to 900, 5 to 800, 5 to 700, 5 to 600, 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 150, 5 to 100, 5 to 75, 5 to 50, 5 to 30, 5 to 20, 10 Up to 8000, 10 to 7000, 10 to 6000, 10 to 5000, 10 to 4000, 10 to 3000, 10 to 2500, 10 to 2000, 10 to 1500, 10 to 1250, 10 to 1000, 10 to 900, 10 to 800, 10 to 700, 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 150, 10 to 100, 10 to 75, 10 to 50, 10 to 30 or 10 to 20 nM.

[0056] In some other embodiments, K at pH 5 DIt can be 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1250, 1500, 1750, or 2000. nM, or any range defined by the aforementioned endpoints, such as 1 to 2000, 1 to 1500, 1 to 1000, 1 to 900, 1 to 800, 1 to 750, 1 to 700, 1 to 650, 1 to 600, 1 to 550, 1 to 500, 1 to 450, 1 to 400, 1 to 350, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 75, 1 to 50, 1 to 40, 1 to 30, 1 To 20, 1 to 10 or to 5, 5 to 2000, 5 to 1500, 5 to 1000, 5 to 900, 5 to 800, 5 to 750, 5 to 700, 5 to 650, 5 to 600, 5 to 550, 5 to 500, 5 to 450, 5 to 400, 5 to 350, 5 to 300, 5 to 250, 5 to 200, 5 to 150, 5 to 100, 5 to 75, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 10 nM.

[0057] Example In some embodiments, the R1 group is selected from the following group (each structure shown below is independent of each other, regardless of whether it is separated from adjacent structures by semicolons):

[0058] ;

[0059] ;

[0060] ;and

[0061] .

[0062] In some embodiments, the compounds disclosed herein have the structure shown in Formula 3:

[0063] Equation 3; and

[0064] The R1 group is selected from the following group (each structure shown below is independent of each other, regardless of whether it is separated from adjacent structures by semicolons):

[0065] .

[0066] X 1 and X 2

[0067] X1 and X2 are independently hydrogen and C, respectively. 1-30 Alkyl, C 1-30 alkenyl, C 1-30 The group may be alkynyl, aryl, aryloxy, or a substituted form thereof, or -(CH2)nX4, where n is 0 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic, or a substituted form thereof, provided that when X4 is a heterocyclic group, the heterocyclic group comprises 1 to 3 heteroatoms selected from the group consisting of O, S, and N, or combinations thereof. Where it is not desired to be bound by theory, at least one of the X1 and X2 groups is designed to provide a compound of this disclosure having the desired hydrophobicity.

[0068] In some embodiments, at least one of X1 and X2 comprises a saturated hydrocarbon chain containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30 carbons, or any range of carbons defined by the aforementioned endpoints, such as 2 to 30, 2 to 28, 2 to 26, 2 to 24, 2 to 20, 2 to 18, 2 to 15, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 30, 3 to 28, 3 to 26, 3 to 24, 3 to 20, 3 to 30. 18, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 30, 4 to 28, 4 to 26, 4 to 24, 4 to 20, 4 to 18, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8, 10 to 30, 10 to 20, 15 to 30, 15 to 28, 15 to 26 or 15 to 20 carbons.

[0069] In some embodiments, X1 and X2 are each independently hydrogen and C. 4-30 Alkyl, C 4-30 alkenyl, C 4-30 Alkyne, aryl, aryloxy or a substituted form thereof, or -(CH2)nX4, where n is 4 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic or a substituted form thereof, provided that when X4 is a heterocyclic group, the heterocyclic group comprises 1 to 3 heteroatoms selected from the group consisting of O, S and N, or combinations thereof.

[0070] In some embodiments, X1 and X2 are each independently hydrogen and C. 8-30 Alkyl, C 8-30 alkenyl, C 8-30Alkyne, aryl, aryloxy or a substituted form thereof, or -(CH2)nX4, where n is 8 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic or a substituted form thereof, provided that when X4 is a heterocyclic group, the heterocyclic group comprises 1 to 3 heteroatoms selected from the group consisting of O, S and N, or combinations thereof.

[0071] In some embodiments, when one of X1 and X2 is hydrogen, the other is not hydrogen. In some embodiments, when one of X1 and X2 is hydrogen, the other comprises a saturated hydrocarbon chain containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30 carbons, or any range of carbons defined by the foregoing endpoints, such as 2 to 30, 2 to 28, 2 to 26, 2 to 24, 2 to 20, 2 to 18, 2 to 15, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 30, 3 to 28, 3 to 26, 3 to 24, 3 to 20. 3 to 18, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 30, 4 to 28, 4 to 26, 4 to 24, 4 to 20, 4 to 18, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8, 10 to 30, 10 to 20, 15 to 30, 15 to 28, 15 to 26, or 15 to 20 carbons. In some embodiments, one of X1 and X2 is C 15-30 Alkyl group, and another is -(CH2)nX4, as defined above.

[0072] In some embodiments, X4 is an aryl, aryloxy, heterocyclic, cycloalkyl, heterocyclic, or combination thereof, and wherein X4 comprises 0 to 6 substituents selected from the group consisting of: C 1-6 Alkyl, halogen, C 1-6 Alkyl halogens and C 1-6 Alkyl group. In some embodiments, X4 contains 1 to 3 substituents. Substituents may be (but are not limited to) CH3, CF3, F, or OCH3.

[0073] In some embodiments, X4 is -R3-O-R4, wherein R3 and R4 are each independently aryl, heterocyclic, cycloalkyl, or heterocyclic, each comprising 0 to 6 substituents selected from the group consisting of: C 1-6 Alkyl, halogen, C 1-6 Alkyl halogens and C 1-6 Alkyl group.

[0074] In some embodiments, X4 is selected from the group consisting of:

[0075] .

[0076] Exemplary compounds disclosed herein

[0077] This section sets forth some exemplary structures of the compounds disclosed herein. However, this disclosure is not limited to the exemplary structures set forth below or in the specification. In some embodiments, the compounds of this disclosure do not contain glycolipid C34 or α-galactosylceramide (α-GalCer).

[0078]

[0079]

[0080]

[0081]

[0082] .

[0083] Compositions for forming lipid nanoparticles

[0084] Another aspect of this disclosure relates to a composition comprising the compounds of this disclosure. In some embodiments, the compounds constitute 1 to 10 mol% of the composition. In some embodiments, the compound constitutes about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol% of the composition, or any range of carbon defined by the aforementioned endpoints, such as 1 to 10 mol%, 1 to 9 mol%, 1 to 8 mol%, 1 to 7 mol%, 1 to 6 mol%, 1 to 5 mol%, 1 to 4 mol%, 1 to 3 mol%, 1 to 2 mol%, 2 to 10 mol%, 2 to 9 mol%, 2 to 8 mol%, 2 to 7 mol%, 2 to 6 mol%, 2 to 5 mol%, 2 to 4 mol%, 2 to 3 mol%, 3 to 10 mol%, 3 to 9 mol%, 3 to 8 mol%, 3 to 7 mol%, 3 to 6 mol%, 3 to 5 mol%, 3 to 4 mol%, 4 to 10 mol%, 4 to 9 mol%, 4 to 8 mol%, 4 to 7 mol%, 4 to 6 mol%, 4 to 5 mol%, 5 to 10 mol%, 5 to 9 mol%. mol%, 5 to 8 mol%, 5 to 7 mol%, or 5 to 6 mol%.

[0085] In some embodiments, the composition may comprise the first compound and the second compound of this disclosure. In some embodiments, the composition may comprise more than one, two, or three compounds, each compound independently according to the compounds of this disclosure. The first compound, the second compound, or any one or two of more than one, two, or three compounds may be designed to have the same targeting portion of the R1 group, different targeting portions of the R1 group targeting the same target, or different targeting portions of the R1 group targeting different targets.

[0086] In some embodiments, the composition is configured to form lipid nanoparticles. In some embodiments, the formed lipid nanoparticles are intended to perform targeted delivery contributed by the compounds of this disclosure. In some embodiments, the composition configured to form lipid nanoparticles may further comprise ionizable lipids, auxiliary lipids, or mixtures thereof. In some embodiments, the ionizable lipids comprise 30 to 60 mol% of the composition, the auxiliary lipids comprise 5 to 60 mol% of the composition, and the remaining percentage is a carrier or solvent.

[0087] In some embodiments, the composition further comprises a payload (i.e., a carrier), which is discussed further below. In some embodiments, the payload is a first payload, and the composition further encapsulates a second payload. The first payload and the second payload may be the same or different.

[0088] Ionizable lipids.

[0089] In some embodiments, the ionizable lipids will be positively charged at low pH. This feature facilitates the encapsulation and endosome escape of charged molecules, such as nucleic acids (e.g., RNA molecules), thereby allowing the efficient release of the carrier / payload into the cytoplasm. In some embodiments, the ionizable lipids will be neutral at physiological pH, thereby reducing potential toxicity to living organisms. In some embodiments, the ionizable lipids constitute 30 to 60 mol% of the composition. In some embodiments, the ionizable lipids comprise about 30, 35, 40, 45, 50, 55, or 60 mol% of the composition, or any range of carbon defined by the aforementioned endpoints, such as 30 to 60 mol%, 30 to 55 mol%, 30 to 50 mol%, 30 to 45 mol%, 30 to 40 mol%, 30 to 35 mol%, 35 to 60 mol%, 35 to 55 mol%, 35 to 50 mol%, 35 to 45 mol%, 35 to 40 mol%, 35 to 40 mol%, 40 to 60 mol%, 40 to 55 mol%, 40 to 50 mol%, 40 to 45 mol%, 45 to 60 mol%, 45 to 55 mol%, 45 to 50 mol%, or 50 to 60 mol%. In some embodiments, the ionizable lipid comprises (but is not limited to) 8-[2-hydroxyethyl-(6-oxo-6-undecyloxyhexyl)amino]octanoic acid heptadecan-9-yl ester (SM-102) TM ), bis(2-hexyldecanoic acid)(4-hydroxybutyl)azonyl)bis(hexane-6,1-diyl) ester (ALC-0315) TM Pfizer or a combination thereof.

[0090] Support lipids.

[0091] Supporting lipids are lipids used to improve the particle stability and flowability of lipid nanoparticles. Supporting lipids can be (but are not limited to) phosphatidylcholine, cholesterol or its derivatives, polyethylene glycol-lipids (PEG-lipids), or mixtures thereof. Where it is not desirable to be bound by theory, phosphatidylcholine can help improve the bilayer stability of lipid nanoparticles and reduce nonspecific binding; cholesterol can fill the gaps between the lipid components forming lipid nanoparticles and thus enhance particle stability by modulating membrane integrity and rigidity; PEGylated lipids (PEG lipids) can enhance the colloidal stability and circulation time of lipid nanoparticles in vivo.

[0092] In some embodiments, the auxiliary lipids comprise 5 to 60 mol% of the composition. In some embodiments, the auxiliary lipids comprise about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 mol% of the composition, or any range of carbon defined by the aforementioned endpoints, such as 5 to 60 mol%, 5 to 55 mol%, 5 to 50 mol%, 5 to 45 mol%, 5 to 40 mol%, 5 to 35 mol%, 5 to 30 mol%, 5 to 25 mol%, 5 to 20 mol%, 5 to 15 mol%, 5 to 10 mol%, 10 to 60 mol%, 10 to 55 mol%, 10 to 50 mol%, 10 to 45 mol%, 10 to 40 mol%, 10 to 35 mol%, 10 to 30 mol%, 10 to 25 mol%, 10 to 20 mol%, 10 to 15 mol%, 15 to 60 mol%, 15 to 55 mol%, 15 to 50 mol%, 15 to 45 mol%. mol%, 15 to 40 mol%, 15 to 35 mol%, 15 to 30 mol%, 15 to 25 mol%, 15 to 20 mol%, 20 to 60 mol%, 20 to 55 mol%, 20 to 50 mol%, 20 to 45 mol%, 20 to 40 mol%, 20 to 35 mol%, 20 to 30 mol%, 20 to 25 mol%, 25 to 60 mol%, 25 to 55 mol%, 25 to 50 mol%, 25 to 45 mol%, 25 to 40 mol%, 25 to 35 mol%, 25 to 30 mol%, 30 to 60 mol%, 30 to 55 mol%, 30 to 50 mol%, 30 to 45 mol%, 30 to 40 mol%, 30 to 35 mol%, 40 to 60 mol%, 40 to 55 mol%, 40 to 50 mol%, 40 to 45 mol%, 50 to 55 mol% or 50 to 60 mol%.

[0093] PhosphatidylcholineIn some embodiments, phosphatidylcholine comprises 5 to 10 mol% of the composition. In some embodiments, phosphatidylcholine comprises about 5, 6, 7, 8, 9, or 10 mol% of the composition, or any range of carbon defined by the foregoing endpoints, such as 5 to 10 mol%, 5 to 9 mol%, 5 to 8 mol%, 5 to 7 mol%, 5 to 6 mol%, 6 to 10 mol%, 6 to 9 mol%, 6 to 8 mol%, 6 to 7 mol%, 7 to 10 mol%, 7 to 9 mol%, 7 to 8 mol%, 8 to 10 mol%, 8 to 9 mol%, or 9 to 10 mol%. Examples of phosphatidylcholine include (but are not limited to) distearate phosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DPOE), or mixtures thereof.

[0094] cholesterol In some embodiments, cholesterol or its derivatives comprise 30 to 40 mol% of the composition. In some embodiments, cholesterol or its derivatives comprise about 30, 32, 34, 36, 38, or 40 mol% of the composition, or any range of carbon as defined by the foregoing endpoints, such as 30 to 40 mol%, 30 to 38 mol%, 30 to 36 mol%, 30 to 34 mol%, 30 to 32 mol%, 32 to 40 mol%, 32 to 38 mol%, 32 to 36 mol%, 32 to 34 mol%, 34 to 40 mol%, 34 to 38 mol%, 34 to 36 mol%, 36 to 40 mol%, 36 to 38 mol%, or 38 to 40 mol%. Examples of cholesterol or its derivatives include (but are not limited to) cholesterol, campesterol, β-phytosterol, brassosterol, ergosterol, dehydroergosterol, stigmasterol, phycosterol, DC-cholesterol HCl, OH-Chol, HAPC-Chol, MHAPC-Chol, DMHAPC-Chol, DMPAC-Chol, cholesterol chloroformate, GL67, cholesterol myristate, cholesterol oleate, cholesterol nervonic acid, LC10, cholesterol hemisuccinate, (3β,5β)-3-hydroxycholene-24-acid, alkyne cholesterol, 27-alkyne cholesterol, E-cholesterol alkyne, trifluoroacetate (Dios-Arg, 2H-Cho-Arg, or Cho-Arg), or mixtures thereof.

[0095] PEG-lipidsIn some embodiments, the PEG-lipid comprises 1 to 10 mol% of the composition. In some embodiments, the PEG-lipid comprises about 1, 2, 4, 6, 8, or 10 mol% of the composition, or any range of carbon defined by the foregoing endpoints, such as 1 to 10 mol%, 1 to 8 mol%, 1 to 6 mol%, 1 to 4 mol%, 1 to 2 mol%, 2 to 10 mol%, 2 to 8 mol%, 2 to 6 mol%, 2 to 4 mol%, 4 to 10 mol%, 4 to 8 mol%, 4 to 6 mol%, 6 to 10 mol%, 6 to 8 mol%, or 8 to 10 mol%. Examples of PEG-lipids include (but are not limited to) DMG-PEG, DSG-PEG, mPEG-DPPE, DOPE-PEG, mPEG-DMPE, mPEG-DOPE, DSPE-PEG-amine, DSPE-PEG, mPEG-DSPE, PEG PE, m-PEG-pentadecylene acid, bromoacetamide-PEG, amine-PEG, azide-PEG, or mixtures thereof. In some embodiments, the function of the PEG-lipid in the composition / lipid nanoparticles may be provided by the compounds of this disclosure, particularly by embodiments of this disclosure in which the linking group of R1 comprises a PEG moiety. In these cases, the composition does not need to contain the PEG-lipid.

[0096] Example The table below lists some exemplary compositions according to this disclosure. However, this disclosure is not limited to the exemplary compositions or the compositions described in this specification.

[0097] Table: Exemplary Examples of Formulations of this Disclosure

[0098]

[0099]

[0100] Lipid nanoparticles (LNP)

[0101] Another aspect of this disclosure relates to lipid nanoparticles (LNPs). The lipid nanoparticles of this disclosure comprise a membrane defining an internal space, wherein the membrane is formed of multiple lipid components comprising compounds of this disclosure. The membrane of the lipid nanoparticles may be a bilayer structure, which may be a single bilayer structure or multiple bilayer structures. In some embodiments, the multiple lipid components may further comprise ionizable lipids, auxiliary lipids, or a combination thereof. Ionizable lipids and auxiliary lipids may be those described above. Where it is not desired to be bound by theory, the membrane is formed via hydrophobic interactions between the multiple lipid components, while in some cases, electrostatic interactions may be involved in the formation of the membrane.

[0102] In some embodiments, the plurality of lipid components do not contain glycolipid C34 or α-galactosylceramide (α-GalCer). In some embodiments, the plurality of lipid compounds contain glycolipid C34 or α-galactosylceramide (α-GalCer), and the molar percentage of glycolipid C34 or α-galactosylceramide (α-GalCer) in the plurality of lipid compounds is less than about 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, or any range defined by the foregoing endpoints, such as 100% to 5%. %, 90% to 5%, 70% to 5%, 70% to 5%, 60% to 5%, 50% to 5%, 40% to 5%, 30% to 5%, 25% to 5%, 20% to 5%, 15% to 5%, 10% to 5%, 100% to 10%, 90% to 10%, 70% to 10%, 70% to 10%, 60% to 10%, 50% to 10%, 40% to 10%, 30% to 10%, 25% to 10%, 20% to 10%, or 15% to 10%.

[0103] In some embodiments, the multiple lipid components of the LNP membrane may comprise the first compound and the second compound of this disclosure. In some embodiments, the multiple lipid components may comprise more than one, two, or three compounds, each compound independently according to the compounds of this disclosure. The first compound, the second compound, or any one or two of more than one, two, or three compounds may be designed to have the same targeting portion having an R1 group, different targeting portions having an R1 group targeting the same target, or different targeting portions having an R1 group targeting different targets.

[0104] In some embodiments, the compounds of this disclosure constitute at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the various lipid components forming the LNP membrane, or any range defined by the aforementioned endpoints, such as 0.1% to 70%, 0.1% to 60%, 0.1% to 50%, 0.1% to 40%, 0.1% to 35%, 0.1% to 30%, 0.1% to 20%, etc. 5%, 0.1% to 20%, 0.1% to 15%, 0.1% to 10%, 0.1% to 5%, 0.1% to 4%, 0.1% to 3%, 0.1% to 2%, 0.1% to 1%, 0.1% to 0.5%, 0.5% to 70%, 0.5% to 60%, 0.5% to 50%, 0.5% to 40%, 0.5% to 35%, 0.5% to 30%, 0.5% to 25%, 0.5% to 20%, 0.5% to 15%, 0.5% to 10%, 0.5% to 5%, 0.5% to 4%. 0.5% to 3%, 0.5% to 2%, 0.5% to 1%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 35%, 1% to 30%, 1% to 25%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 1% to 4%, 1% to 3%, 1% to 2%, 5% to 70%, 5% to 60%, 5% to 50%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, 5% to 15%, 5% to 10% %, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, 10% to 15%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 50% to 70%, 50% to 65%, 50% to 60%, 60% to 70%, or 60% to 65%.

[0105] LNP dimensions .

[0106] In some embodiments, the diameter of the LNP of this disclosure is 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 micrometers, or any range defined by the aforementioned endpoints, such as 0.00 to 5, 0.01 to 4, 0.00 to 3, 0.01 to 2, 0.01 to 1, 0.01 to 0.8, 0.01 to 0.6, 0.01 to 0.4, 0.01 to 0.2, 0.01 to 0.1, 0.01 to 0.05, 0. 0.01 to 0.01, 0.05 to 5, 0.05 to 4, 0.05 to 3, 0.05 to 2, 0.05 to 1, 0.05 to 0.8, 0.05 to 0.6, 0.05 to 0.4, 0.05 to 0.2, 0.05 to 0.1, 0.1 to 5, 0.1 to 4, 0.1 to 3, 0.1 to 2, 0.1 to 1, 0.1 to 0.8, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.5 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1, 0.5 to 0.8, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 micrometers. The size of the LNP can be determined by using (but not limited to) dynamic light scattering (DLS). In some embodiments, the polydispersity index (PDI) of the LNP of this disclosure is about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1, or any range defined by the aforementioned endpoints, such as 0.01 to 1, 0.01 to 0.9, 0.01 to 0.8, 0.01 to 0.7, 0.01 to 0.6, 0.01 to 0.5, 0.01 to 0.4, 0.01 to 0.3, 0.01 to 0.2, 0.01 to 0.1, 0.01 to 0.05, 0.1 to 1, 0.1 to 0.9, 0.1 to 0.8, 0.1 to 0.7, 0.1 to 0.6, 0.1 to 0.5, 0.1 to 0.4, 0.1 to 0.3 or 0.1 to 0.2.

[0107] ζ-potential and molecular weight .

[0108] Without being bound by theory, the zeta potential and molecular weight of LNPs may influence cellular uptake of LNPs. In some embodiments, exemplary targeting of LNPs of this disclosure comprises about -50, -40, -30, -20, -15, -10, -5, 0, +5, +10, +15, +20, +30, +40, or +50, or any range defined by the aforementioned endpoints, such as -50 to +50, -50 to +40, -50 to +30, -50 to +20, -50 to +15, -50 to +10, -50 to +5, -50 to -5, -50 to -10, -50 to -15, -50 to -20, -20 to +5 ζ potentials of 0, -20 to +40, -20 to +30, -20 to +20, -20 to +15, -20 to +10, -20 to +5, -20 to -5, -20 to -10, -20 to -15, -15 to +50, -15 to +40, -15 to +30, -15 to +20, -15 to +15, -15 to +10, -15 to +5, -15 to -5, -15 to -10, +5 to +50, +5 to +40, +5 to +30, +5 to +20, +5 to +15, or +5 to +10. In some embodiments, the exemplary targeted LNPs of this disclosure comprise approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50 kDa, or any range defined by the aforementioned endpoints, such as 1 to 50 kDa, 1 to 40 kDa, 1 to 30 kDa, 1 to 20 kDa, 1 to 15 kDa, 1 to 10 kDa, 1 to 5 kDa, 2 to 50 kDa, 2 to 40 kDa, 2 to 30 kDa, 2 to 20 kDa, 2 to 15 kDa, 2 to 10 kDa, 2 to 5 kDa, 5 to 50 kDa, 5 to 40 kDa, 5 to 30 kDa, 5 to 20 kDa, 5 to 15 kDa, 5 to 10 kDa, 8 to 50 kDa, 8 to 45 kDa, 8 to 40 kDa, 8 to 35 kDa, 8 to 30 kDa, 8 to 25 kDa, 8 to 25 kDa. Molecular weights of kDa, 8 to 20 kDa, 8 to 15 kDa, 8 to 10 kDa, 12 to 50 kDa, 12 to 45 kDa, 12 to 35 kDa, 12 to 25 kDa, 12 to 15 kDa, 25 to 50 kDa, 25 to 40 kDa, or 25 to 30 kDa.

[0109] Payload

[0110] In some embodiments, the membrane of the LNP defines an internal space configured to encapsulate or carry a payload (i.e., a carrier). As described herein, “encapsulating a payload” or “carrying a payload” refers to the situation where the payload is held within the LNP by its membrane. The payload may be contained within the internal space defined by the LNP or may be embedded within the membrane (e.g., embedded within a bilayer structure). The payload may be able to move freely within the internal space or be covalently or non-covalently connected to the membrane. Encapsulation may be substantial, complete, or partial, and does not exclude the possibility that a portion of the payload may be exposed to the external environment of the LNP. In partially encapsulated embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the payload is held, sealed, or surrounded by the membrane of the LNP. In some embodiments, the payload may be a biomolecule, such as a nucleic acid, compound, peptide, protein, glycan head, or a combination thereof.

[0111] In some embodiments, the payload is ribonucleic acid (RNA, such as mRNA) or deoxyribonucleic acid (DNA, such as double-stranded or single-stranded DNA), which can encode polypeptides or proteins in vivo after delivery to target cells using the LNP of this disclosure. Nucleic acids, such as the mRNA molecules used in this disclosure, can be prepared by in vitro transcription from a reference nucleic acid. In vitro transcription can be performed as described in PCT patent publication WO2014 / 152027, filed March 13, 2014, which is incorporated herein by reference in its entirety.

[0112] In some embodiments, the peptide or protein is immunogenic (e.g., antigenic) to the organism to which the exemplary targeted LNP is administered. In such embodiments, the exemplary targeted LNP of this disclosure is used to encapsulate and carry an immunogenic protein or a nucleic acid, such as an mRNA molecule in an RNA vaccine, configured to encode an immunogenic protein in vivo. The immunogenic protein may be a protein of a pathogen derived from a virus (e.g., severe acute respiratory syndrome coronavirus (SARS-CoV, including SARS-CoV-2), influenza (flu), respiratory syncytial virus (RSV), EBV, dengue fever (DENGUE), VZV, HIV, ZIKA, or NIPAH), bacteria, or fungi. In some embodiments, the immunogenic protein may be a viral spike protein. In some embodiments, the spike protein may be of coronavirus (CoV) origin (e.g., SARS-CoV, MERS-CoV, and SARS-CoV-2). In some embodiments, examples of coronaviruses (CoV) described herein include (but are not limited to) α-SARS-CoV2, β-SARS-CoV2, γ-SARS-CoV2, δ-SARS-CoV2, o-SARS-CoV2, and variants thereof.

[0113] In some embodiments, the payload is a nucleic acid, which may be a polynucleotide having an open reading frame configured to encode a polypeptide or protein in vivo. These polynucleotides can be 5' capped, which is generated during in vitro transcription using the following chemical RNA cap analogs: 3"-O-Me-m7G(5)ppp(5')G [ARCA cap], G(5)ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, or m7G(5')ppp(5')G (New England BioLabs, Ipswich, Massachusetts). The 5'-capping of the modified polynucleotide can be completed post-transcriptionally using a vaccinia virus capping enzyme to generate the "Cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, Massachusetts). The Cap 1 structure can be generated using a vaccinia virus capping enzyme and a 2'-O methyltransferase to produce m7G(5')ppp(5')G-2'-O-methyl. The Cap 2 structure can be generated from the Cap 1 structure, followed by 2'-O-methylation of the 5'-last nucleotide using a 2'-O-methyltransferase. The Cap 3 structure can be generated from the Cap 2 structure, followed by 2'-O-methylation of the 5'-last nucleotide using a 2'-O-methyltransferase. The enzyme can be derived from recombinant sources. After transfection into mammalian cells, the modified polynucleotide exhibits stability for 12 to 18 hours, or greater than 18 hours, such as 24, 36, 48, 60, 72, or greater than 72 hours.

[0114] In some embodiments, nucleic acids may be modified. In some embodiments, nucleic acids may have several (more than one) modifications that are the same as or different from each other. In some embodiments, nucleic acids contain one, two or more (optionally different) nucleoside or nucleotide modifications in a specific region. In some embodiments, modified nucleic acids (e.g., modified mRNA polynucleotides) exhibit reduced degradation in cells or organisms relative to unmodified nucleic acids. In some embodiments, modified nucleic acids may exhibit reduced immunogenicity (e.g., reduced innate response) in organisms.

[0115] In some embodiments, the modification may include chemical modifications. In some embodiments, the modification may be naturally occurring, non-natural, or both. Some exemplary modifications applicable to this disclosure include, but are not limited to, modifications of sugars, nucleobases, nucleoside linkages (e.g., linkages to phosphate esters, phosphodiester linkages, or phosphodiester backbones), or combinations thereof. In some embodiments, the nucleic acid (e.g., RNA) used as the payload of this disclosure may be codon-optimized. For example, the nucleic acid may be modified to enhance its G / C content. The G / C content of a nucleic acid can affect its stability. Nucleic acids with increased amounts of guanine (G) and / or cytosine (C) residues may be functionally more stable than nucleic acids containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. For example, WO2002 / 098443 discloses a pharmaceutical composition containing mRNA stabilized by sequence modifications in the translation region. Due to the degeneracy of the genetic code, the modification works by replacing existing codons with codons that contribute to greater RNA stability without altering the resulting amino acids.

[0116] In some embodiments, the nucleic acid may also include a sequence encoding a signal peptide. The signal peptide may comprise three regions: (1) an N-terminal region of varying lengths, which typically contains positively charged amino acids, (2) a hydrophobic region, and (3) a short C-terminal peptide region. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) guides the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across the membrane. The signal peptide is not typically responsible for the final destination of the mature protein, but is not limited thereto in this disclosure. The signal peptide is typically cleaved from the precursor protein by an ER-resident signal peptidase. It may remain unclewd and act as a membrane anchor. In some embodiments, the signal peptide may be programmed to fuse with a polypeptide or protein encoded by the payload at its C-terminus or N-terminus.

[0117] In some embodiments, the payload may be a therapeutic or preventive agent for treating or preventing diseases such as cancer or infectious diseases. For example, the payload can be an antiviral agent, including (but not limited to) ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine, remdesivir (GS-5734) and favipiravir (T-705), interferon, adefovir, tenofovir, acyclovir, brivudin, cidofovir, fomivirsen, foscarnet, ganciclovir, amantadine, rimantadine, zanamivir, remdesivir, molnupiravir, and paxlovid. In other instances, the payload may be an anticancer agent. In some embodiments, the payload is a nucleic acid configured to encode a therapeutic or preventative agent.

[0118] In some embodiments, the N / P ratio (positively charged amine (nitrogen atom of ionizable lipid, N=nitrogen) group to negatively charged nucleic acid phosphate (P) group) of the exemplary targeted LNP of the encapsulated nucleic acid is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 3 0, 35, 40, 45, or 50, or any range defined by the aforementioned endpoints (including or excluding the endpoints), such as 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 10, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 8 to 40, 8 to 20, 8 to 12, 9 to 50, 9 to 30, or 9 to 15. In another embodiment, the exemplary targeting LNP nanoparticle / mRNA (N / P) ratio of the encapsulated mRNA is about 10 or about 20.

[0119] In some embodiments where the payload of the LNP is a nucleic acid configured to encode a peptide or protein in a target cell, after being taken up by the target cell, the LNP is configured to encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 copies of the peptide or protein in vivo, or any range defined by the aforementioned endpoints (including or excluding the endpoints), such as 1 to 50, 1 to 40, or 1 to 30. 1 to 20, 1 to 10, 1 to 5, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 5 to 8, 4 to 50, 4 to 45, 4 to 35, 4 to 25, 4 to 15, 4 to 9, 4 to 6, 7 to 50, 7 to 45, 7 to 35, 7 to 25, 7 to 15, or 7 to 9 replicates. In some embodiments, after being taken up by target cells, the LNP is configured to continuously and in real-time encode a polypeptide or protein in vivo until the nucleic acid (i.e., the payload) is deactivated in vivo.

[0120] The payload encoding a low-glucose immunogenic peptide (low-glucose universal vaccine) Without being bound by theory, it has been documented that removing the glycan shield of immunogenic peptides can expose the epitopes masked by the glycan shield, thereby increasing antigen presentation and the corresponding immune response induced by the immunogenic peptide.

[0121] Compared to wild-type spike proteins, such as the Wuhan strain and delta strains (e.g., SEQ ID NO: 2, 16, 18, and 20), the modified spike proteins described herein contain the deletion or addition of one or more amino acids at the N-linked glycosylation sequence (NXS / T) to eliminate the N-linked glycan sequence. Alternatively, the modified spike proteins described herein contain the substitution of one or more amino acids from S / T to alanine (A) at the O-linked glycosylation site to eliminate the O-linked glycosylation site.

[0122] In the first aspectA demonstrative mRNA of the coronavirus spike protein can be used as a demonstrative coronavirus vaccine, having a mutation or variant thereof having one or more sugar sites in the receptor-binding domain (RBD), subunit 1 (S1), or subunit 2 (S2) domain. When the mRNA is properly formulated in a targeted LNP formulation of this disclosure, it can be effectively used for dendritic cell-targeted delivery. As described herein, the mutation of CoV or its variants can be a deletion, addition, or substitution. In some embodiments, the coronavirus spike protein mRNA has a mutation of one or more sugar sites in the RBD, S1, or S2 having one or more substitutions of N to Q or S / T to A or combinations thereof. The mutation of the N-sugar site is changing the putative sequence NXS / T to QXS / T and / or changing the S / T of the O-sugar site to A. Sugar sites with N to Q substitutions include (but are not limited to) the following:

[0123] S-(deg-RBD) is an S protein that has all two N-glycan sites in the RBD that have been mutated from N to Q and two O-glycan sites that have been mutated from S / T to A (e.g., SEQ ID NO:4, 22, 24 or 26).

[0124] S-(deg-S2) is an S protein that has all nine sugar sites in S2 that have been mutated from N to Q (e.g., SEQ ID NO: 6, 28, 30 or 32);

[0125] S-(deg-S2-1194) is an S protein (e.g., SEQ ID NO:8 or 34) that has eight sugar sites other than sugar site 1194 in S2, which are mutated from N to Q.

[0126] S-(deg-RBD-801) is an S protein (e.g., SEQ ID NO:10 or 36) that has all two N-glycan sites in the RBD that are mutated from N to Q, two O-glycan sites that are mutated from S / T to A, and a sugar site 801 that is mutated from N to Q.

[0127] S-(deg-RBD-1194), is an S protein (e.g., SEQ ID NO: 12 or 38) containing all two N-glycosites in the RBD mutated from N to Q, two O-glycosites mutated from S / T to A, and glycosite 1194 mutated from N to Q; and

[0128] S-(deg-RBD-122-165-234) is an S protein (e.g., SEQ ID NO: 14 or 40) that has all two N-glycan sites in the RBD that are mutated from N to Q, two O-glycan sites that are mutated from S / T to A, and glycan sites 122, 165 and 234 that are mutated from N to Q.

[0129] In another embodiment, the mRNA or DNA of S-(deg-RBD) has the sequence of SEQ ID NO:3, 21, 23 or 25, the mRNA or DNA of S-(deg-S2) has the sequence of SEQ ID NO:5, 27, 29 or 31, the mRNA or DNA of S-(deg-S2-1194) has the sequence of SEQ ID NO:7 or 33, the mRNA or DNA of S-(deg-RBD-801) has the sequence of SEQ ID NO:9 or 35, the mRNA or DNA of S-(deg-RBD-1194) has the sequence of SEQ ID NO:11 or 37, and the mRNA or DNA of S-(deg-RBD-122-165-234) has the sequence of SEQ ID NO:13 or 39.

[0130] This disclosure also provides a linear DNA comprising a promoter, a 5' untranslated region, a 3' untranslated region, an expression plasmid with or without S-2P, and a poly(A) tail signal sequence, wherein the putative sequence NXS / T is changed to QXS / T, and the O-glycan site on the expression plasmid is changed from S / T to A. In one embodiment, the S-2P expression plasmid comprises the S gene of SARS-CoV-2, which encodes a pre-fusion state of S with proline substitutions at K968 and V969.

[0131] In some embodiments, the cell-targeting formulations of this disclosure may contain exemplary immunogenic peptides that remove glycan shielding, and may further include (but are not limited to) immunogenic peptides comprising at least one amino acid sequence selected from the group consisting of: TESIVRFPNITNL (SEQ ID NO:41), NITNLCPFGEVFNATR (SEQ ID NO:42), LYNSASFSTFK (SEQ ID NO:43), LDSKVGGNYN (SEQ ID NO:44), KSNLKPFERDIST (SEQ ID NO:45), KPFERDISTEIYQAG (SEQ ID NO:46), GPKKSTNLVKNKC (SEQ ID NO:47), NCDVVIGIV[N]NTVY (SEQ ID NO:48), PELDSFKEELDKYFK[N]HTS (SEQ ID NO:49), VNIQKEIDRLNEVA (SEQ ID NO:50), NL[N]ESLIDLQ (SEQ ID NO:51), and LGKYEQYIKWP (SEQ ID NO:49). SEQ ID NO:52), or an amino acid sequence that is at least about 99%, 98%, 97%, 96%, 95% or 90% identical to any one of SEQ ID NO:41 to 52.

[0132] In some embodiments, the cell-targeting formulation payload comprises RNA encoding a modified protein comprising (1) one or more amino acid substitutions from asparagine (N) to glutamine (Q) at an N-linked glycosylation sequence (NXS / T), wherein X is any amino acid residue other than proline, and S / T represents a serine or threonine residue; and / or (2) one or more amino acid substitutions from S / T to alanine (A) at an O-glycan site. In some embodiments, the payload comprises RNA encoding an immunogenic peptide comprising at least one amino acid sequence selected from the group consisting of: TESIVRFPNITNL (SEQ ID NO:41), NITNLCPFGEVFNATR (SEQ ID NO:42), LYNSASFSTFK (SEQ ID NO:43), LDSKVGGNYN (SEQ ID NO:44), KSNLKPFERDIST (SEQ ID NO:45), KPFERDISTEIYQAG (SEQ ID NO:46), GPKKSTNLVKNKC (SEQ ID NO:47), NCDVVIGIV[N]NTVY (SEQ ID NO:48), PELDSFKEELDKYFK[N]HTS (SEQ ID NO:49), VNIQKEIDRLNEVA (SEQ ID NO:50), NL[N]ESLIDLQ (SEQ ID NO:51), and LGKYEQYIKWP (SEQ ID NO:52), or with SEQ ID NO. Any one of NO:41 to 52 has an amino acid sequence with at least about 99%, 98%, 97%, 96%, 95%, or 90% homology.

[0133] In some embodiments, the formulation payload targeting cells is a nucleic acid comprising the nucleotide sequences set forth in SEQ ID NO:01, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, or having at least about 99%, 98%, 97%, 96%, 95%, or 90% similarity thereto; or the payload is configured to encode a peptide comprising the following: SEQ ID NO:02, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:39. The amino acid sequences described in SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:40, or amino acid sequences that are at least about 99%, 98%, 97%, 96%, 95%, or 90% identical to them.

[0134] In the second aspectExemplary immunogenic peptides for removing glycan shielding include (but are not limited to) immunogenic peptides comprising at least one amino acid sequence selected from the group consisting of: SSANNCTFEYVSQ (SEQ ID NO:58), TESIVRFPNITNL (SEQ ID NO:59), KPFERDISTEIYQAG (SEQ ID NO:60), GPKKSTNLVKNKC (SEQ ID NO:61), TEVPVAIHADQ (SEQ ID NO:62), RVYSTGSNVFQTR (SEQ ID NO:63), RRARSVASQS (SEQ ID NO:64), DPSKPSKRSF (SEQ ID NO:65), FIKQYGDCLGDI (SEQ ID NO:66), ENQKLIANQFNS (SEQ ID NO:67), GKIQDSLSSTA (SEQ ID NO:68), NCDVVIGIVNNTVY (SEQ ID NO:69), PELDSFKEELDKYFKNHTS (SEQ ID NO:68). NO:70), TSPDVDLGDISGINA (SEQ ID NO:71), VNIQKEIDRLNEVA (SEQ ID NO:72), NLNESLIDLQ (SEQ ID NO:73) and LGKYEQYIKWP (SEQ ID NO:74).

[0135] In some embodiments, the cell-targeting formulation payload comprises RNA encoding a modified protein comprising (1) one or more amino acid substitutions from asparagine (N) to glutamine (Q) at an N-linked glycosylation sequence (NXS / T), wherein X is any amino acid residue other than proline, and S / T represents a serine or threonine residue; and / or (2) one or more amino acid substitutions from S / T to alanine (A) at an O-glycan site. In some embodiments, the payload comprises RNA encoding an immunogenic peptide comprising at least one amino acid sequence selected from the group consisting of: SSANNCTFEYVSQ (SEQ ID NO:58), TESIVRFPNITNL (SEQ ID NO:59), KPFERDISTEIYQAG (SEQ ID NO:60), GPKKSTNLVKNKC (SEQ ID NO:61), TEVPVAIHADQ (SEQ ID NO:62), RVYSTGSNVFQTR (SEQ ID NO:63), RRARSVASQS (SEQ ID NO:64), DPSKPSKRSF (SEQ ID NO:65), FIKQYGDCLGDI (SEQ ID NO:66), ENQKLIANQFNS (SEQ ID NO:67), GKIQDSLSSTA (SEQ ID NO:68), NCDVVIGIVNNTVY (SEQ ID NO:69), PELDSFKEELDKYFKNHTS (SEQ ID NO:70), TSPDVDLGDISGINA (SEQ ID NO:71), VNIQKEIDRLNEVA (SEQ ID NO:72), NLNESLIDLQ (SEQ ID NO:73), and LGKYEQYIKWP (SEQ ID NO:74).

[0136] In some embodiments, the formulation payload targeting cells is configured to encode a peptide comprising the amino acid sequence set forth in SEQ ID NO: 53, 54, 55, 56 or 57, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95% or 90% similarity thereto.

[0137] As described herein, the exemplary low-sugar universal vaccine (LSUV) is also described in WO2022 / 221835 (wherein the mRNA contains sequences selected from SEQ ID NO 1-52), WO2022 / 221837A2 (wherein the mRNA contains sequences selected from SEQ ID NO 1-21), and US20200046826A1 (wherein the mRNA contains sequences selected from SEQ ID NO 1-20), all of which are incorporated herein by reference in their entirety.

[0138] Kit for preparing lipid nanoparticles .

[0139] One aspect of this disclosure relates to a kit / reagent mixture for preparing targeted LNP formulations of this disclosure. The kit comprises a first reagent and a second reagent, wherein the first reagent comprises a compound of this disclosure, and the second reagent comprises an ionizable lipid, an auxiliary lipid, or a mixture thereof. The ionizable lipid and the auxiliary lipid can be the ionizable lipid and auxiliary lipid described herein. In some embodiments, the second reagent comprises an ionizable lipid, and in some embodiments, the kit further comprises a third reagent comprising an auxiliary lipid. In some embodiments, the kit further comprises a fourth reagent comprising a payload, wherein the payload can be the payload described herein.

[0140] Package All components of the kit disclosed herein can be packaged separately in physical containers. In some embodiments, the first and second reagents are contained in the same container; in other words, in a ready-to-use package. In some other embodiments, the first and second reagents are contained in separate containers, so that the user can decide whether and when to mix the first and second reagents.

[0141] Composition / Formulation

[0142] One aspect of this disclosure relates to a pharmaceutically acceptable vaccine composition comprising a targeting LNP of this disclosure. The LNP of the composition can encapsulate a payload and is configured to deliver the payload to a target region of an organism. The payload may, as described herein, comprise nucleic acids, compounds, peptides, proteins, glycan heads, or combinations thereof. In some embodiments, the payload may be an immunogenic protein or a nucleic acid configured to encode an immunogenic protein in vivo. In some embodiments, the formulation further comprises a pharmaceutically acceptable excipient, adjuvant, or combination thereof. In some embodiments, the composition is a pharmaceutical composition or a pharmaceutical formulation.

[0143] In some embodiments, the composition comprises 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 95% (w / w) of the LNP of this disclosure, encapsulated or unencapsulated payload, or any range defined by the aforementioned endpoints, such as (including or excluding the endpoints) 0.01% to 95% (w / w), 0.01% to 90% (w / w), 0.01% to 80% (w / w), 0.01% to 70% (w / w), 0.01% to 60% (w / w), 0.01% to 50% (w / w), 0.01% to 40% (w / w), 0.01% to 30% (w / w), 0.01% to 20% (w / w), or 0.01% to 10%. (w / w), 0.01% to 5% (w / w), 0.01% to 1% (w / w), 0.01% to 0.1% (w / w), 0.1% to 95% (w / w), 0.1% to 90% (w / w), 0.1% to 80% (w / w), 0.1% to 70% (w / w), 0.1% to 60% (w / w), 0.1% to 50% (w / w), 0.1% to 40% (w / w), 0.1% to 30% (w / w), 0.1% to 20% (w / w), 0.1% to 10% (w / w), 0.1% to 5% (w / w), 0.1% to 1% (w / w), 1% to 95% (w / w), 1% to 90% (w / w), 1% to 80% (w / w), 1% to 70% (w / w), 1% to 60% (w / w), 1% to 50% (w / w), 1% to 40% (w / w), 1% to 30% (w / w), 1% to 20% (w / w), 1% to 10% (w / w), 1% to 5% (w / w), 5% to 95% (w / w), 5% to 90% (w / w), 5% to 80% (w / w), 5% to 70% (w / w), 5% to 60% (w / w), 5% to 50% (w / w), 5% to 40% (w / w), 5% to 30% (w / w), 5% to 20% (w / w), or 5% to 10% (w / w). The remaining percentage of the composition may be excipients as described herein.

[0144] In some embodiments, the composition is an mRNA vaccine, wherein the LNP encapsulation is configured to encode an immunogenic protein in vivo. The immunogenic protein may be a viral spike protein or other antigenic molecules of a pathogen. In some embodiments, the compositions of the present invention may be a COVID-19 mRNA vaccine.

[0145] The exemplary COVID-19 mRNA vaccines described herein can be designed based on mRNA technology to remove the glycan shielding of coronavirus (e.g., SARS-CoV-2) spike proteins, thereby better exposing conserved regions of the spike protein. Compared to unmodified mRNA, coronavirus spike protein mRNA vaccines have the deletion of glycosylation sites in the receptor-binding domain (RBD) or subunit 2 (S2) domain to expose highly conserved epitopes and elicit antibody and CD8 T cell responses, providing broader protection against α, β, γ, δ, O, and various variants. Vaccines may be low sugar universal vaccines (LSUVs) as described in WO2022 / 221835 (where the mRNA contains sequences selected from SEQ ID NO 1-52), WO2022 / 221837A2 (where the mRNA contains sequences selected from SEQ ID NO 1-21), and US20200046826A1 (where the mRNA contains sequences selected from SEQ ID NO 1-20), which are incorporated herein by reference in their entirety.

[0146] In some embodiments, the compositions / formulations of the present invention are configured to treat or prevent diseases (e.g., cancer). In such embodiments, the payload carried by the LNP may be a therapeutic agent, a preventative agent, or a nucleic acid configured to encode a therapeutic or preventative agent in vivo. For example, the composition may be a personalized cancer vaccine (e.g., melanoma), a KRAS vaccine (KRAS-driven), or a checkpoint vaccine (e.g., PD-1, PDL-1 related).

[0147] In some embodiments, the compositions / formulations of the present invention may be administered together with another composition (e.g., a vaccine or a drug). Examples of the other composition may be (but are not limited to) influenza (flu) vaccines, adenovirus vaccines, anthrax vaccines, cholera vaccines, diphtheria vaccines, hepatitis A or B vaccines, HPV vaccines, measles vaccines, mumps vaccines, smallpox vaccines, rotavirus vaccines, tuberculosis vaccines, pneumococcal vaccines, and Haemophilus influenzae type b vaccines.

[0148] Combination composition

[0149] In some embodiments, the composition may be a combination composition (e.g., a combination vaccine) comprising a first LNP encapsulating a first payload and a second LNP encapsulating a second payload. The first and second LNPs may be in the form of LNPs described herein, but differ from each other in terms of the structure or properties of their copolymers. For example, the first and second LNPs may differ in size, the copolymers in which they form a film, the payloads encapsulated within the LNPs, or combinations thereof.

[0150] For example, the first LNP contains a glycan head configured to bind DC-SIGN, while the second LNP contains a glycan head configured to bind CD1d. In another example, the first LNP contains a glycan configured to target antigen-presenting cells, while the second LNP contains a glycan configured to target cancer cells.

[0151] In some embodiments, the first payload and the second payload are different from each other. For example, the first payload may be a protein or peptide, while the second payload may be a nucleic acid. In some embodiments, both the first and second payloads may be mRNA molecules that encode different proteins. For example, the first payload may be mRNA configured to encode the spike protein of δ-SARS-CoV2, while the second payload may be mRNA configured to encode the spike protein of o-SARS-CoV2.

[0152] Additional components of the composition

[0153] In some embodiments, the compositions disclosed herein may further comprise adjuvants and / or inactive substances, such as pharmaceutically acceptable excipients. In some embodiments, the adjuvant may be (but is not limited to) C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide (α-GalCer), aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts), squalene, MF59, QS-21, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), CpG 1018 (Dynavax), or combinations thereof.

[0154] In some embodiments, pharmaceutically acceptable excipients may comprise solvents, dispersion media, diluents, dispersions, suspending agents, surfactants, isotonics, thickeners or emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, or mixtures thereof. Various excipients used to formulate pharmaceutical compositions and techniques used to prepare compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 22nd edition, edited by Allen, Loyd V., Jr., Pharmaceutical Press). The use of conventional excipient media is within the scope of this disclosure unless any conventional excipient medium may be incompatible with the substance or its derivatives, for example, producing any undesirable biological effects or otherwise interacting in a harmful manner with any other component of the pharmaceutical composition. The formulation of pharmaceutically acceptable excipients can be performed using conventional methods in pharmaceutical technology (see Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Eastern Pennsylvania, USA).

[0155] In some embodiments, the composition further comprises a phosphate ester conjugate. Where it is not desired to be bound by theory, the phosphate ester conjugate may increase in vivo circulation time and / or increase targeted delivery of the LNP of this disclosure. The phosphate ester conjugate for use in this disclosure may be prepared using the methods described in PCT Publication No. WO2013 / 033438, filed August 30, 2012, or U.S. Publication No. US2013 / 0196948, filed June 23, 2011, the contents of each of which are incorporated herein by reference in their entirety. As a non-limiting example, the phosphate ester conjugate may comprise compounds of any of the formulas described in PCT Publication No. WO2013 / 033438, filed August 30, 2012, which is incorporated herein by reference in its entirety.

[0156] In some embodiments, the composition further comprises a conjugate to enhance the delivery of the LNPs of this disclosure. Where it is not desired to be bound by theory, the conjugate chosen may inhibit phagocytic clearance of LNPs in an individual. In some instances, the conjugate may be the human membrane protein CD47 or a “self” peptide derived therefrom (e.g., the “self” particle described by Rodriguez et al. (Science 2013, 339, 971-975), which is incorporated herein by reference in its entirety).

[0157] In some embodiments where the payload is an immunogenic agent or a nucleic acid configured to encode an immunogenic agent, the composition further comprises an immunostimulant to enhance the immune response induced by the immunogenic agent. As a non-limiting example, the composition may comprise a Th1 immunostimulant that can enhance a Th1-based response of the immune system (see PCT Publication WO2010 / 123569 and U.S. Publication 2011 / 0223201, each of which is incorporated herein by reference in its entirety).

[0158] In some embodiments, the composition does not include viral components (e.g., viral capsid, viral enzymes, or other viral proteins, such as those required for viral replication), nor is the composition packaged within, encapsulated within, linked to, or otherwise bound to a virus or viral particle.

[0159] How to use

[0160] One aspect of this disclosure relates to methods of using the LNPs or targeted formulations of this disclosure. Specifically, the methods are performed to achieve a desired effect, such as targeted delivery of a payload, prevention or treatment of disease, or enhancement of an adaptive immune response in an individual. In some embodiments, the individual may be (but is not limited to) an animal or human for which the payload is designed to demonstrate its efficacy, an animal or human requiring treatment or prevention of disease, or an animal or human requiring enhancement of their adaptive immune response.

[0161] Methods for targeted payload delivery in individuals

[0162] In some embodiments, a method of targeted delivery of a payload to an individual is provided, comprising administering to the individual an effective amount of an LNP or a targeted formulation of the present disclosure. The LNP and payload are as described herein, and the payload is encapsulated by the LNP. Without wishing to be bound by any theory, targeted delivery is achieved through the compounds of the present disclosure. Specifically, the R1 group of the compounds of the present disclosure provides the desired binding affinity / specificity to a desired region of the individual via its glycosidic group.

[0163] Methods for preventing or treating diseases in an individual

[0164] In some embodiments, a method for preventing or treating a disease in an individual is provided, comprising administering to the individual an effective amount of an LNP or a formulation of the present disclosure. The LNP in this method encapsulates an effective load, and the effective load is a therapeutic agent or derivative configured to prevent and / or treat a disease.

[0165] Without wishing to be bound by any theory, the LNPs or formulations of this disclosure provide targeted delivery via the compounds of this disclosure. Therefore, by using the LNPs of this disclosure to deliver the payload, the efficacy of the payload can be more effectively utilized. For example, in embodiments where the R1 group of the compound comprises a structure that specifically binds to DC-SIGN on dendritic cells, antigenic or immunogenic payloads can be effectively delivered to dendritic cells to elicit an immune response, thereby preventing the disease of interest. This strategy is beneficial for delivering antigens or nucleic acids encoding antigens in vaccines. Some other examples include having an R1 group designed to target cancer cells, allowing antitumor agents to be effectively delivered to the cancer microenvironment. This strategy can increase the efficacy of antitumor agents and reduce the side effects of treatment.

[0166] In some embodiments, the disease is characterized by a dysfunction or abnormality in the activity of proteins or peptides. For example, diseases are selected from the group consisting of: rare diseases, infectious diseases, cancers and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0167] In some embodiments, the disease may be cancer or an infectious disease. In some embodiments, the disease may be a virus-related infection, including (but not limited to) human parainfluenza virus 3, respiratory syncytial virus (RSV), cell cytomegalovirus (CMV), human interstitial pneumonia virus (hMPV), or SARS-CoV-2 (COVID-19)-related infection.

[0168] Methods to enhance adaptive immune response

[0169] In some embodiments, a method for enhancing an adaptive immune response is provided, comprising administering an effective amount of the LNP of this disclosure to an individual. The LNP of this method encapsulates a payload within an internal space defined by a membrane of the LNP, and the payload is a therapeutic agent or a derivative of a therapeutic agent configured to elicit an adaptive immune response in the individual.

[0170] Without wishing to be bound by any theory, the LNP of this disclosure provides targeted delivery to immune cells via the compounds of this disclosure. In some embodiments, the R1 group of the compound comprises a structure that binds to antigen-presenting cells with desired specificity or affinity. For example, the R1 group may comprise a structure that specifically binds to DC-SIGN on dendritic cells, enabling the LNP to specifically deliver an immunogenic payload to dendritic cells to promote an adaptive immune response.

[0171] In some embodiments, the enhanced adaptive immune response is directed against a disease, including (but not limited to) cancer or an infectious disease. For example, an infectious disease can be a virus-associated infection, including (but not limited to) human parainfluenza virus 3, respiratory syncytial virus (RSV), cell cytomegalovirus (CMV), human interstitial pneumonia virus (hMPV), or SARS-CoV-2 (COVID-19)-associated infection.

[0172] application

[0173] Regarding the methods of this disclosure, in some embodiments, a single dose of the LNP or targeted formulation of this disclosure is administered to an individual, with or without encapsulation of the payload. However, in some embodiments, an initial dose of the LNP is administered to an individual, followed by at least one follow-up dose, such as one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more, wherein the interval between each dose is approximately 1, 2, 3, 4, 5, 6, 7 days, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or any range defined by the aforementioned endpoints, including or excluding the endpoints. 1 to 7 days, 1 to 5 days, 1 to 3 days, 1 to 10 weeks, 1 to 8 weeks, 1 to 6 weeks, 1 to 4 weeks, 1 to 2 weeks, 1 to 12 months, 1 to 8 months, 1 to 6 months, 1 to 4 months, 1 to 2 months, or 6 to 12 months. In some embodiments, the encapsulated effective load of the LNP of this disclosure is administered twice at the same or different doses, with the interval between the two administrations being 1 day, 3 days, 5 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1 to 5 days, 1 to 2 weeks, 1 to 3 months, 1 to 6 months, 1 month to 1 year, 3 months to 1 year, or 6 months to 1 year.

[0174] Application routeThe LNPs or formulations described herein can be administered via any route. Suitable routes include, but are not limited to, oral, nasal, mucosal, submucosal, intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, percutaneous, and buccal routes. Some practical topical applications include, but are not limited to, drops, sprays, aerosols, gels, or ointments for the mucosal epithelium of the eyes, nose, mouth, anus, or vagina. Other possible routes of administration are via inhalation of sprays, aerosols, or powders.

[0175] Effective dosage The effective amount described herein refers to an amount sufficient to provide the desired effect. In embodiments where the purpose of applying the LNP of this disclosure is to treat a disease, the effective amount refers to a therapeutically effective amount, while in some other embodiments where the purpose is to prevent a disease, the effective amount refers to a preventatively effective amount.

[0176] However, in some other embodiments where the purpose of administering LNP and the payload is to enhance an adaptive immune response, the effective amount can be determined as an amount sufficient to induce an antigen-specific immune response in individuals administered LNP and the payload. An antigen-specific immune response can be characterized by measuring the antibody titer against the anti-antigenic peptide (i.e., the payload or the product of the payload) produced in individuals administered LNP and the payload. In some embodiments, the measurement can be performed using an enzyme-linked immunosorbent assay (ELISA).

[0177] In some embodiments, antibody titer is used to assess whether an individual has an infection or to determine whether immunization is required. In some embodiments, antibody titer is used to determine the strength of an autoimmune response, to determine whether booster immunization is needed or has been administered, to determine the effectiveness of a previous vaccine, and / or to identify any current or previous infections.

[0178] The effective amount of the method disclosed herein can be determined based on several factors, including (but not limited to) individual conditions (age, sex, species, weight, health status, etc.), the progression of the disease to be treated, the route of administration, the dosage and interval of administration, and the nature of the payload. Regarding the nature of the payload, for example, in embodiments where the LNP of this disclosure is used to carry mRNA, such as in an mRNA vaccine, the effective amount can be determined based on the effective amount of mRNA required to elicit a sufficient immune response in the individual. Therefore, in some embodiments where the payload is mRNA, the effective amount of the method disclosed herein is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 micrograms (μg or ug), or any range defined by the foregoing endpoints.For example (including or excluding endpoints): 5 micrograms to 1000 micrograms, 5 micrograms to 900 micrograms, 5 micrograms to 800 micrograms, 5 micrograms to 700 micrograms, 5 micrograms to 600 micrograms, 5 micrograms to 500 micrograms, 5 micrograms to 400 micrograms, 5 micrograms to 300 micrograms, 5 micrograms to 200 micrograms, 5 micrograms to 175 micrograms, 5 micrograms to 150 micrograms, 5 micrograms to 125 micrograms, 5 micrograms to 100 micrograms, 5 micrograms to 90 micrograms, 5 micrograms to 80 micrograms, 5 micrograms to 70 micrograms, 5 micrograms to 60 micrograms, 5 micrograms to 50 micrograms, 5 micrograms to 40 micrograms, 5 micrograms to 30 micrograms, 5 micrograms to 20 micrograms, 5 micrograms to 10 micrograms, 10 micrograms to 1000 micrograms, 10 micrograms to 900 micrograms. 0.00 micrograms, 10 micrograms to 800 micrograms, 10 micrograms to 700 micrograms, 10 micrograms to 600 micrograms, 10 micrograms to 500 micrograms, 10 micrograms to 400 micrograms, 10 micrograms to 300 micrograms, 10 micrograms to 200 micrograms, 10 micrograms to 175 micrograms, 10 micrograms to 150 micrograms, 10 micrograms to 125 micrograms, 10 micrograms to 100 micrograms, 10 micrograms to 90 micrograms, 10 micrograms to 80 micrograms, 10 micrograms to 70 micrograms, 10 micrograms to 60 micrograms, 10 micrograms to 50 micrograms, 10 micrograms to 40 micrograms, 10 micrograms to 30 micrograms, 10 micrograms to 20 micrograms, 50 micrograms to 1000 micrograms, 50 micrograms to 900 micrograms, 50 Micrograms to 800 micrograms, 50 micrograms to 700 micrograms, 50 micrograms to 600 micrograms, 50 micrograms to 500 micrograms, 50 micrograms to 400 micrograms, 50 micrograms to 300 micrograms, 50 micrograms to 200 micrograms, 50 micrograms to 175 micrograms, 50 micrograms to 150 micrograms, 50 micrograms to 125 micrograms, 50 micrograms to 100 micrograms, 50 micrograms to 90 micrograms, 50 micrograms to 80 micrograms, 50 micrograms to 70 micrograms or 50 micrograms to 60 micrograms, 100 micrograms to 1000 micrograms, 100 micrograms to 900 micrograms, 100 micrograms to 800 micrograms, 100 micrograms to 700 micrograms, 100 micrograms to 600 micrograms, 100 micrograms to 500 micrograms 100 micrograms to 400 micrograms, 100 micrograms to 300 micrograms, 100 micrograms to 200 micrograms, 100 micrograms to 175 micrograms, 100 micrograms to 150 micrograms, 300 micrograms to 1000 micrograms, 300 micrograms to 900 micrograms, 300 micrograms to 800 micrograms, 300 micrograms to 700 micrograms, 300 micrograms to 600 micrograms, 300 micrograms to 500 micrograms, 300 micrograms to 400 micrograms, 500 micrograms to 1000 micrograms, 500 micrograms to 900 micrograms, 500 micrograms to 800 micrograms, 500 micrograms to 700 micrograms, 500 micrograms to 600 micrograms, 600 micrograms to 800 micrograms, or 700 micrograms to 900 micrograms.

[0179] However, given the targeted delivery provided by the LNP of this disclosure, it is anticipated that the effective amount required in the method of this disclosure may be lower than that required in other non-targeted delivery methods. For example, the effective amount required in the method of this disclosure may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95, or 99% lower than that required in other non-targeted delivery methods, or any range defined by the aforementioned endpoints, such as (including or excluding the endpoints). 1 to 99%, 1 to 95%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, 1 to 10%, 1 to 5%, 5 to 99%, 5 to 95%, 5 to 90%, 5 to 80%, 5 to 70%, 5 to 60%, 5 to 50%, 5 to 40%, 5 to 30%, 5 to 20%, 5 to 10%, 10 to 90%, 10 to 80%, 10 to 70%, 10 to 60%, 10 to 50%, 10 to 40% %, 10 to 30%, 10 to 20%, 30 to 99%, 30 to 95%, 30 to 90%, 30 to 80%, 30 to 70%, 30 to 60%, 30 to 50%, 30 to 40%, 50 to 99%, 50 to 95%, 50 to 90%, 50 to 80%, 50 to 70%, 50 to 60%, 70 to 99%, 70 to 95%, 70 to 90%, 70 to 80%, 80 to 99%, 80 to 95%, 80 to 90%, 90 to 99% or 95 to 99%.

[0180] Furthermore, in some embodiments in which the LNP of this disclosure is used to deliver an antigen or a nucleic acid encoding an antigen to induce an antibody against the antigen, the titer of the antibody induced by this disclosure is increased by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 logs, or any range defined by the aforementioned endpoints, such as (including or excluding the endpoints) 1 to 10 logs, 1 to 8 logs, 1 to 6 logs, 1 to 4 logs, 2 to 9 logs, 2 to 7 logs, 2 to 5 logs, 3 to 10 logs, 3 to 8 logs, 3 to 5 logs or 4 to 6 logs.

[0181] In some other embodiments, when the LNP of this disclosure is used to deliver an antigen or a nucleic acid encoding an antigen to induce an immune response against the antigen, the antibody titer induced by this disclosure is 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than that induced by non-targeted delivery methods, or any range defined by the aforementioned endpoints, for example (including or excluding the endpoints). 0.1 to 10, 0.1 to 9, 0.1 to 8, 0.1 to 7, 0.1 to 6, 0.1 to 5, 0.1 to 4, 0.1 to 3, 0.1 to 2, 0.1 to 1, 0.1 to 0.5, 0.5 to 10, 0.5 to 9, 0.5 to 8, 0.5 to 7, 0.5 to 6, 0.5 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 7 to 10, 7 to 9, 7 to 8 or 8 to 10.

[0182] However, in some embodiments in which the LNP of this disclosure is used to deliver an antigen or a nucleic acid encoding an antigen to induce an immune response against the antigen, the immune response is induced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 days earlier than an immune response induced by a non-targeted delivery method, or any range defined by the aforementioned endpoints, such as (including or excluding the endpoints) 1 to 20, 1 to 18, 1 to 14, 1 to 10, 1 to 6, 2 to 20, 2 to 18, 2 to 14, 2 to 10, 2 to 6, 5 to 20, 5 to 18, 5 to 14, or 5 to 10 days earlier.

[0183] In some embodiments, LNP as described herein in the methods of this disclosure may be administered once or more daily at dose levels sufficient to deliver a payload of about 0.0001 mg to about 100 mg, about 0.001 mg to about 0.05 mg, about 0.005 mg to about 0.05 mg, about 0.001 mg to about 0.005 mg, about 0.05 mg to about 0.5 mg, about 0.01 mg to about 50 mg, about 0.1 mg to about 40 mg, about 0.5 mg to about 30 mg, about 0.01 mg to about 10 mg, about 0.1 mg to about 10 mg, or about 1 mg to about 25 mg per kg of individual body weight to obtain the desired in vivo effect.

[0184] definition

[0185] Unless otherwise expressly defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise stated, the techniques employed or covered herein are standard methods well-known to one of ordinary skill in the art. Unless otherwise instructed, the practice of this disclosure will employ conventional techniques of microbiology, tissue culture, molecular biology, chemistry, biochemistry, and recombinant DNA technology, within the skill of the art. Materials, methods, and examples are illustrative and non-limiting only. The following is presented in an illustrative manner and is not intended to limit the scope of this disclosure.

[0186] The figures used to describe and claim certain embodiments of this disclosure representing the amount, characteristics (e.g., molecular weight, reaction conditions, and results, etc.) of components should be understood to be modified by the term "about" in some cases. Those skilled in the art will understand the meaning of the term "about" in its defined value. The numerical values ​​presented in some embodiments of this disclosure may contain some error resulting from the standard deviation in their corresponding test measurements. For example, as used herein, the term "about" refers to a measurable value, such as an amount, duration, etc., and means to cover variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from a specified value, provided such variations are appropriate.

[0187] As used herein, "substantially" means sufficient to achieve the intended purpose. Therefore, the term "substantially" allows for small, insignificant variations in absolute or perfect state, dimensions, measurements, results, etc., that would be expected by someone of ordinary skill in the art but would not significantly affect overall performance. When used with respect to numerical values ​​or parameters or characteristics that are numerical, "substantially" means within ten percent.

[0188] As used herein, “treat,” “treatment,” and “treating” refer to a method for achieving a beneficial or desired outcome, such as a clinical outcome. For the purposes of this disclosure, a beneficial or desired outcome may include inhibiting or suppressing the onset or progression of an infection or disease; alleviating symptoms of an infection or disease or reducing its development; or a combination thereof.

[0189] As used herein, “preventing” and “prevention” are used interchangeably with “prophylaxis” and can mean complete prevention of infection or prevention of the development of symptoms of said infection, delaying the onset of disease or its symptoms, or reducing the severity of subsequently developed infection or its symptoms.

[0190] As used herein, “polysaccharide” or “glycoside” refers to a polysaccharide, oligosaccharide, or monosaccharide. Polysaccharides can be monomers or polymers of sugar residues and can be linear or branched. Polysaccharides can include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, mannose phosphate, 6'-sulfonic acid N-acetylglucosamine, etc.).

[0191] As used herein, "alkyl" refers to a straight or branched, saturated or unsaturated hydrocarbon chain containing a specified number of carbon atoms. For example, C 1-6 The indicator group may have 1 to 6 (inclusive) carbon atoms. Non-limiting examples include methyl, ethyl, isopropyl, tert-butyl, and n-hexyl. "Heteroalkyl" is an alkyl group in which at least one carbon atom of the chain has been replaced by a heteroatom. In some embodiments, the heteroalkyl group has 1 to 20 carbon atoms. The term "alkoxy" is intended to mean the part-OR, where R is an alkyl group. The term "aryloxy" is intended to mean the part-OR, where R is an aryl group.

[0192] As used herein, "alkenyl" refers to a hydrocarbon chain comprising at least one double bond, which may be straight or branched and contains a specified number of carbon atoms. For example, C 2-6 The indicator group may have 2 to 6 (inclusive) carbon atoms. Non-limiting examples include vinyl and propylene-1-en-2-yl.

[0193] As used herein, "alkynyl" refers to a hydrocarbon chain that includes at least one triple bond, which may be straight or branched, and contains a specified number of carbon atoms. For example, C 2-6 The indicator group may have 2 to 6 (inclusive) carbon atoms. Non-limiting examples include ethynyl and 3,3-dimethylbut-1-yn-1-yl.

[0194] As used herein, “cycloalkyl” refers to a non-aromatic cyclic, bicyclic, fused, or spirocyclic hydrocarbon group having 3 to 10 carbons, such as 3 to 8 carbons, such as 3 to 7 carbons, wherein the cycloalkyl group may optionally be substituted. Examples of cycloalkyl groups include five-, six-, and seven-membered rings. A cycloalkyl group may include one or more unsaturated elements; cycloalkyl groups including unsaturated elements are also referred to herein as “cycloalkenyl”. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0195] As used herein, “heterocyclic alkyl” refers to a non-aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic fused or spirocyclic system group, having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, wherein the heteroatoms are selected from O, N, or S (e.g., carbon atom and 1-3, 1-6, or 1-9 N, O, or S heteroatoms respectively for monocyclic, bicyclic, or tricyclic), wherein 0, 1, 2, or 3 atoms of each ring may be substituted with substituents. Heterocyclic alkyl groups may also include oxidized ring members, such as -N(O)-, -S(O)-, and -S(O)2-. Examples of heterocyclic alkyl groups include five-, six-, and seven-membered heterocycles. Examples include piperazine, pyrrolyl, dioxanehexyl, morpholinyl, tetrahydrofuranyl, etc.

[0196] As used herein, “aryl” or “aryl group” refers to the portion formed by removing one or more hydrogen atoms (“H”) or deuterium atoms (“D”) from an aromatic compound. An aryl group can be a single ring (monocyclic) or multiple rings fused together or covalently linked (bicyclic or more rings). A “carbocyclic aryl” has only carbon atoms in one or more aromatic rings. A “heteroaryl” is intended to mean an aromatic ring system containing 5 to 14 aromatic ring atoms, which can be a single ring, two fused rings, or three fused rings, wherein at least one aromatic ring atom is a heteroatom selected from (but not limited to) the group consisting of O, S, and N. Heteroaryl groups may also include oxidized ring members, such as -N(O)-, -S(O)-, and -S(O)2-. Examples include furanyl, thiopheneyl, pyrroleyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, etc. Examples also include carbazolyl, quinazinyl, quinolinyl, isoquinolinyl, nitrolinyl, phthalazinyl, quinazolinyl, quinolinyl, triazinyl, indoleyl, isoindoleyl, indazoleyl, indoleazinyl, purineyl, naphridinyl, pteridinyl, carbazolyl, acridineyl, phenazinyl, phenothiazinyl, phenothiazinyl, benzoxazolyl, benzothiazolyl, 1H-benzimidazolyl, imidazopyridyl, benzothiopheneyl, benzofuranyl, isobenzofuran, etc.

[0197] As used herein, “amine” refers to a compound containing a basic nitrogen atom with a lone pair of electrons. The term “amino” refers to a functional group or part of -NH2, -NHR, or -NR2, where R may be the same or different each time it appears and may be alkyl or aryl.

[0198] As used herein, "halogen" or "halogen group" refers to fluorine, bromine, chlorine, or iodine. Specifically, when attached to an alkyl group, it generally refers to fluorine or chlorine, and when attached to an aryl or heteroaryl group, it also includes bromine or iodine.

[0199] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more halogen groups as defined herein. Halogenated groups can be monohalogenated, dihalogenated, trihalogenated, or polyhalogenated, including perhalogenated groups. Monohalogenated groups may have a chlorine or fluorine atom within the alkyl group. Chlorine and fluorine are typically present as substituents on the alkyl or cycloalkyl group; fluorine, chlorine, and bromine are typically present on the aryl or heteroaryl group. Dihalogenated and polyhalogenated groups may have two or more identical halogen atoms or combinations of different halogen groups on the alkyl group. Typically, polyhalogenated groups contain up to 12, 10, 8, 6, 4, 3, or 2 halogen groups. Non-limiting examples of halogenated groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhaloalkyl refers to an alkyl group in which all hydrogen atoms are replaced by halogen atoms, such as trifluoromethyl.

[0200] As used herein, unless otherwise specified, the term "heteroatom" refers to a nitrogen (N), oxygen (O), or sulfur (S) atom.

[0201] Example

[0202] Example 1: Synthesis of the exemplary compounds of this disclosure

[0203] Chemical Materials and Methods

[0204] For chemical synthesis, unless otherwise specified, all starting materials and commercially available reagents were purchased from Sigma-Aldrich and used as is. All reactions were carried out under a nitrogen atmosphere using anhydrous solvents in dried glassware. 1 H and 13 The 10⁻¹⁴ NMR spectra were recorded on a Brucker AV-600 spectrometer, and referenced to the solvent used (for...). 1 H and 13 C represents CDCl3 at δ 7.24 and 77.23, CD3OD at δ 3.31 and 49.2, D2O at δ 4.80, and DMSO-d6 at δ 2.5 and 39.51, respectively. Chemical shifts (δ) are reported in ppm using the following conventions: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiply), integral, and coupling constant. J ),in J Reported in Hz. High-resolution mass spectra were recorded under ESI-TOF mass spectrometry conditions. Silica gel (Merck, Inc.) was used for rapid chromatography. IMPACT TMThe system (containing an Intein-Mediated Purification with Affinity Chitinbinding Tag) was purchased from New England Biolabs. His-tag purification resin was purchased from Roche. The HiTrap IMAC column (5 mL) was purchased from GE Healthcare Life Sciences. Gel permeation chromatography (GPC) was performed at 30°C using THF as eluent with an Ultimate 3000 liquid chromatography system equipped with a 101 refractive index detector and a Shodex column at 1 mL / min. -1 Flow rate analysis was performed on the polymer products. Calibration was performed based on the narrow-linear poly(styrene) Shodex standard (SM-105). The molecular weight (Mw) and dispersion of the polymer products were calculated using DIONX Chromeleon software. Transmission electron microscopy (TEM) images were obtained using a FEI Tecnai G2 F20 S-Twin microscope.

[0205] The chemical materials and methods described herein apply to all instances described in this disclosure.

[0206] Synthesis and Results

[0207] The exemplary compounds described herein were synthesized according to the following synthetic schemes 1, 2, and 3. Detailed synthetic procedures are described below.

[0208] Option 1

[0209]

[0210] Option 2

[0211]

[0212] Option 3

[0213]

[0214] Compounds 1 to 5

[0215] Compounds 1 to 5 were synthesized and characterized according to the published protocol (ACS Nano 2021, 15, 309-321).

[0216] Compound 8

[0217]

[0218] (11-Carboxynonyl)triphenylphosphonium-6 bromide (2.5 g, 10 mmol) was prepared by reflux of triphenylphosphine (10 mmol) and 11-bromoundecanoic acid (10 mmol). It was then dissolved in 50 mL of tetrahydrofuran (THF) and cooled to 0 °C. Lithium bis(trimethylsilyl)amide (LHMDS; 1 M in THF, 20 mmol) was added to the solution to produce an orange ylide. Subsequently, 20 mL of THF containing 4-(4-fluorophenoxy)benzaldehyde (12 mmol) was added dropwise to the solution and stirred at room temperature for 4 hours. The reaction mixture was quenched with methanol and concentrated. The residue was extracted with EA and brine, and then dried over MgSO4. After solvent removal, the mixture was subjected to silica gel (EA-hexane = 1:2) chromatography to give unsaturated fatty acid 7. Saturated fatty acids were prepared by catalytic hydrogenation in 50 ml of methanol containing 10 mol% palladium / charcoal (Pd / C). The reaction mixture was stirred overnight at room temperature under H2. The hydrogenation product was filtered through diatomaceous earth, and the resulting solution was concentrated and subjected to silica gel chromatography (EA-hexane = 1:2) to give a product (66%) as a yellow solid.

[0219] Compound 9

[0220]

[0221] Compound 9. EDC (1.5 mmol), HOBt (1.5 mmol), DMAP (0.1 mmol), trimethylamine (2 mmol), and phytosphoamine (1.2 mmol) were added to THF (10 mL) containing compound 8 (1 mmol), and the resulting solution was stirred at room temperature under nitrogen for 12 hours. The solvent was then removed by evaporation, followed by extraction with EA / H2O. The collected organic layer was washed with saturated NaHCO3 (aqueous solution), water, and brine, and dried over MgSO4. The crude product was purified by silica gel column chromatography (EA / hexane 1:1) to give 9 (74%).

[0222] Compound 10

[0223]

[0224] 4-Nitrophenylchloroformate (2 mmol) and trimethylamine (2 mmol) were added to THF (10 mL) containing compound 9 (1 mmol), and the resulting solution was stirred at room temperature under nitrogen for 12 hours. The solvent was then removed by evaporation, and the crude compound was used directly in the next step without further purification.

[0225] Compound 11

[0226]

[0227] 10 (1 mmol) and trimethylamine (2 mmol) were added to THF (10 mL) containing compound 5 (1 mmol), and the resulting solution was stirred at room temperature under nitrogen for 2 hours. The solvent was then removed by evaporation, followed by extraction with EA / H₂O. The collected organic layer was washed with saturated NaHCO₃ (aqueous solution), water, and brine, and dried over MgSO₄. The crude product was purified by silica gel column chromatography (EA / hexane 1:1 + 10% MeOH) to give 11 (59%).

[0228] Compound 12

[0229]

[0230] NaOMe (0.2 equivalents) was added to MeOH containing compound 11, and the resulting solution was stirred at room temperature under nitrogen for 2 hours. The mixture was neutralized by IR-120, then filtered and concentrated to dryness under vacuum to give compound 12 (quantitative).

[0231] Compound 13

[0232]

[0233] NaOMe (0.2 equivalents) was added to MeOH containing compound 4, and the resulting solution was stirred at room temperature under nitrogen for 2 hours. The mixture was neutralized by IR-120, then filtered and concentrated to dryness under vacuum to give compound 13 (quantitative).

[0234] Compound 14

[0235]

[0236] NaOMe (0.2 equivalents) was added to MeOH containing compound 13 (1 mmol), and the resulting solution was stirred at room temperature under nitrogen for 2 hours. The mixture was neutralized by IR-120, then filtered and concentrated to dryness under vacuum. It was then dissolved in anhydrous DCM (10 mL) and treated with imidazole (1.5 mmol) at 0 °C, followed by the addition of TBDPSCl (1.2 mmol). The mixture was stirred at room temperature under nitrogen for 2.5 hours. The reaction was quenched by the addition of MeOH. After stirring at room temperature for 10 minutes, the solvent was removed under reduced pressure to give a dry residue, which was purified by column chromatography with MeOH / DCM (1 / 10) to give compound 14 (82%).

[0237] Compound 15

[0238]

[0239] Trimethyl orthobenzoate (3 mmol) was added to a solution of compound 14 (1 mmol) and a catalytic amount of CSA (0.1 mmol) in CH3CN (20 mL) at room temperature under atmospheric nitrogen. After stirring for 30 min, Et3N was added to quench the reaction, and the resulting mixture was dried under reduced pressure. The residue was purified by column chromatography with EA / hexane (1 / 2) to give compound 15 (79%).

[0240] Compound 16

[0241]

[0242] Compound 15 (1 mmol) was dissolved in DCM (10 mL) and mixed sequentially with DIPEA (2 mmol), benzoic anhydride (2 mmol), and DMAP (0.1 mmol). After stirring for 2 hours, the solvent was evaporated under reduced pressure to obtain a dry residue, which was then poured into EA (20 mL) and 2 N HCl (10 mL) and stirred vigorously for 30 minutes. The solvent was then removed by evaporation, followed by extraction with EA / H2O. The collected organic layer was washed with ice-cold saturated NaHCO3 (aqueous solution), water, and brine, and dried over MgSO4. The dry residue was purified by column chromatography with EA / hexane (1 / 2) to give compound 16 (71%).

[0243] Compound 17

[0244]

[0245] At 0 °C, AcOH (4 mmol) and 1 M TBAF (2.4 mmol in THF) were added to compound 16 (1 mmol). The resulting mixture was gradually heated to room temperature and stirred for 2 hours, followed by dilution with EA. The organic layer was washed with saturated NaHCO3 (aqueous solution), water, and brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The dried residue was purified by column chromatography with EA / hexane (1 / 2) to give compound 47 (88%).

[0246] Compound 18

[0247]

[0248] A stirred solution of 17 (1 mmol) and 4 A molecular sieve (0.1 g) in anhydrous DCM (10 mL) was cooled to -40 °C, and then BF3(OEt)2 (0.1 mmol) was added dropwise to the solution. A solution of 3 in anhydrous DCM was added dropwise to the above mixture and stirred at -40 °C for 1 hour. The reactants were then gradually heated to room temperature and stirred for another 1 hour. The solution was quenched by adding triethylamine, then filtered, and extracted with DCM after adding a saturated aqueous solution of NaHCO3. The organic layer was dried over MgSO4 and evaporated to dryness. The residue was purified by silica gel column chromatography to obtain the trisaccharide product. The product was then dissolved in MeOH, and NaOMe (0.2 equivalents) was added, and the resulting solution was stirred at room temperature for 2 hours. The mixture was neutralized by IR-120, then filtered and concentrated to dryness under vacuum. The deacetylated mixture was purified by Bio-Gel P-2 gel electrophoresis with H2O as the eluent to obtain pure trisaccharide. The compound was freeze-dried to dryness to give compound 18 (39%).

[0249] Compound 19

[0250]

[0251] NaOMe (0.2 equivalents) was added to MeOH containing compound 13 (1 mmol), and the resulting solution was stirred at room temperature under nitrogen for 2 hours. The mixture was neutralized by IR-120, then filtered and concentrated to dryness under vacuum.

[0252] Compound 20

[0253]

[0254] 10 (1 mmol) and trimethylamine (2 mmol) were added to THF (10 mL) containing compound 19 (1 mmol), and the resulting solution was stirred at room temperature under nitrogen for 2 hours. The solvent was then removed by evaporation, followed by extraction with EA / H₂O. The collected organic layer was washed with saturated NaHCO₃ (aqueous solution), water, and brine, and dried over MgSO₄. The crude product was purified by silica gel column chromatography (EA / hexane 1:1 + 10% MeOH) to give 20.

[0255] Compound 21

[0256]

[0257] NaOMe (0.2 equivalents) was added to MeOH containing compound 20, and the resulting solution was stirred at room temperature under nitrogen for 2 hours. The mixture was neutralized by passing it through an IR-120 filter, then filtered and concentrated to dryness under vacuum to give compound 21 (quantitative). Compound 21 was detected using LCMS spectroscopy, and the LCMS spectrum showed peaks at 1236.13, 1245.16, 1247.64, 1268.77, 1279.86, 1305.99, 1308.13, 1311.57, 1313.93, 1343.46, 1354.29, 1355.60, 1358.33, 1379.12, 1403.82, 1408.57, 1425.66, 1448.31, 1453.30, 1458.39, 1467.71, 1471.33, and 1491.66. Figure 12 ).

[0258] Compound 22

[0259]

[0260] EtOH / H2O (0.5 / 0.5 mL) containing arylmannoside 22S (0.1 mmol) was added to DSPE-NHS (0.1 mmol) and trimethylamine (2 mmol), and the resulting solution was stirred at room temperature for 12 hours. The solvent was removed by evaporation, and the crude product was purified by Bio-Gel P-2 gel electrophoresis with H2O as the eluent to give 22S (79%).

[0261] Compound 23

[0262]

[0263] DSPE-NHS (0.1 mmol) and trimethylamine (2 mmol) were added to EtOH / H2O (0.5 / 0.5 mL) containing aryl trimannoside 23S (0.1 mmol), and the resulting solution was stirred at room temperature for 12 hours. The solvent was removed by evaporation, and the crude product was purified by Bio-Gel P-2 gel electrophoresis with H2O as the eluent to give 23S (76%).

[0264] Example 2: Preparation and characterization of the LNP disclosed herein

[0265] Preparation of LNP

[0266] A lipid mixture solution in EtOH (10 mg / ml) was prepared with a molar ratio of 50% SM-102, 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG2000. LNP formulations were prepared by mixing the disclosed compounds with the lipid mixture solution (where the molar ratio was 45% SM-102, 9% DSPC, 34.5% cholesterol, 1.5% DMG-PEG2000, and 10% of the disclosed compounds). The LNP formulation was added to a 1.5 mL tube. Subsequently, the mRNA payload diluted with citrate buffer (10 mM, pH 4) before use was added to the tube at a final concentration of 0.18 μg / μL. The aqueous mRNA solution in the tube was then rapidly added to the ethanol solution and thoroughly mixed by vortexing for 1 min. The resulting solution was then dialyzed overnight against PBS at 4°C using a micro-float-A-Lyzer (8–10 kD) to obtain the LNP of this example. The resulting LNP can be stored at 4°C for several days before use.

[0267] Characterization of LNP

[0268] Size measurement The size of the LNPs prepared above was measured using dynamic light scattering (DNP). First, 5 µL of LNP solution was transferred to a clean 1.5 mL tube and diluted with 95 µL of PBS. The mixture was then transferred to a colorimetric tube, and the particle size of the LNPs was measured using a Nano ZS instrument. The table below shows the size and polydispersity index (PDI) of the prepared LNP samples.

[0269] Table: Dimensions and PDI Measurements

[0270]

[0271] ζ potential and encapsulation efficiencyNext, the encapsulation efficiency of the LNPs disclosed herein was evaluated using the Quant-it Ribogreen assay. A 2000-fold dilution of quant-it Ribogreen reagent was prepared using 1×TE (working solution). Subsequently, a standard dilution series of 0–50 ng / mL RNA (100 µL) was prepared to obtain a standard curve. 5 µL of the LNP solution prepared above was transferred to a clean tube and diluted to a final volume of 100 µL. The working solution of quant-it Ribogreen reagent (100 µL) was then added to the LNP sample. The fluorescence signal of the sample was then detected using a microplate reader (ex / em 485 / 535). Based on the standard curve, the concentration of unencapsulated mRNA in the solution (ng / mL) was calculated using the fluorescence signal. For zeta potential measurements, 0.75 mL of DP-intermediate was introduced into a capillary cell and measured using a Malvern Zetasizer Pro device at 25°C.

[0272] Table: Zeta potential and encapsulation efficiency

[0273]

[0274] Example 3: In vitro uptake and transfection of mRNA-LNP in dendritic cells

[0275] Experiment 3-1

[0276] Spleen cell preparation and BMDC culture. This example tests the uptake of several exemplary LNPs (shown in the table below) in bone marrow-derived dendritic cells (BMDCs) and spleen cells according to embodiments of this disclosure. To prepare spleen cells, mouse spleens were homogenized with the ground end of a glass slide and treated with RBC lysis buffer (Sigma) to consume red blood cells (RBCs), followed by passing through a cell filter (BD Biosciences). Bone marrow was isolated from the mouse femur and tibia and treated with RBC lysis buffer (Sigma-Aldrich) to consume RBCs. Cells were then grown at 2 × 10⁻⁶ cells / day. 5Cells were cultured at a density of 100 cells / mL in RPMI-1640 containing 10% heat-inactivated FBS (Thermo Fisher Scientific), 1% penicillin / streptomycin (Thermo Fisher Scientific), 50 μM 2-mercaptoethanol (Thermo Fisher Scientific), and 20 ng / mL recombinant mouse GM-CSF (eBioscience). On day 3, cells were replenished with an equal volume of complete culture medium (RPMI-1640, 100 U / mL penicillin / streptomycin, 55 µM 2-mercaptoethanol, and 10% FBS), and replenished with half the volume of medium on day 6. Suspension cells were collected on day 8.

[0277] The table of exemplary LNPs tested in this experiment.

[0278]

[0279] LNP treatment of spleen cells and BMDC. Spleen cells or BMDCs were co-cultured with different FITC-labeled LNP formulations in RPMI-1640 at 37°C for 1 hour. Cells were blocked for 20 minutes with an Fc receptor binding inhibitor (clone: ​​93, EB Biosciences). Spleen cells were stained with antibodies against CD3 (clone: ​​17A2, BV421-binding, Biolegend) and CD19 (clone: ​​1D3, PECy7-binding, BD Biosciences). BMDCs were stained with an antibody against CD11c (clone N418 APC-binding, Biolegend). The labeled cells were analyzed using FACSC and flow cytometry (BD Biosciences).

[0280] Flow cytometry After incubation with different mRNA-LNPs, BMDCs were washed with ice-cold FACS buffer (1×DPBS containing 1% FBS with 0.1% sodium azide) and incubated on ice with purified anti-mouse CD16 / 32 antibody (Bio-Rad Laboratories) in FACS buffer for 20 min, followed by washing with FACS buffer. BMDCs were stained with APC anti-mouse CD11c antibody (Bio-Rad Laboratories) at 4°C for 30 min and washed with FACS buffer. Finally, BMDCs were stained with propidium iodide (Sigma-Aldrich). Flow cytometry was performed on a FACS Canto™ flow cytometer (BD Biosciences).

[0281] result FACS results are displayed on Figure 1 and Figure 2 And in the table below. Figure 1 The results show that, compared to non-BMDC, BMDC exhibits specific uptake of LNPs prepared using the compounds of this disclosure. Conventional LNPs (i.e., those not using the compounds of this disclosure) show slightly higher uptake of BMDC than non-BDMC, but their specificity is not significantly different compared to the specificity of the LNPs of this disclosure (56.1 / 8.62 or 55.2 / 6.64 vs. 0.48 / 0.12). Similarly, in Figure 2 In this study, dendritic cells (DCs) exhibited at least 3-fold higher specific uptake of the LNPs disclosed herein compared to B cells (30.6 / 10.7 and 38.3 / 11.9) and at least 6-fold higher than T cells (30.6 / 0.5 and 38.3 / 0.68). DCs also showed higher uptake of conventional LNPs, but this tendency was not as significant as that of the LNPs disclosed herein.

[0282] Table of intake results (arbitrary units of FITC signal)

[0283]

[0284] Experiment 3-2

[0285] In this experiment, exemplary LNPs (shown in the table below) prepared using different formulations according to embodiments of the present disclosure were tested. Uptake and transfection were tested to assess whether the payload delivered by the LNPs of the present disclosure was properly expressed in the targeted cells. Bone marrow-derived dendritic cells (BMDCs) were isolated from the tibia and femur of 57BL / 6 mice. Bone marrow cells were stimulated for 8 days with 20 ng / mL GM-CSF in RPMI medium (RPMI-1640, 100 U / ml penicillin / streptomycin, 55 µM 2-mercaptoethanol, and 10% FBS). After 8 days of culture, 1×10⁻⁶ cells were added to the LNPs. 6 One BMDC (centrifuged at 400 g for 5 minutes and replaced with 1 ml Opti-MEM) was seeded in a 6-well plate, and different samples of LNPs encapsulated with mRNA were diluted with 0.25 mL Opti-MEM and incubated with BMDC.

[0286] for intake Analysis showed that FITC-labeled LNPs encapsulated with mRNA encoding the SARS-CoV-2 spike protein were incubated with BMDC at 37°C for 2 hours. For transfection For analysis, LNPs encapsulating eGFP mRNA were incubated with BMDC at 37°C for 4 hours. Four hours after transfection, 1.25 ml of complete RPMI medium was added to the BMDC, and the mixture was incubated at 37°C for 48 hours. Experiments were performed using FACS similar to those described above.

[0287] The table of exemplary LNPs tested in this experiment.

[0288]

[0289] result FACS results are displayed on Figure 3 and Figure 4 And as shown in the table below. LNPs prepared using the compounds of this disclosure at different molar ratios all exhibited higher uptake than the negative control ("conventional" LNPs without the compounds of this disclosure). The data also confirm that the LNPs of this disclosure not only deliver the payload to the targeted cells, but also transfect and allow the targeted cells to express the payload. Considering the higher specificity to the targeted cells, the transfection signal detected in the group using the LNPs of this disclosure was also significantly higher than that detected in the conventional group. This result indicates that using the LNPs of this disclosure allows for lower doses of payload to achieve similar results.

[0290] A table showing the results of uptake and transfection (arbitrary units of FITC signal).

[0291]

[0292] Experiment 3-3

[0293] To evaluate the binding of DC-SIGN to the LNP of this disclosure, ELISA plates were plated overnight in PBS at 4°C with the exemplary LNP. The plates were incubated for 1 hour at room temperature with diluted DC-SIGN ECD (15 to 0.075 nM in HEPES buffer containing 20 mM HEPES, 150 mM NaCl, 10 mM CaCl2, and 0.1% BSA) at pH 7.4, 6.0, and 5.0. The bound DC-SIGN ECD was detected using an HRP-bound anti-DC-SIGN (B2) IgG antibody (Santa Cruz Biotechnology). After incubation at room temperature for 1 hour, the plates were treated with tetramethylbenzidine (TMB) for 10 minutes. Optical density was measured at 450 nm after adding 0.5 M sulfuric acid to the plates using a microplate reader. Apparent Kd was calculated using a GraphPad Prism with nonlinear regression curve fitting.

[0294] Example 4: In vivo delivery of luciferase mRNA-LNP

[0295] This experiment tested the in vivo targeted delivery of the disclosed LNPs (shown in the table below). The LNPs tested in this experiment carry mRNA encoding luciferase. Mice were intravenously injected with LNPs (200 µL) and maintained for one or six hours, followed by in vivo imaging system (IVIS) testing. ® Measurements were performed. For IVIS measurements, animals were first anesthetized using a rodent anesthesia system with isoflurane (2.5% (volume / volume) in a 0.2 L / min O2 stream). Subsequently, D-fluorescein solution (dissolved in 1×PBS; 150 mg / kg body weight) was injected intravenously. Three minutes after injection, the animals were scanned using an IVIS imaging system (data not shown). After imaging, the animals were euthanized in a CO2 chamber. Organs (heart, lungs, liver, spleen, kidneys, and lymph nodes) were collected and analyzed and quantified using an IVIS system.

[0296] The table of exemplary LNPs tested in this experiment.

[0297]

[0298] result .result( Figure 5 Both compounds 22-LNP and 12-LNP tend to accumulate in the spleen and lymph nodes. While compound 12-LNP also accumulates in the liver, compound 22-LNP exhibits a high level of specificity targeting the spleen and lymph nodes. These results demonstrate the targeted delivery capability of the lipid nanoparticle formulations disclosed herein, consistent with the observations from the aforementioned experiments.

[0299] Furthermore, according to this disclosure, exemplary LNPs are prepared to carry not only mRNA encoding luciferase, but also mRNA encoding either the broad-spectrum SARS-CoV-2 spike protein or the low-glycemic-spectrum SARS-CoV-2 spike protein. LNPs (200 µL) are administered intravenously to mice and maintained for one or six hours, followed by in vivo imaging system (IVIS) testing. ® Measurements were performed. For IVIS measurements, animals were first anesthetized using a rodent anesthesia system with isoflurane (2.5% (volume / volume) in a 0.2 L / min O2 stream). Subsequently, D-fluorescein solution (dissolved in 1×PBS; 150 mg / kg body weight) was injected intravenously. Three minutes after injection, the animals were scanned using an IVIS imaging system (data not shown). After imaging, the animals were euthanized in a CO2 chamber. Organs (heart, lungs, liver, spleen, kidneys, and lymph nodes) were collected and analyzed and quantified using an IVIS system.

[0300] Example 6: Immunization

[0301] animal Balb / c mice (8 weeks old) were maintained in a specific pathogen-free environment. Eight-week-old Balb / c mice were immunized twice IM-1, 2 weeks apart. Each immunization contained PBS (100 μl). Serum collected from immunized mice was analyzed by ELISA 10 days after the last immunization.

[0302] LNP For the neutralization assay, according to one embodiment of this disclosure, an LNP was prepared for this experiment. Two control LNPs were also prepared to compare the performance of the LNPs of this disclosure. The first control LNP was formed using SM-102 and DSPC (“L1+L2”) without the compounds of this disclosure. The second control LNP was Spikevax’s Moderna product (“LNP(M)”). All tested LNPs carried mRNA carriers encoding the SARC-CoV-2 spike protein. For the IgG titer assay, the LNPs of this disclosure were prepared to carry mRNA encoding either wild-type SARC-CoV-2 spike protein or wild-type SARC-CoV-2 spike protein with low-glycemic modification.

[0303] Animal immunization BALB / c mice aged 6 to 8 weeks (n = 5) were intramuscularly immunized with 15 μg of phosphate-buffered saline (PBS) containing LNP. Animals were immunized at week 0 and given a booster immunization at week 2. Serum samples were collected from each mouse 2 weeks after the second immunization.

[0304] pseudovirus neutralization assayThe pseudoviruses were constructed using a procedure similar to that described previously. In short, pseudolentiviruses carrying the SARS-CoV-2 spike protein were generated by transiently transfecting HEK-293T cells with pCMV-ΔR8.91 and pLAS2w.Fluc.Ppuro. HEK-293T cells were seeded one day prior to transfection, and the specified plasmid was delivered into the cells using the TransITR-LT1 transfection reagent (Mirus). The culture medium was refreshed 16 hours post-transfection, and cells were collected at 48 and 72 hours. Cell debris was removed by centrifugation at 4,000 × g for 10 min, and the supernatant was passed through a 0.45 μm syringe filter (Pall Corporation). The pseudolentiviruses were aliquoted and subsequently stored at -80°C. Lentiviral titers were estimated using the AlarmaBlue assay (Thermo Scientific), and transduction units (TUs) of the SARS-CoV-2 pseudolentiviruses were estimated by reacting the limited dilutions of lentivirus with a cell viability assay. In short, HEK-293T cells stably expressing the human ACE2 gene were seeded in 96-well plates one day prior to lentiviral transduction. To titrate pseudotyped lentivirus, varying amounts of lentivirus were added to medium containing polybrene (final concentration 8 μg / ml). Spin infection was performed at 1,100 × g for 30 min in 96-well plates at 37°C. After culturing cells at 37°C for 16 h, the medium containing virus and polybrene was removed and replaced with fresh, complete DMEM containing 2.5 μg / ml puromycin. After 48 h of puromycin treatment, the medium was removed, and cell viability was assessed using 10% AlamarBlue reagent according to the manufacturer's instructions. The viability of uninfected cells (without puromycin treatment) was set at 100%. Viral titer (transduction units) was determined by plotting the relationship between surviving cells and diluted virus doses. For the neutralization assay, heat-inactivated serum or antibody was serially diluted and incubated in DMEM at 37°C for 1 hour with 1,000 TU of SARS-CoV-2 pseudotyped lentivirus. The mixture was then seeded in 96-well plates with 10,000 HEK-293T cells stably expressing the human ACE2 gene. Sixteen hours post-infection, the medium was replaced with fresh, complete DMEM supplemented with 10% FBS and 100 U / mL penicillin / streptomycin and continuously incubated for another 48 hours. Luciferase gene expression levels were determined using the Bright-Glo luciferase assay system (Promega). Relative light units (RLU) were detected using the Tecan i-control (Infinite 500). The formula (RLU) was used. 对照 -RLU血清 The RLU / RLU control is calculated as the ratio of RLU reduction in the presence of diluted serum to the RLU value in the serum-free control.

[0305] Measurement of serum IgG titer ELISA was used to determine the IgG titer in mouse serum. The wells of a 96-well ELISA plate (Greiner Bio-One) were plated overnight at 4°C with 100 ng of SARS-CoV-2 spike protein (ACROBiosystems, wild-type, δ, or O) in 100 mM sodium bicarbonate at pH 8.8. The wells were then blocked at 37°C with 200 µl of 1×PBS containing 5% skim milk for 1 hour and washed three times with 200 µl of PBST (1×PBS, 0.05% Tween 20, pH 7.4). Serially diluted mouse serum samples (2-fold) were added to the wells and incubated at 37°C for 2 hours, followed by six washes with 200 µl of PBST. At 37°C, the wells were incubated for 1 hour with 100 µl of HRP-bound anti-mouse secondary antibody (1:10000, in PBS) and washed six times with 200 µl of PBST. 100 µl of horseradish peroxidase substrate (1-Step™ Ultra TMB-ELISA substrate solution) (Thermo Scientific™) was added to the wells, followed by 100 µl of 1 M H2SO4. After 30 minutes of incubation, absorbance (OD 450 nm) was measured using a SpectraMax M5.

[0306] result . Figure 9 All tested LNPs carrying mRNA delivery vehicles were able to deliver and express mRNA in vivo, thereby triggering an immune response leading to neutralization inhibition. However, the inhibitory effect of the tested LNPs varied with increasing dilution factor. Compared to the negative control, L1+L2 LNPs and LNP (M) showed only slightly higher inhibitory effects at a 1:5000 dilution, but the LNPs of this disclosure maintained approximately 40% inhibition. The data indicate that the LNPs of this disclosure can elicit an immune response at concentrations much lower than those of other LNPs tested in this experiment.

[0307] Figure 10 The LNP of this disclosure was verified to induce antigen-specific IgG in vivo. LNP induction carrying mRNA encoding a broad spike protein still resulted in IgG recognition of both the δ and O variant spike proteins at a good level. Data show that the targeted delivery characteristics of the LNP can at least partially overcome immune escape due to spike protein variations between variants.

[0308] Furthermore, LNPs carrying mRNA encoding the wild-type SARS-CoV-2 spike protein (“WT LNP”) and LNPs carrying mRNA encoding a low-glycemic modified spike protein (“Low-glycemic LNP”) were observed to induce comparable IgG titers against broad-spectrum viruses. WT LNPs showed lower IgG titers against delta and o virus strains, indicating immune evasion. In contrast, low-glycemic LNPs maintained high IgG titers against both variant virus strains. These results suggest that removing the glycan shield improves the immunogenicity of LNP formulations.

[0309] Figure 11 The results of additional experiments are presented. In this experiment, Moderna LNPs were prepared using Moderna's proprietary formulation. Additionally, LNPs were prepared using Moderna formulations with the addition of the compounds disclosed herein to test whether the compounds of this disclosure improve the performance of the Moderna formulations. LNPs were administered to animals, and IgG titer and neutralization assays were performed in this example as described above. The results showed that the compounds of this disclosure increased spike protein-specific IgG. Serum obtained from mice administered LNPs containing the compounds of this disclosure also showed better neutralization. This experiment confirms the targeted delivery characteristics of the compounds of this disclosure and validates their applicability to commercially available LNP formulations.

[0310] Exemplary Examples

[0311] Example A1. A pharmaceutical formulation comprising lipid nanoparticles and a pharmaceutically acceptable excipient; wherein the lipid nanoparticles comprise a membrane defining an internal space, wherein the membrane is formed of multiple lipid components; wherein the multiple lipid components comprise a bifunctional compound, the bifunctional compound comprising:

[0312] Formula 1; or

[0313] Formula 2;

[0314] R1 contains substituted or unsubstituted glycosidic groups; X1 and X2 are each independently hydrogen and C. 1-30 Alkyl, C 1-30 alkenyl, C 1-30 Alkyne, aryl, aryloxy, or a substituted form thereof, or -(CH2)nX4, where n is 0 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic, or a substituted form thereof, provided that when X4 is a heterocyclic group, the heterocyclic group comprises 1 to 3 heteroatoms selected from the group consisting of O, S, and N, or combinations thereof; and wherein X3 is hydrogen, C 1-6 Alkyl or hydroxyl; wherein the membrane encapsulates a payload encoding the spike protein of SARS-CoV-2, wherein the spike protein has a reduced and / or missing glycan profile.

[0315] Example A2. The pharmaceutical formulation according to Example 1, wherein the spike protein comprises an amino acid substitution from asparagine (N) to glutamine (Q) at an N-linked glycosylation sequence (NXS / T), wherein X is any amino acid residue other than proline, and S / T represents a serine or threonine residue.

[0316] Example A3. A pharmaceutical formulation according to Example A1 or Example A2, wherein the spike protein comprises an amino acid deletion or addition at an N-linked glycosylation sequence (NXS / T) to eliminate the N-linked glycan sequence.

[0317] Example A4. A pharmaceutical formulation according to any one of Examples A1 to A3, wherein the spike protein comprises an amino acid substitution of S / T to alanine (A) at the O-linked glycosylation site to eliminate the O-linked glycosylation site.

[0318] Example A5. A pharmaceutical formulation according to any one of Examples A1 to A4, wherein the spike protein comprises the amino acid sequence set forth in SEQ ID NO:2, 16, 18 or 20, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% homology with the amino acid sequence set forth in SEQ ID NO:2, 16, 18 or 20; or the payload encoding the spike protein is RNA comprising the nucleotide sequence set forth in SEQ ID NO:1, 15, 17 or 19, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% homology with the nucleotide sequence set forth in SEQ ID NO:1, 15, 17 or 19, respectively.

[0319] Example A6. A pharmaceutical formulation according to any one of Examples A1 to A5, wherein the spike protein comprises the amino acid sequence set forth in SEQ ID NO:4, 22, 24 or 26, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% homology with the amino acid sequence set forth in SEQ ID NO:4, 22, 24 or 26, and wherein the spike protein comprises a receptor-binding domain (RBD) lacking at least one glycosylation site; or the payload encoding the spike protein is RNA comprising the nucleotide sequence set forth in SEQ ID NO:3, 21, 23 or 25, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% homology with the nucleotide sequence set forth in SEQ ID NO:3, 21, 23 or 25, respectively.

[0320] Example A7. A pharmaceutical formulation according to any one of Examples A1 to A6, wherein the spike protein comprises the amino acid sequence set forth in SEQ ID NO:6, 28, 30 or 32, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% homology with the amino acid sequence of said SEQ ID NO:6, 28, 30 or 32, and wherein the spike protein comprises an S2 subunit lacking at least one glycosylation site; or the payload encoding the spike protein is RNA comprising the nucleotide sequence set forth in SEQ ID NO:5, 27, 29 or 31, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% homology with the nucleotide sequence set forth in SEQ ID NO:5, 27, 29 or 31, respectively.

[0321] Example A8. A pharmaceutical formulation according to any one of Examples A1 to A7, wherein the spike protein comprises the amino acid sequence set forth in SEQ ID NO: 8 or 34, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology with the amino acid sequence of SEQ ID NO: 8 or 34, and wherein the spike protein comprises an S2 subunit comprising a single glycosylation site at N1194; or the payload encoding the spike protein is RNA comprising the nucleotide sequence set forth in SEQ ID NO: 7 or 33, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology with the nucleotide sequence set forth in SEQ ID NO: 7 or 33, respectively.

[0322] Example A9. A pharmaceutical formulation according to any one of Examples A1 to A8, wherein the spike protein comprises the amino acid sequence set forth in SEQ ID NO: 10 or 36, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology with the amino acid sequence of said SEQ ID NO: 10 or 36, and wherein the spike protein comprises a receptor-binding domain (RBD) lacking at least one glycosylation site and an amino acid substitution of N801Q; or the payload encoding the spike protein is RNA comprising the nucleotide sequence set forth in SEQ ID NO: 9 or 35, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology with the nucleotide sequence set forth in said SEQ ID NO: 9 or 35, respectively.

[0323] Example A10. A pharmaceutical formulation according to any one of Examples A1 to A9, wherein the spike protein comprises the amino acid sequence set forth in SEQ ID NO:12 or 38, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology with the amino acid sequence of said SEQ ID NO:12 or 38, and wherein the spike protein comprises a receptor-binding domain (RBD) lacking at least one glycosylation site and an amino acid substitution of N1194Q; or the payload encoding the spike protein is RNA comprising the nucleotide sequence set forth in SEQ ID NO:11 or 37, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology with the nucleotide sequence set forth in said SEQ ID NO:11 or 37, respectively.

[0324] Example A11. A pharmaceutical formulation according to any one of Examples A1 to A10, wherein the spike protein comprises the amino acid sequence set forth in SEQ ID NO:14 or 40, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology with the amino acid sequence of SEQ ID NO:14 or 40, and wherein the spike protein comprises a receptor-binding domain (RBD) lacking at least one glycosylation site and amino acid substitutions of N122Q, N165Q, N234Q, or combinations thereof; or the payload encoding the spike protein is RNA comprising the nucleotide sequence set forth in SEQ ID NO:13 or 39, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology with the nucleotide sequence set forth in SEQ ID NO:13 or 39, respectively.

[0325] Example A12. A pharmaceutical formulation according to any one of Examples A1 to A11, wherein the payload encodes an immunogenic peptide, the immunogenic peptide comprising an amino acid sequence selected from the group consisting of: TESIVRFPNITNL (SEQ ID NO:41), NITNLCPFGEVFNATR (SEQ ID NO:42), LYNSASFSTFK (SEQ ID NO:43), LDSKVGGNYN (SEQ ID NO:44), KSNLKPFERDIST (SEQ ID NO:45), KPFERDISTEIYQAG (SEQ ID NO:46), GPKKSTNLVKNKC (SEQ ID NO:47), NCDVVIGIVNNTVY (SEQ ID NO:48), PELDSFKEELDKYFKNHTS (SEQ ID NO:49), VNIQKEIDRLNEVA (SEQ ID NO:50), NLNESLIDLQ (SEQ ID NO:51), and LGKYEQYIKWP (SEQ ID NO:52), or with SEQ ID NO. Any one of NO:41 to 52 has an amino acid sequence with at least about 99%, 98%, 97%, 96%, 95%, or 90% homology.

[0326] Example A13. A pharmaceutical formulation according to any one of Examples A1 to A11, wherein the spike protein comprises the amino acid sequence set forth in SEQ ID NO: 53, 54, 55, 56 or 57, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% homology with the amino acid sequence of SEQ ID NO: 53, 54, 55, 56 or 57.

[0327] Example A14. The pharmaceutical formulation according to Example A13, wherein the payload encodes an immunogenic peptide, the immunogenic peptide comprising an amino acid sequence selected from the group consisting of:

[0328]

[0329] Or an amino acid sequence that is at least about 99%, 98%, 97%, 96%, 95% or 90% identical to any one of SEQ ID NO:58 to 74.

[0330] Example A15. A pharmaceutical compound according to any one of Examples A1 to A14, wherein R1 includes the formula R2-R A - where R2 is the substituted or unsubstituted glycosidic group, R A It is a linking group, and said linking group includes aryl, alkyl, amide, alkylamide, its substituted form, its combination or covalent bond.

[0331] Example A16. The pharmaceutical compound according to Example A15, wherein R A Contains an aryl group having 0 to 3 substituents (e.g., 1 to 3 substituents), wherein said substituents are C 1-6 Alkyl, halide or C 1-6 Alkyl halides.

[0332] Example A17. The pharmaceutical compound according to Example A16, wherein R A It also contains polyethylene glycol (PEG) moieties with 2 to 72 (OCH2CH2) subunits.

[0333] Example A18. The pharmaceutical formulation according to Example A16 or Example A17, wherein the PEG portion is linear.

[0334] Example A19. A pharmaceutical formulation according to any one of Examples A1 to A18, wherein the glycoside comprises mannoside, fucoidan, or a combination thereof.

[0335] Example A20. A pharmaceutical formulation according to any one of Examples A1 to A19, wherein the glycoside comprises terminal mannoside, terminal fucoside, or both.

[0336] Example A21. A pharmaceutical formulation according to any one of Examples A1 to A20, wherein the glycoside comprises monomannoside, dimannoside, or trimannoside.

[0337] Example A22. The pharmaceutical formulation according to Example A21, wherein the trimannoside is a straight-chain or branched-chain trimannoside.

[0338] Example A23. The pharmaceutical formulation according to Example A22, wherein the branched trimannoside is α-1,3-α-1,6-trimannoside.

[0339] Example A24. A pharmaceutical formulation according to any one of Examples A1 to A23, wherein R1 is a substituted glycoside.

[0340] Example A25. The pharmaceutical formulation according to Example A24, wherein the glycosidic group comprises 1 to 6 substituents, wherein the substituents are C 1-6 Alkyl, C 1-6 alkenyl, halogen, C 1-6 Alkyl halides, C 1-6 Alkoxy, amine, nitro, C 1-6 Alkylamines, amides, azides, aryls, cycloalkyls, heterocycloalkyls, sulfites, or their substituted forms, or combinations thereof.

[0341] Example A26. The pharmaceutical formulation according to Example A25, wherein the substituents of the glycosidic group are selected from the group consisting of: aryl, 5-membered cycloalkyl, 6-membered cycloalkyl, 5-membered heterocycloalkyl, and 6-membered heterocycloalkyl and their substituted forms, and the glycosidic group comprises 1 to 6 substituents selected from the group consisting of: C 1-6 Alkyl, halogen, C 1-6 Alkyl halides, C 1-6 Alkoxy, amine, nitro, C 1-6 Alkylamines, amides, azides, carboxyl groups, hydroxyl groups, aryl groups, cycloalkyl groups, heterocycloalkyl groups, or their substituted forms, or combinations thereof.

[0342] Example A27. A pharmaceutical formulation according to Example A25 or Example A26, wherein the substituent of the glycosidic group is a substituted or unsubstituted aryl group, optionally, the substituent of the glycosidic group is a phenyl group substituted with OH, CH3, NH2, CF3, OCH3, F, Br, Cl, NO2, N3 or a combination thereof.

[0343] Example A28. A pharmaceutical formulation according to Example A25 or Example A26, wherein the heterocyclic alkyl group comprises 1 to 3 heteroatoms selected from the group consisting of O, S and N.

[0344] Example A29. A pharmaceutical preparation according to any one of Examples A1 to A28, wherein R1 is selected from the group consisting of:

[0345] ;

[0346] ;

[0347] ;

[0348] .

[0349] Example A30. A pharmaceutical formulation according to any one of Examples A1 to A29, wherein the compound has Formula 1.

[0350] Example A31. A pharmaceutical formulation according to any one of Examples A1 to A30, wherein the compound has Formula 2.

[0351] Example A32. A pharmaceutical formulation according to Example A31, wherein the compound has Formula 3:

[0352] Equation 3; and

[0353] R1 is selected from the following groups:

[0354] .

[0355] Example A33. A pharmaceutical formulation according to any one of Examples A1 to A32, wherein at least one of X1 and X2 comprises a saturated hydrocarbon chain containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28 or 30 carbons.

[0356] Example A34. A pharmaceutical formulation according to any one of Examples A1 to A33, wherein X1 and X2 are each independently hydrogen and C. 4-30 Alkyl, C 4-30 alkenyl, C 4-30Alkyne, aryl, aryloxy or a substituted form thereof, or -(CH2)nX4, where n is 4 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic or a substituted form thereof, provided that when X4 is a heterocyclic group, the heterocyclic group comprises 1 to 3 heteroatoms selected from the group consisting of O, S and N, or combinations thereof.

[0357] Example A35. The pharmaceutical compound according to Example A34, wherein X1 and X2 are each independently hydrogen and C. 8-30 Alkyl, C 8-30 alkenyl, C 8-30 Alkyne, aryl, aryloxy or a substituted form thereof, or -(CH2)nX4, where n is 8 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic or a substituted form thereof, provided that when X4 is a heterocyclic group, the heterocyclic group comprises 1 to 3 heteroatoms selected from the group consisting of O, S and N, or combinations thereof.

[0358] Example A36. A pharmaceutical compound according to any one of Examples A1 to A35, provided that when one of X1 and X2 is hydrogen, the other is not hydrogen.

[0359] Example A37. A pharmaceutical formulation according to any one of Examples A1 to A36, wherein X4 is an aryl, aryloxy, heterocyclic, cycloalkyl, heterocyclic, or combination thereof, and wherein X4 comprises 0 to 6 substituents selected from the group consisting of: C 1-6 Alkyl, halogen, C 1-6 Alkyl halogens and C 1-6 Alkyl group.

[0360] Example A38. The pharmaceutical formulation according to Example A37, wherein the substituent is CH3, CF3, F or OCH3.

[0361] Example A39. The pharmaceutical compound according to Example A37 or Example A38, wherein X4 comprises 1 to 3 substituents.

[0362] Example A40. A pharmaceutical formulation according to any one of Examples A37 to A39, wherein X4 is -R3-O-R4, wherein R3 and R4 are each independently aryl, heterocyclic, cycloalkyl, or heterocyclic, each comprising 0 to 6 substituents selected from the group consisting of: C 1-6 Alkyl, halogen, C 1-6 Alkyl halogens and C 1-6 Alkyl group.

[0363] Example A41. A pharmaceutical preparation according to any one of Examples A1 to A40, wherein one of X1 and X2 is C 15-30Alkyl group, and another one is -(CH2)nX4.

[0364] Example A42. A pharmaceutical preparation according to any one of Examples A1 to A41, wherein X4 is selected from the group consisting of:

[0365] .

[0366] Example A43. A pharmaceutical formulation according to any one of Examples A1 to A42, wherein the compound is selected from the group consisting of:

[0367]

[0368]

[0369]

[0370] .

[0371] Example A44. A pharmaceutical formulation according to any one of Examples A1 to A43, wherein the component is not glycolipid C34 or α-galactosylceramide.

[0372] Example A45. A pharmaceutical formulation according to any one of Examples A1 to A44, comprising 0.01 to 95% (w / w) of the lipid nanoparticles.

[0373] Example A46. A pharmaceutical formulation according to any one of Examples A1 to A45, wherein the lipid nanoparticles are first lipid nanoparticles, and the composition further comprises second lipid nanoparticles.

[0374] Example A47. A pharmaceutical formulation according to Example A46, wherein the first lipid nanoparticles and the second lipid nanoparticles differ in size, membrane composition, effective load encapsulated within the lipid nanoparticles, or a combination thereof.

[0375] Example A48. A pharmaceutical formulation according to any one of Examples A1 to A47, wherein the excipient comprises a solvent, a dispersion medium, a diluent, a dispersion, a suspending agent, a surfactant, an isotonic agent, a thickener or emulsifier, a preservative, a polymer, a peptide, a protein, a cell, hyaluronidase, or a mixture thereof.

[0376] Example A49. The pharmaceutical formulation according to any one of Examples A1 to A48 further comprises an adjuvant.

[0377] Example A50. A pharmaceutical formulation according to Example A49, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, aluminum salt, squalene, MF59, or QS-21. Other examples of adjuvants that can be used in some vaccines in the compositions of this disclosure are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), and CpG1018 (Dynavax) or combinations thereof.

[0378] Example A51. A method for targeted delivery of a payload in an individual, comprising administering to the individual an effective amount of a pharmaceutical preparation according to any one of Examples A1 to A50.

[0379] Example A52. The method according to Example A51, wherein the pharmaceutical preparation is administered at an initial dose, and subsequently administered once, twice, three times, four times, five times or more additional doses.

[0380] Example A53. The method according to Example A51 or Example A52, wherein the additional dose is administered approximately one month, two months, three months, four months, five months, or six months or longer after the initial dose.

[0381] Example A54. The method according to any one of Examples A51 to A53, wherein the effective amount is in the range of about 5 μg to 1000 μg.

[0382] Example A55. A method for preventing or treating a disease in an individual, comprising administering to the individual an effective amount of a pharmaceutical preparation according to any one of Examples A1 to A50.

[0383] Example A56. The method according to Example A55, wherein the lipid nanoparticles are administered at an initial dose, and subsequently administered once, twice, three times, four times, five times or more of additional doses.

[0384] Example A57. The method according to Example A55 or Example A56, wherein the additional dose is administered approximately one month, two months, three months, four months, five months, or six months or longer after the initial dose.

[0385] Example A58. The method according to any one of Examples A55 to A57, wherein the effective amount is in the range of about 5 μg to 1000 μg.

[0386] Example A59. A method for enhancing an adaptive immune response, comprising administering to an individual an effective amount of a pharmaceutical formulation according to any one of Examples A1 to A50.

[0387] Example A60. The method according to Example A59, wherein the lipid nanoparticles are administered at an initial dose, and subsequently administered one, two, three, four, five or more additional doses.

[0388] Example A61. The method according to Example A59 or Example A60, wherein the additional dose is administered approximately one month, two months, three months, four months, five months, or six months or longer after the initial dose.

[0389] Example A62. The method according to any one of Examples A59 to A61, wherein the effective amount is in the range of about 5 μg to 1000 μg.

[0390] Example B1. A compound for forming lipid nanoparticles, wherein the components comprise the following formula:

[0391] Formula 1; or

[0392] Formula 2;

[0393] R1 contains substituted or unsubstituted glycosidic groups; X1 and X2 are each independently hydrogen and C. 1-30 Alkyl, C 1-30 alkenyl, C 1-30 Alkyne, aryl, aryloxy, or a substituted form thereof, or -(CH2)nX4, where n is 0 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic, or a substituted form thereof, provided that when X4 is a heterocyclic group, the heterocyclic group comprises 1 to 3 heteroatoms selected from the group consisting of O, S, and N, or combinations thereof; and wherein X3 is hydrogen, C 1-6 Alkyl or hydroxyl.

[0394] Example B2. The compound according to Example B1, wherein R1 comprises formula R2-R A -, where R A R1 is a linking group and R2 is the substituted or unsubstituted glycosidic group, wherein the linking group comprises aryl, alkyl, amide, alkylamide, its substituted form, its combination or covalent bond.

[0395] Example B3. The compound according to Example B2, wherein R A It contains an aryl group having 0 to 3 substituents, wherein said substituents are C 1-6 Alkyl, halide or C1-6 Alkyl halides.

[0396] Example B4. The compound according to Example B3, wherein R A It also contains polyethylene glycol (PEG) moieties with 2 to 72 (OCH2CH2) subunits.

[0397] Example B5. The copolymer according to Example B3 or Example B4, wherein the PEG portion is linear.

[0398] Example B6. The compound according to any one of Examples B1 to B5, wherein the glycosidic group comprises mannoside, fucoidan, or a combination thereof.

[0399] Example B7. The compound according to any one of Examples B1 to B6, wherein the glycosidic group comprises terminal mannoside, terminal fucoside, or both.

[0400] Example B8. The compound according to any one of Examples B1 to B7, wherein the glycosidic group comprises monomannoside, dimannoside or trimannoside.

[0401] Example B9. The compound according to Example B8, wherein the trimannoside is a straight-chain or branched-chain trimannoside.

[0402] Example B10. The compound according to Example B9, wherein the branched trimannoside is α-1,3-α-1,6-trimannoside.

[0403] Example B11. The compound according to any one of Examples B1 to B10, wherein R1 is a substituted glycosidic group.

[0404] Example B12. The compound according to Example B11, wherein the glycosidic group comprises 1 to 6 substituents, wherein the substituents are C 1-6 Alkyl, C 1-6 alkenyl, halogen, C 1-6 Alkyl halides, C 1-6 Alkoxy, amine, nitro, C 1-6 Alkylamines, amides, azides, aryls, cycloalkyls, heterocycloalkyls, sulfites, or their substituted forms, or combinations thereof.

[0405] Example B13. The compound according to Example B12, wherein the substituents of the glycosidic group are selected from the group consisting of: aryl, 5-membered cycloalkyl, 6-membered cycloalkyl, 5-membered heterocycloalkyl, and 6-membered heterocycloalkyl and their substituted forms, and the glycosidic group comprises 1 to 6 substituents selected from the group consisting of: C 1-6 Alkyl, halogen, C1-6 Alkyl halides, C 1-6 Alkoxy, amine, nitro, C 1-6 Alkylamines, amides, azides, carboxyl groups, hydroxyl groups, aryl groups, cycloalkyl groups, heterocycloalkyl groups, or their substituted forms, or combinations thereof.

[0406] Example B14. The compound according to Example B12 or Example B13, wherein the substituent of the glycosidic group is a substituted or unsubstituted aryl group, and optionally, the substituent of the glycosidic group is a phenyl group substituted with OH, CH3, NH2, CF3, OCH3, F, Br, Cl, NO2, N3 or a combination thereof.

[0407] Example B15. The compound according to Example B12 or Example B13, wherein the heterocyclic alkyl group comprises 1 to 3 heteroatoms selected from the group consisting of O, S and N.

[0408] Example B16. The compound according to any one of Examples B1 to B15, wherein R1 is selected from the group consisting of:

[0409] ;

[0410] ;

[0411] ;

[0412] .

[0413] Example B17. The compound according to any one of Examples B1 to B16, wherein the compound has Formula 1.

[0414] Example B18. The compound according to any one of Examples B1 to B16, wherein the compound has Formula 2.

[0415] Example B19. The compound according to Example B18, wherein the compound has Formula 3:

[0416] Equation 3; and

[0417] R1 is selected from the following groups:

[0418] .

[0419] Example B20. A compound according to any one of Examples B1 to B19, wherein at least one of X1 and X2 comprises a saturated hydrocarbon chain containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28 or 30 carbons.

[0420] Example B21. A compound according to any one of Examples B1 to B20, wherein X1 and X2 are each independently hydrogen and C. 4-30 Alkyl, C 4-30 alkenyl, C 4-30 Alkyne, aryl, aryloxy or a substituted form thereof, or -(CH2)nX4, where n is 4 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic or a substituted form thereof, provided that when X4 is a heterocyclic group, the heterocyclic group comprises 1 to 3 heteroatoms selected from the group consisting of O, S and N, or combinations thereof.

[0421] Example B22. The compound according to Example B21, wherein X1 and X2 are each independently hydrogen and C. 8-30 Alkyl, C 8-30 alkenyl, C 8-30 Alkyne, aryl, aryloxy or a substituted form thereof, or -(CH2)nX4, where n is 8 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic or a substituted form thereof, provided that when X4 is a heterocyclic group, the heterocyclic group comprises 1 to 3 heteroatoms selected from the group consisting of O, S and N, or combinations thereof.

[0422] Example B23. The compound according to any one of Examples B1 to B22, provided that when one of X1 and X2 is hydrogen, the other is not hydrogen.

[0423] Example B24. A compound according to any one of Examples B1 to B23, wherein X4 is an aryl, aryloxy, heterocyclic, cycloalkyl, heterocyclic, or combination thereof, and wherein X4 comprises 0 to 6 substituents selected from the group consisting of: C 1-6 Alkyl, halogen, C 1-6 Alkyl halogens and C 1-6 Alkyl group.

[0424] Example B25. The compound according to Example B24, wherein the substituent is CH3, CF3, F or OCH3.

[0425] Example B26. The compound according to Example B24 or Example B25, wherein X4 comprises 1 to 3 substituents.

[0426] Example B27. The compound according to any one of Examples B24 to B26, wherein X4 is -R3-O-R4, wherein R3 and R4 are each independently aryl, heterocyclic, cycloalkyl, or heterocyclic, each comprising 0 to 6 substituents selected from the group consisting of: C 1-6 Alkyl, halogen, C 1-6 Alkyl halogens and C 1-6 Alkyl group.

[0427] Example B28. The compound according to any one of Examples B1 to B27, wherein one of X1 and X2 is C. 15-30 Alkyl group, and another one is -(CH2)nX4.

[0428] Example B29. The compound according to any one of Examples B1 to B28, wherein X4 is selected from the group consisting of:

[0429] .

[0430] Example B30. The compound according to any one of Examples B1 to B29, selected from the group consisting of:

[0431]

[0432]

[0433]

[0434] .

[0435] Example B31. The component according to any one of Examples B1 to B30, wherein the component is not glycolipid C34 or α-galactosylceramide.

[0436] Example B32. A formulation for forming lipid nanoparticles comprising a compound according to any one of Examples B1 to B31, wherein the compound comprises 1 to 10 mol of the composition.

[0437] Example B33. The formulation according to Example B32 further comprises ionizable lipids, auxiliary lipids, or mixtures thereof, wherein the ionizable lipids comprise 30 to 60 mol% of the composition, the auxiliary lipids comprise 5 to 60 mol% of the composition, and the remaining percentage is a carrier or solvent.

[0438] Example B34. A formulation according to Example B33, wherein the ionizable lipid comprises 8-[2-hydroxyethyl-(6-oxo-6-undecyloxyhexyl)amino]octanoic acid heptadecan-9-yl ester (SM-102) TM (4-Hydroxybutyl)azonide)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) TM (Pfizer Inc.) or a combination thereof.

[0439] Example B35. A formulation according to Example B33 or Example B34, wherein the auxiliary lipid comprises phosphatidylcholine, cholesterol or a derivative thereof, polyethylene glycol-lipid (PEG-lipid) or a mixture thereof, wherein the phosphatidylcholine comprises 5 to 10 mol% of the composition, the cholesterol or a derivative thereof comprises 30 to 40 mol% of the composition, and the polyethylene glycol-lipid (PEG-lipid) comprises 1 to 10 mol% of the composition.

[0440] Example B36. The formulation according to Example B35, wherein the phosphatidylcholine comprises distearate phosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DPOE), or a mixture thereof.

[0441] Example B37. A formulation according to Example B35 or Example B36, wherein the cholesterol or its derivative is cholesterol, rapeseed sterol, β-phytosterol, brassosterol, ergosterol, dehydroergosterol, stigmasterol, phycosterol, DC-cholesterol HCl, OH-Chol, HAPC-Chol, MHAPC-Chol, DMHAPC-Chol, DMPAC-Chol, cholesterol chloroformate, GL67, cholesterol myristate, cholesterol oleate, cholesterol nervonic acid, LC10, cholesterol hemisuccinate, (3β,5β)-3-hydroxycholene-24-acid, alkyne cholesterol, 27-alkyne cholesterol, E-cholesterol alkyne, trifluoroacetate (Dios-Arg, 2H-Cho-Arg, or Cho-Arg), or a mixture thereof.

[0442] Example B38. A formulation according to any one of Examples B35 to B37, wherein the PEG-lipid is DMG-PEG, DSG-PEG, mPEG-DPPE, DOPE-PEG, mPEG-DMPE, mPEG-DOPE, DSPE-PEG-amine, DSPE-PEG, mPEG-DSPE, PEG PE, m-PEG-pentadecylene acid, bromoacetamido-PEG, amine-PEG, azide-PEG, or a mixture thereof.

[0443] Example B39. The formulation according to any one of Examples B33 to B38 further comprises an effective load.

[0444] Example B40. The formulation according to Example B39, wherein the effective load is a nucleic acid, compound, polypeptide, protein, polysaccharide or a combination thereof.

[0445] Example B41. The formulation according to Example B40, wherein the nucleic acid is RNA or DNA.

[0446] Example B42. The formulation according to Example B41, wherein the payload encodes a polypeptide.

[0447] Example B43. A formulation according to any one of Examples B39 to B42, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0448] Example B44. A formulation according to any one of Examples B39 to B43, wherein the effective load is a first effective load, and the composition further encapsulates a second effective load, wherein the second effective load is a nucleic acid, a compound, a polypeptide, a protein, a polysaccharide, or a combination thereof.

[0449] Example B45. The formulation according to Example B44, wherein the first effective load and the second effective load are different.

[0450] Example B46. A formulation according to any one of Examples B32 to B45, further comprising a pharmaceutically acceptable excipient, adjuvant, or combination thereof.

[0451] Example B47. A lipid nanoparticle comprising a membrane defining an internal space, wherein the membrane is formed of a plurality of lipid components comprising any one of Examples B1 to B31.

[0452] Example B48. The lipid nanoparticles according to Example B47, wherein the plurality of lipid components further comprise ionizable lipids, auxiliary lipids, or combinations thereof.

[0453] Example B49. Lipid nanoparticles according to Example B47 or Example B48, wherein the membrane is formed via hydrophobic interactions between the plurality of lipid components.

[0454] Example B50. Lipid nanoparticles according to Example B49, wherein the ionizable lipid comprises 8-[2-hydroxyethyl-(6-oxo-6-undecyloxyhexyl)amino]octanoic acid heptadecan-9-yl ester (SM-102) TM(4-Hydroxybutyl)azonide)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) TM (Pfizer) or a combination thereof.

[0455] Example B51. Lipid nanoparticles according to Example B49 or Example B50, wherein the auxiliary lipid comprises phosphatidylcholine, cholesterol or a derivative thereof, polyethylene glycol-lipid (PEG-lipid) or a mixture thereof.

[0456] Example B52. The lipid nanoparticles according to Example B51, wherein the phosphatidylcholine comprises distearate phosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DPOE), or a mixture thereof.

[0457] Example B53. Lipid nanoparticles according to Example B51 or Example B52, wherein the cholesterol or its derivative is cholesterol, rapeseed sterol, β-phytosterol, brassin, ergosterol, dehydroergosterol, stigmasterol, phycosterol, DC-cholesterol HCl, OH-Chol, HAPC-Chol, MHAPC-Chol, DMHAPC-Chol, DMPAC-Chol, cholesterol chloroformate, GL67, cholesterol myristate, cholesterol oleate, cholesterol nervonic acid, LC10, cholesterol hemisuccinate, (3β,5β)-3-hydroxycholene-24-acid, alkyne cholesterol, 27-alkyne cholesterol, E-cholesterol alkyne, trifluoroacetate (Dios-Arg, 2H-Cho-Arg, or Cho-Arg), or mixtures thereof.

[0458] Example B54. Lipid nanoparticles according to any of Examples B51 to B53, wherein the PEG-lipid is DMG-PEG, DSG-PEG, mPEG-DPPE, DOPE-PEG, mPEG-DMPE, mPEG-DOPE, DSPE-PEG-amine, DSPE-PEG, mPEG-DSPE, PEG PE, m-PEG-pentadecylene acid, bromoacetamido-PEG, amine-PEG, azide-PEG, or a mixture thereof.

[0459] Example B55. Lipid nanoparticles according to any one of Examples B47 to B54, wherein the membrane encapsulates the effective load.

[0460] Example B56. The lipid nanoparticles according to Example B55, wherein the effective load is a nucleic acid, compound, peptide, protein, polysaccharide or a combination thereof.

[0461] Example B57. The lipid nanoparticles according to Example B56, wherein the nucleic acid is RNA or DNA.

[0462] Example B58. The lipid nanoparticles according to Example B57, wherein the payload encodes a polypeptide.

[0463] Example B59. Lipid nanoparticles according to any one of Examples B55 to B58, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0464] Example B60. Lipid nanoparticles according to any one of Examples B55 to B59, wherein the effective load is a first effective load, and the membrane further encapsulates a second effective load.

[0465] Example B61. The lipid nanoparticles according to Example B60, wherein the second effective load is a nucleic acid, compound, polypeptide, protein, polysaccharide or a combination thereof.

[0466] Example B62. Lipid nanoparticles according to Example B60 or Example B61, wherein the first effective load and the second effective load are different.

[0467] Example B63. Lipid nanoparticles according to any one of Examples B47 to B62, which are made from the composition according to any one of Examples B29 to B43.

[0468] Example B64. Lipid nanoparticles according to any one of Examples B47 to B63, wherein the membrane is a bilayer structure.

[0469] Example B65. The lipid nanoparticles according to any one of Examples B47 to B64 have a diameter of 0.01 to 5 micrometers.

[0470] Example B66. Lipid nanoparticles according to any one of Examples B47 to B65, wherein the plurality of lipid components do not contain glycolipid C34 or α-galactosylceramide (α-GalCer).

[0471] Example B67. A formulation comprising lipid nanoparticles according to any one of Examples B47 to B66.

[0472] Example B68. The formulation according to Example B67, comprising 0.01 to 95% (w / w) of the lipid nanoparticles.

[0473] Example 69. The formulation according to Example 67 or Example 68, wherein the lipid nanoparticles are first lipid nanoparticles, and the composition further comprises second lipid nanoparticles.

[0474] Example B70. A formulation according to Example B69, wherein the first lipid nanoparticles and the second lipid nanoparticles differ in size, membrane composition, effective load encapsulated within the lipid nanoparticles, or a combination thereof.

[0475] Example B71. The formulation according to any one of Examples B67 to B70 further comprises an excipient, an adjuvant, or a combination thereof.

[0476] Example B72. The formulation according to Example B71, wherein the excipient comprises a solvent, dispersion medium, diluent, dispersion, suspending agent, surfactant, isotonic agent, thickener or emulsifier, preservative, polymer, peptide, protein, cell, hyaluronidase or mixture thereof.

[0477] Example B73. A formulation according to Example B71 or Example B72, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, aluminum salt, squalene, MF59, or QS-21. Other examples of adjuvants that can be used in some vaccines in the compositions of this disclosure are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), and CpG 1018 (Dynavax) or combinations thereof.

[0478] Example B74. A kit for preparing lipid nanoparticles, comprising: a first reagent comprising a compound according to any one of Examples B1 to B31; and a second reagent comprising an ionizable lipid, an auxiliary lipid, or a mixture thereof.

[0479] Example B75. The kit according to Example B74, wherein the second reagent comprises the ionizable lipid.

[0480] Example B76. The kit according to Example B75, wherein the kit further comprises a third reagent comprising an auxiliary lipid.

[0481] Example B77. A kit according to any one of Examples B74 to B76, wherein the ionizable lipid comprises 8-[2-hydroxyethyl-(6-oxo-6-undecyloxyhexyl)amino]octanoic acid heptadecan-9-yl ester (SM-102) TM(4-Hydroxybutyl)azonide)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) TM (Pfizer Inc.) or a combination thereof.

[0482] Example B78. A kit according to any one of Examples B74 to B77, wherein the auxiliary lipid comprises phosphatidylcholine, cholesterol or a derivative thereof, polyethylene glycol-lipid (PEG-lipid) or a mixture thereof.

[0483] Example B79. The kit according to Example B78, wherein the phosphatidylcholine comprises distearate phosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DPOE), or a mixture thereof.

[0484] Example B80. The kit according to Example B78 or Example B79, wherein the cholesterol or its derivative is cholesterol, rapeseed sterol, β-phytosterol, brassin, ergosterol, dehydroergosterol, stigmasterol, phycosterol, DC-cholesterol HCl, OH-Chol, HAPC-Chol, MHAPC-Chol, DMHAPC-Chol, DMPAC-Chol, cholesterol chloroformate, GL67, cholesterol myristate, cholesterol oleate, cholesterol nervonic acid, LC10, cholesterol hemisuccinate, (3β,5β)-3-hydroxycholene-24-acid, alkyne cholesterol, 27-alkyne cholesterol, E-cholesterol alkyne, trifluoroacetate (Dios-Arg, 2H-Cho-Arg or Cho-Arg), or mixtures thereof.

[0485] Example B81. The kit according to any one of Examples B78 to B80, wherein the PEG-lipid is DMG-PEG, DSG-PEG, mPEG-DPPE, DOPE-PEG, mPEG-DMPE, mPEG-DOPE, DSPE-PEG-amine, DSPE-PEG, mPEG-DSPE, PEG PE, m-PEG-pentadecylene acid, bromoacetamido-PEG, amine-PEG, azide-PEG, or a mixture thereof.

[0486] Example B82. The kit according to any one of Examples B74 to B81 further comprises a fourth reagent, the fourth reagent comprising a payload, wherein the payload is a nucleic acid, a compound, a polypeptide, a protein, a polysaccharide, or a combination thereof.

[0487] Example B83. The kit according to Example B82, wherein the nucleic acid is RNA or DNA.

[0488] Example B84. The kit according to Example B83, wherein the payload encodes a polypeptide.

[0489] Example B85. A kit according to any one of Examples B82 to B84, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0490] Example B86. A method for targeted delivery of a payload in an individual, comprising administering to the individual an effective amount of lipid nanoparticles according to any one of Examples 47 to 54, wherein the payload is a nucleic acid, compound, peptide, protein, polysaccharide, or combination thereof, and the payload is encapsulated by the lipid nanoparticles.

[0491] Example B87. The method according to Example B86, wherein the nucleic acid is RNA or DNA.

[0492] Example B88. The method according to Example B87, wherein the payload encodes a polypeptide.

[0493] Example B89. The method according to any one of Examples B86 to B88, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0494] Example B90. The method according to any one of Examples B86 to B89, wherein the effective load is a first effective load, and the membrane further encapsulates a second effective load, wherein the second effective load is a nucleic acid, a compound, a polypeptide, a protein, a polysaccharide, or a combination thereof.

[0495] Example B91. The method according to Example B90, wherein the first payload and the second payload are different.

[0496] Example B92. The method according to any one of Examples B86 to B91, wherein the lipid nanoparticles are applied together with excipients, adjuvants, or combinations thereof.

[0497] Example B93. The method according to Example B92, wherein the excipient comprises a solvent, dispersion medium, diluent, dispersion, suspending agent, surfactant, isotonic agent, thickener or emulsifier, preservative, polymer, peptide, protein, cell, hyaluronidase or mixture thereof.

[0498] Example B94. The method according to Example B92 or Example B93, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, aluminum salt, squalene, MF59, or QS-21. Other examples of adjuvants that can be used in some vaccines of the compositions of this disclosure are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), and CpG 1018 (Dynavax) or combinations thereof.

[0499] Example B95. A method for preventing or treating a disease in an individual, comprising administering to the individual an effective amount of lipid nanoparticles according to any one of Examples B47 to B54, wherein the effective load is a nucleic acid, compound, polypeptide, protein, polysaccharide, or combination thereof, and said effective load is encapsulated within said lipid nanoparticles; and wherein said effective load is a therapeutic agent or a derivative therapeutic agent.

[0500] Example B96. The method according to Example B95, wherein the nucleic acid is RNA or DNA.

[0501] Example B97. The method according to Example B96, wherein the payload encodes a polypeptide.

[0502] Example B98. The method according to any one of Examples B95 to B97, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0503] Example B99. The method according to any one of Examples B95 to B98, wherein the effective load is a first effective load, and the membrane further encapsulates a second effective load, wherein the second effective load is a nucleic acid, a compound, a polypeptide, a protein, a polysaccharide, or a combination thereof.

[0504] Example B100. The method according to Example B99, wherein the first payload and the second payload are different.

[0505] Example B101. The method according to any one of Examples B95 to B100, wherein the lipid nanoparticles are applied together with excipients, adjuvants, or combinations thereof.

[0506] Example B102. The method according to Example B101, wherein the excipient comprises a solvent, a dispersion medium, a diluent, a dispersion, a suspending agent, a surfactant, an isotonic agent, a thickener or emulsifier, a preservative, a polymer, a peptide, a protein, a cell, hyaluronidase, or a mixture thereof.

[0507] Example B103. The method according to Example B101 or Example B102, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, aluminum salt, squalene, MF59, or QS-21. Other examples of adjuvants that can be used in some vaccines in the compositions of this disclosure are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), and CpG 1018 (Dynavax) or combinations thereof.

[0508] Example B104. The method according to any one of Examples B95 to B103, wherein the lipid nanoparticles are administered at an initial dose, and subsequently administered once, twice, three times, four times, five times or more of additional doses.

[0509] Example B105. The method according to Example B104, wherein the additional dose is administered approximately one month, two months, three months, four months, five months, or six months or longer after the initial dose.

[0510] Example B106. The method according to any one of Examples B95 to B105, wherein the effective amount is in the range of about 5 μg to 1000 μg.

[0511] Example B107. A method for enhancing an adaptive immune response, comprising administering to an individual an effective amount of lipid nanoparticles according to any one of Examples B47 to B54, wherein the effective load is a nucleic acid, compound, polypeptide, protein, polysaccharide, or combination thereof, and said effective load is encapsulated within said lipid nanoparticles; and wherein said effective load is an immunogenic or derived immunogenic biomolecule.

[0512] Example B108. The method according to Example B107, wherein the nucleic acid is RNA or DNA.

[0513] Example B109. The method according to Example B108, wherein the payload encodes a polypeptide.

[0514] Example B110. The method according to any one of Examples B107 to B109, wherein the effective load is a first effective load, and the membrane further encapsulates a second effective load, wherein the second effective load is a nucleic acid, a compound, a polypeptide, a protein, a polysaccharide, or a combination thereof.

[0515] Example B111. The method according to Example B110, wherein the first payload and the second payload are different.

[0516] Example B112. The method according to any one of Examples B107 to B111, wherein the lipid nanoparticles are applied together with excipients, adjuvants, or combinations thereof.

[0517] Example B113. The method according to Example B112, wherein the excipient comprises a solvent, dispersion medium, diluent, dispersion, suspending agent, surfactant, isotonic agent, thickener or emulsifier, preservative, polymer, peptide, protein, cell, hyaluronidase or mixture thereof.

[0518] Example B114. The method according to Example B112 or Example B113, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, aluminum salt, squalene, MF59, or QS-21. Other examples of adjuvants that can be used in some vaccines of the compositions of this disclosure are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), and CpG 1018 (Dynavax) or combinations thereof.

[0519] Example B115. The method according to any one of Examples B107 to B114, wherein the lipid nanoparticles are administered at an initial dose, and subsequently administered once, twice, three times, four times, five times or more of additional doses.

[0520] Example B116. The method according to Example B115, wherein the additional dose is administered approximately one month, two months, three months, four months, five months, or six months or longer after the initial dose.

[0521] Example B117. The method according to any one of Examples B107 to B116, wherein the effective amount is in the range of about 5 μg to 1000 μg.

[0522] sequence

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Claims

1. A pharmaceutical formulation comprising a lipid nanoparticle and a pharmaceutically acceptable excipient; wherein the lipid nanoparticle is formed from a plurality of lipid components; wherein the plurality of lipid components comprises a bifunctional compound comprising: wherein R1comprises a substituted or unsubstituted glycoside group; wherein each of X1and X2is independently hydrogen, C 1-30 alkyl, C 1-30 alkenyl, C 1-30 alkynyl, aryl, aryloxy, or a substituted version thereof, or -(CH2)nX4, n is 0 to 30, and X4is hydrogen, aryl, aryloxy, heterocyclyl, or a substituted version thereof, provided that when X4is heterocyclyl, the heterocyclyl comprises 1 to 3 heteroatoms selected from the group consisting of O, S, and N, or a combination thereof; and wherein X3is hydrogen, C 1-6 alkyl or hydroxyl; wherein the lipid nanoparticle encapsulates a payload encoding a spike protein of a severe acute respiratory syndrome coronavirus (SARS-CoV), wherein the spike protein has a reduced and / or absent glycan profile.

2. The pharmaceutical formulation of claim 1, wherein the spike protein comprises an amino acid substitution of an asparagine acid (N) to glutamine (Q) at an N-linked glycosylation sequon (N-X-S / T), wherein X is any amino acid residue except proline, and S / T denotes a serine or threonine residue.

3. The pharmaceutical formulation of claim 1, wherein the spike protein comprises an amino acid deletion or addition at an N-linked glycosylation sequon (N-X-S / T) to eliminate an N-linked glycan sequon.

4. The pharmaceutical formulation of claim 1, wherein the spike protein comprises an amino acid substitution of S / T to alanine (A) at an O-linked glycosylation site to eliminate an O-linked glycosylation site.

5. The pharmaceutical formulation of claim 1, wherein the spike protein comprises the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, or 20, or an amino acid sequence that is at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, or 20; or the payload encoding a spike protein is an RNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 15, 17, or 19, respectively, or a nucleotide sequence that is at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence set forth in SEQ ID NO: 1, 15, 17, or 19, respectively.

6. The pharmaceutical formulation of claim 1, wherein the spike protein comprises the amino acid sequence set forth in SEQ ID NO: 4, 22, 24, or 26, or an amino acid sequence that is at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acid sequence of SEQ ID NO: 4, 22, 24, or 26, and wherein the spike protein comprises a receptor binding domain (RBD) that lacks at least one glycosylation site; or the payload encoding a spike protein is an RNA comprising the nucleotide sequence set forth in SEQ ID NO: 3, 21, 23, or 25, respectively, or a nucleotide sequence that is at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence set forth in SEQ ID NO: 3, 21, 23, or 25, respectively. The payload encoding the spike protein is RNA comprising the nucleotide sequence described in SEQ ID NO:3, 21, 23 or 25, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence described in SEQ ID NO:3, 21, 23 or 25, respectively.

7. The pharmaceutical preparation according to claim 1, wherein... The spike protein comprises the amino acid sequence set forth in SEQ ID NO:6, 28, 30, or 32, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology with the amino acid sequence of SEQ ID NO:6, 28, 30, or 32, and wherein the spike protein comprises an S2 subunit lacking at least one glycosylation site; or The payload encoding the spike protein is RNA comprising the nucleotide sequence described in SEQ ID NO:5, 27, 29 or 31, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence described in SEQ ID NO:5, 27, 29 or 31, respectively.

8. The pharmaceutical preparation according to claim 1, wherein... The spike protein comprises the amino acid sequence set forth in SEQ ID NO:8 or 34, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology with the amino acid sequence of SEQ ID NO:8 or 34, and wherein the spike protein comprises an S2 subunit, the S2 subunit comprising a single glycosylation site at N1194; or The payload encoding the spike protein is RNA comprising the nucleotide sequence described in SEQ ID NO:7 or 33, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence described in SEQ ID NO:7 or 33, respectively.

9. The pharmaceutical preparation according to claim 1, wherein... The spike protein comprises the amino acid sequence set forth in SEQ ID NO:10 or 36, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology with the amino acid sequence of SEQ ID NO:10 or 36, and wherein the spike protein comprises a receptor-binding domain (RBD) lacking at least one glycosylation site and an amino acid substitution of N801Q; or The payload encoding the spike protein is RNA comprising the nucleotide sequence described in SEQ ID NO:9 or 35, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence described in SEQ ID NO:9 or 35, respectively.

10. The pharmaceutical preparation according to claim 1, wherein... The spike protein comprises the amino acid sequence set forth in SEQ ID NO:12 or 38, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology with the amino acid sequence of SEQ ID NO:12 or 38, and wherein the spike protein comprises a receptor-binding domain (RBD) lacking at least one glycosylation site and an amino acid substitution of N1194Q; or The payload encoding the spike protein is RNA comprising the nucleotide sequence described in SEQ ID NO:11 or 37, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence described in SEQ ID NO:11 or 37, respectively.

11. The pharmaceutical preparation according to claim 1, wherein... The spike protein comprises the amino acid sequence set forth in SEQ ID NO:14 or 40, or has at least about 99%, 98%, 97%, 96%, 95%, 90%, or 85% of the amino acid sequence of SEQ ID NO:14 or 40. Or an 80% identical amino acid sequence, wherein the spike protein comprises a receptor-binding domain (RBD) lacking at least one glycosylation site and amino acid substitutions of N122Q, N165Q, N234Q, or combinations thereof; or The payload encoding the spike protein is RNA comprising the nucleotide sequence described in SEQ ID NO:13 or 39, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence described in SEQ ID NO:13 or 39, respectively.

12. The pharmaceutical compound of claim 1, wherein the payload encodes an immunogenic peptide, the immunogenic peptide comprising an amino acid sequence selected from the group consisting of: TESIVRFPNITNL (SEQ ID NO:41), NITNLCPFGEVFNATR (SEQ ID NO:42), LYNSASFSTFK (SEQ ID NO:43), LDSKVGGNYN (SEQ ID NO:44), KSNLKPFERDIST (SEQ ID NO:45), KPFERDISTEIYQAG (SEQ ID NO:46), GPKKSTNLVKNKC (SEQ ID NO:47), NCDVVIGIVNNTVY (SEQ ID NO:48), PELDSFKEELDKYFKNHTS (SEQ ID NO:49), VNIQKEIDRLNEVA (SEQ ID NO:50), NLNESLIDLQ (SEQ ID NO:51), and LGKYEQYIKWP (SEQ ID NO:52), or with SEQ ID NO. Any one of NO:41 to 52 has an amino acid sequence with at least about 99%, 98%, 97%, 96%, 95%, or 90% homology.

13. The pharmaceutical formulation of claim 1, wherein the spike protein comprises the amino acid sequence set forth in SEQ ID NO: 53, 54, 55, 56 or 57, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% homology with the amino acid sequence of SEQ ID NO: 53, 54, 55, 56 or 57.

14. The pharmaceutical formulation of claim 13, wherein the payload encodes an immunogenic peptide, the immunogenic peptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:58-SSANNCTFEYVSQ; SEQ ID NO:59-TESIVRFPNITNL; SEQ ID NO:60-KPFERDISTEIYQAG; SEQ ID NO:61-GPKKSTNLVKNKC; SEQ ID NO:62-TEVPVAIHADQ; SEQ ID NO:63-RVYSTGSNVFQTR; SEQ ID NO:64-RRARSVASQS; SEQ ID NO:65-DPSKPSKRSF; SEQ ID NO:66-FIKQYGDCLGDI; SEQ ID NO:67-ENQKLIANQFNS; SEQ ID NO:68-GKIQDSLSSTA; SEQ ID NO:69-NCDVVIGIVNNTVY; SEQ ID NO:70-PELDSFKEELDKYFKNHTS; SEQ ID NO:71-TSPDVDLGDISGINA; SEQ ID NO:72-VNIQKEIDRLNEVA; SEQ ID NO:73-NLNESLIDLQ; and SEQ ID NO:74-LGKYEQYIKWP; Or an amino acid sequence that is at least about 99%, 98%, 97%, 96%, 95% or 90% identical to any one of SEQ ID NO:58 to 74.

15. The pharmaceutical formulation of claim 1, wherein R1 comprises the formula R2-R A wherein R2 is the substituted or unsubstituted glycosyl group, and R A is a linking group, and wherein the linking group comprises an aryl group, an alkyl group, an amide, an alkylamide, substituted versions thereof, combinations thereof, or a covalent bond.

16. The pharmaceutical formulation of claim 15, wherein R A comprising an aryl group having 1 to 3 substituents, wherein the substituents are C 1-6 alkyl, halide, or C 1-6 alkyl halide.

17. The pharmaceutical formulation of claim 16, wherein R A Also included are polyethylene glycol (PEG) moieties having from 2 to 72 (OCH2CH2) subunits.

18. The pharmaceutical formulation according to claim 1, wherein the glycoside comprises mannoside, fucoidan, or a combination thereof.

19. The pharmaceutical formulation according to claim 1, wherein the glycoside comprises monomannoside, dimannoside, or trimannoside.

20. The pharmaceutical formulation of claim 1, wherein R1 is a substituted glycoside group, wherein the glycoside group comprises 1 to 6 substituents, wherein the substituents are C 1-6 alkyl, C 1-6 alkenyl, halogen, C 1-6 alkyl halide, C 1-6 alkoxy, amine, nitro, C 1-6 alkyl amine, amide, azido, aryl, cycloalkyl, heterocycloalkyl, sulfite, or a substituted version thereof, or a combination thereof.

21. The pharmaceutical compound according to claim 1, wherein R1 is selected from the group consisting of:

22. The pharmaceutical formulation of claim 1, wherein the compound has Formula 3: and R1 is selected from the following groups:

23. The pharmaceutical formulation according to claim 1, wherein at least one of X1 and X2 comprises a saturated hydrocarbon chain, said saturated hydrocarbon chain comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28 or 30 carbons.

24. The pharmaceutical formulation of claim 1, wherein X1and X2are each independently hydrogen, C 4-30 alkyl, C 4-30 alkenyl, C 4-30 alkynyl, aryl, aryloxy, or a substituted version thereof, or -(CH2)nX4, where n is 4 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic group or a substituted form thereof, provided that when X4 is a heterocyclic group, the heterocyclic group comprises 1 to 3 heteroatoms selected from the group consisting of O, S and N, or combinations thereof.

25. The pharmaceutical formulation of claim 1, wherein X4 is aryl, aryloxy, heterocyclyl, cycloalkyl, heterocycloalkyl, or combinations thereof, and wherein X4 comprises 0 to 6 substituents selected from the group consisting of C 1-6 alkyl, halogen, C 1-6 alkyl halogen, and C 1-6 alkoxy.

26. The pharmaceutical formulation of claim 25, wherein X4 is -R3-O-R4, wherein R3 and R4 are each independently aryl, heterocyclyl, cycloalkyl, heterocycloalkyl, each of which comprises 0 to 6 substituents selected from the group consisting of C 1-6 alkyl, halogen, C 1-6 alkyl halogen, and C 1-6 alkoxy.

27. The pharmaceutical preparation according to claim 1, wherein X4 is selected from the group consisting of:

28. The pharmaceutical compound according to any one of claims 1 to 27, wherein the compound is selected from the group consisting of:

29. The pharmaceutical formulation according to claim 1, wherein the component is not glycolipid C34 or α-galactosylceramide.

30. The pharmaceutical formulation according to claim 1, wherein the excipient comprises a solvent, a dispersion medium, a diluent, a dispersion liquid, a suspending agent, a surfactant, an isotonic agent, a thickener or emulsifier, a preservative, a polymer, a peptide, a protein, a cell, hyaluronidase, or a mixture thereof.

31. The pharmaceutical formulation of claim 1, further comprising an adjuvant, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, an aluminum salt, squalene, MF59, or QS-21. Other examples of adjuvants that can be used in some vaccines in the compositions of this disclosure are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), and CpG 1018 (Dynavax) or combinations thereof.

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